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Electronic Theses, Treatises and Dissertations
The Graduate School
2013
Effects of Vinegar Treatment on
Detectability and Allergenicity of Finfish
Ye Wang
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THE FLORIDA STATE UNIVERSITY
COLLEGE OF HUMAN SCIENCES
EFFECTS OF VINEGAR TREATMENT ON DETECTABILITY AND ALLERGENICITY OF
FINFISH
By
YE WANG
A Thesis submitted to the
Department of Nutrition, Food and Exercise Sciences
in partial fulfillment of the
requirements for the degree of
Master of Science
Degree Awarded:
Summer Semester, 2013
Ye Wang defended this thesis on June 24th, 2013.
The members of the supervisory committee were:
Yun-Hwa Peggy Hsieh
Professor Directing Thesis
Shridhar Sathe
Committee member
Ming Cui
Committee member
The Graduate School has verified and approved the above-named committee members, and
certifies that the thesis has been approved in accordance with university requirements.
ii
ACKNOWLEDGMENTS
I would like to express my sincerest appreciation to my advisor Dr. Yun-Hwa Peggy Hsieh for
all the opportunities she afforded throughout the past two years, without her support this work
would not have been completed. Her constant inspiration and insightful comments were
invaluable to my personal and professional development.
I also would like to thank Dr. Shridhar Sathe and Dr. Ming Cui for being on my thesis committee
despite their extremely busy schedule. Additionally, I would like to thank Dr. Jack Ofori, Dr. YiTien Chen, Dr. Yuhong Wang, Behnam Keshavarz, and William Fredericks for their willingness
to advise and help me in all areas throughout my course of study. And also thank you to my dear
friends Yitong Zhao and Jingjie Xiao for their kindness and help in my life.
Finally, I express my greatest thank to my mother Yonghua Wu for her unconditional love and
support. Without her, I would not have had the opportunity to study in the U.S and have this
wonderful life.
iii
TABLE OF CONTENTS
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
Abstract .......................................................................................................................................... ix
CHAPTER ONE: INTRODUCTION ..............................................................................................1
CHAPTER TWO: LITERATURE REVIEW ..................................................................................4
2.1
2.2
Fish Allergy ...................................................................................................................4
Fish Allergens ................................................................................................................5
2.2.1 Fish Parvalbumins ..............................................................................................5
2.2.2 Other Fish Allergens ..........................................................................................6
2.3 Effects of Processing on Food Proteins ..........................................................................7
2.3.1 Thermal Processing .............................................................................................7
2.3.2 Acid Processing .................................................................................................8
2.4 Detection Methods..........................................................................................................9
2.4.1 Diagnosis of Food Allergies ...............................................................................9
2.4.2 Detection of Allergenic Foods ..........................................................................11
CHAPTER THREE: HYPOTHESES AND OBJECTIVES..........................................................13
3.1 Hypotheses ...................................................................................................................13
3.2 Objectives .....................................................................................................................13
CHAPTER FOUR: MATERIALS AND METHODS ...................................................................14
4.1
4.2
Materials ......................................................................................................................14
Methods ........................................................................................................................15
4.2.1 Sample Preparation ...........................................................................................15
4.2.2 Protein Extraction .............................................................................................16
4.2.3 Indirect ELISA ..................................................................................................16
4.2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blot..........................................................................................................17
CHAPTER FIVE: RESULTS AND DISCUSSION ......................................................................18
5.1 Effects of Different Types of Vinegar on the Detectability of Whiting .......................18
5.2 Effects of Vinegar Treatment Time on the Detectability of Three Common Species of
Finfish ....................................................................................................................................19
5.3 Effects of Vinegar Treatment on Protein Banding Patterns of Finfish ........................20
5.4 Effects of Vinegar Treatment on Antigenic Protein of MAb 8F5 in Finfish ...............21
5.5 Effects of Vinegar Induced Chemical Reactions on the Detectability of Finfish ........22
5.6 Effects of Vinegar Treatment on the Allergenicity of Finfish .....................................24
5.7 Effects of Vinegar Induced Chemical Reactions on the Allergenicity of Finfish ........26
CHAPTER SIX: CONCLUSIONS ................................................................................................29
iv
APPENDIX ...................................................................................................................................30
A. TABLES AND FIGURES ........................................................................................................30
REFERENCES ..............................................................................................................................48
BIOGRAPHICAL SKETCH .........................................................................................................56
v
LIST OF TABLES
Table 1. Fish allergens ...................................................................................................................30
Table 2. Total soluble protein concentration of whiting, cod and red grouper ..............................31
Table 3. Clinical features of patients .............................................................................................31
Table 4. Immunoreactivity of MAb 8F5 against vinegar-treated fish samples determined by
iELISA ...........................................................................................................................................32
vi
LIST OF FIGURES
Figure 1. Effects of different types of vinegar on the detectability of whiting by MAb 8F5 basediELISA. ..........................................................................................................................................32
Figure 2. Effects of vinegar treatment on the detectability of whiting, cod and red grouper by
MAb 8F5-based iELISA ................................................................................................................33
Figure 3. SDS-PAGE profiles of vinegar-treated, water-treated and control samples of whiting,
cod and red grouper .......................................................................................................................34
Figure 4. Antigenic protein banding patterns of vinegar-treated, water-treated and control
samples of whiting, cod and red grouper .......................................................................................35
Figure 5. Study of the effects of vinegar induced chemical reactions on the detectability of
whiting, cod and red grouper (Group A) .......................................................................................36
Figure 6. Study of the effects of vinegar induced chemical reactions on the detectability of
whiting, cod and red grouper (Group B) .......................................................................................37
Figure 7. Study of the effects of vinegar induced chemical reactions on the detectability of
whiting, cod and red grouper (Group C) .......................................................................................38
Figure 8. Screen for IgE-binding reactivity of raw and cooked salmon and cod ..........................39
Figure 9. Effects of vinegar treatment on the IgE-immunoreactivity of whiting by human plasmabased iELISA .................................................................................................................................40
Figure 10. Effects of vinegar treatment on the IgE-immunoreactivity of cod by human plasmabased iELISA .................................................................................................................................41
Figure 11. Effects of vinegar treatment on the IgE-immunoreactivity of red grouper by human
plasma-based iELISA ....................................................................................................................42
Figure 12. Study of the effects of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group A) ..............43
Figure 13. Study of the effects of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group B) .............44
Figure 14. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting by human plasma-based iELISA (Group C) ...............................................45
Figure 15. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of cod by human plasma-based iELISA (Group C) ......................................................46
vii
Figure 16. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of red grouper by human plasma-based iELISA (Group C) .........................................47
viii
ABSTRACT
Fish provides a valuable source of many essential nutrients. However, it is also one of the “Big
Eight” allergenic foods that account for more than 90% of food allergic reactions, so reliable
detection of its presence is crucial. The addition of acid ingredients such as vinegar, lemon juice,
and tomato sauce has shown to markedly reduce the detectability of fish in an immunoassay using
a previously developed fish-specific monoclonal antibody (MAb 8F5), whose antigenic protein is
fish tropomyosin-a 36 kDa myofibrilar protein, and acid ingredients may also reduce fish’s
allergenicity. This study, therefore, focused on studying the effects of vinegar on the detectability
(assay immunoreactivity) and allergenicity of three commonly consumed fish species (whiting,
cod, and red grouper).
MAb 8F5 [Immunoglobulin G (IgG)] and human plasma [Immunoglobulin E (IgE)] from three
fish allergic patients were individually used to investigate the effects of vinegar on the detectability
and allergenicity of each fish sample, using indirect enzyme-linked immunosorbent assay
(iELISA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western
blot were used to reveal changes in the overall and antigenic protein banding patterns in vinegartreated samples.
The results of iELISA with MAb 8F5 demonstrated that vinegar dramatically reduced the
detectability of fish samples (up to 90% of the OD reading) when compared with water-treated
and non-treated control samples. SDS-PAGE results showed that the intensity of bands of vinegartreated samples became lighter than those of controls in all three fish species. The vinegar-treated
samples in western blot showed little or no band at 36 kDa, which agreed with the results of the
MAb-8F5 based iELISA. Considerable reductions of the OD readings were also apparent in all the
fish samples cooked (100 °C) with vinegar for 60 min when tested by IgE-based iELISA. However,
there were variations among species and subjects: of the three fish species tested, red grouper was
more resistant to vinegar treatment and the subject with a higher IgE concentration in plasma was
less affected by vinegar-induced alterations in the fish allergens. Moreover, the chemical reactions
that attribute to the vinegar’s effects on antigenic tropomyosin-IgG binding and fish allergen-IgE
binding are distinctively different. These results indicate that vinegar treatment of fish decreased
ix
the detectability of finfish using MAb 8F5-based iELISA via acidic precipitation of the antigenic
protein-tropomyosin, while the decreased allergenicity caused by vinegar was due to the acid
denaturation of the allergen.
x
CHAPTER ONE
INTRODUCTION
Fish, eggs, wheat, tree nuts, peanuts, soy beans, cow’s milk, and crustacean shellfish are the “Big
Eight” allergenic foods that account for 90% of allergic reactions to food in humans. Food allergy
is an adverse immunological response that is usually mediated by IgE and directed against a
specific protein or part of a protein in food. Although most food allergies cause relatively mild
symptoms, some can induce severe reactions and may even be life-threatening. In recent years, the
number of fish allergy incidences has increased worldwide, probably as a result of the increase in
fish consumption over same period.
The current treatment for a food allergy involves strict avoidance of the offending allergen. It is
usually suggested that people who are found to be allergic to one kind of fish avoid eating all kinds
of fish for the rest of their lives. However, eliminating fish from the daily diet is difficult. Fish and
fish ingredients are commonly found in many food products, including some unexpected sources.
For example, surimi (processed fish meat) is sometimes used as a substitute for beef or pork
(Musmand and others 1996); capsulates often contain fish gelatin; and isinglass, which is mainly
composed of fish collagen, is widely used as a fining agent in beer, wines, and champagnes (Taylor
and others 2004). Foods like salad dressing, Worcestershire sauce, bouillabaisse, and barbecue
sauce, may also be unexpected sources of fish allergens (FAAN website 2012). The unintentional
contamination of foods by fish and fish ingredients during manufacturing and household food
preparation can also cause reactions for individuals with a fish allergy. To help ensure the safety
of individuals with food allergies, the Food Allergen Labeling and Consumer Protection Act
(FALCPA) was enacted by Congress in August 2004 and became effective on January 1, 2006
(Public law 108-282). It requires manufacturers to declare on the label of packaged foods that
contain an ingredient that is or contains protein from the “Big Eight”. This new regulation has
highlighted the need for rapid tests capable of detecting the eight foods or food groups listed in
FALCPA as potential sources of allergens in food products. The monoclonal antibody (MAb) 8F5
has therefore been developed by Dr. Peggy Hsieh to detect fish protein in food products
(unpublished data). This antibody reacts with protein extracts of 55 common food fish species
without cross-reactivity with non-fish animal species and food protein additives. The antigenic
1
component recognized by MAb 8F5 is a heat stable protein with molecular weight of 36 kDa, later
identified as tropomyosin (Chen and Hsieh 2012). Preliminary data have indicated that acid
ingredients such as vinegar, lemon juice, and tomato sauce could reduce the immunoreactivity,
and thus the detectability, of cod in the MAb 8F5-based ELISA (Hou and Hsieh 2012). However,
there have been no reports of research into the effects of acid ingredients on the detectability of
finfish, which prompted us to initiate this study. MAb 8F5-based ELISA and immunoblot tests
were performed in order to determine the effects of vinegar on the immunoreactivity of three
common food fish species: whiting (Merlangius merlangus), cod (Gadus morhua), and red grouper
(Epinephelus morio). Whiting is the most commonly used inexpensive fish in surimi products, red
grouper is a popular fish, in high demand by consumers for its taste and texture, while cod is the
most commonly used fish species in allergen related scientific studies. These three species can be
easily obtained from a local market and are also habitually eaten with acid ingredients, which led
to their selection for this study.
Acid ingredients used as daily food additives may have the potential to decrease the allergenicity
of several allergens. For example, in skin prick tests administered to 18 pediatric and 26 adult
patients, shrimp soaked in vinegar prior to cooking produced a smaller wheal compared to shrimp
prepared conventionally (Perez-Macalalag and others 2007). Armentia and others (2010) reported
that the wheal areas of skin prick tests decreased significantly when the test foods (in this case,
egg and lentil) were treated with vinegar. This may indicate that adding vinegar during cooking
decreases the allergenicity of egg and lentil somewhat. Kim and others (2012) also reported a
reduced IgE binding capacity for the major peanuts allergens Ara h 1, Ara h 2, and Ara h 3 after
being treated with pH 1.0 acetic acid and commercial vinegar. However, the effect of acid
ingredients on the allergenicity of finfish are as yet unknown.
Finfish play an important role in many Americans’ daily diet, especially in coastal areas where
fish are more frequently consumed. Eliminating fish from the diet may lead to malnutrition and/or
eating disorders, so it is important to find an effective way to reduce the allergenicity of fish and
thus benefit those suffering from a fish allergy. Reduced allergenic finfish could be a good
candidate for oral immunotherapy for individuals with severe fish allergic reactions. It is also
possible that sensitive individuals who take anti-acid medication might face a particularly high
2
risk of fish allergy. In these sensitive individuals, proteins that are normally degradable might act
as food allergens in the presence of drugs that hinder peptic digestion (Pali-Schöll and JensenJarolim 2011). If the addition of acidic ingredients reduces the allergenicity of finfish, it might also
lower the risk of fish allergy for these patients. According to Thomas and others (2006), IgEbinding methods (e.g., IgE-based ELISA) are useful for investigating the allergenicity of food
before and after processing. Hence in this study plasma (IgE) from three fish allergic individuals
was used to compare the allergenicity of vinegar -treated and non-treated finfish.
3
CHAPTER TWO
LITERATURE REVIEW
2.1 Fish Allergy
An allergy is a hypersensitive disorder of the immune system. An allergic reaction is induced by
an allergen, which can be any kind of foreign substance, for example pollen, iron, or latex. Food
allergens are defined as those food components that induce the production of, and react with, IgE
to cause a release of mediators from mast cells and basophiles, resulting in immediate
hypersensitive reactions (Taylor and Lehrer 1996). The symptoms of food allergy can vary
considerably and include oral allergy syndrome (e.g., itching and angioedema of the lips, mouth
and pharynx), skin disorders (e.g., pruritus, angioedema, morbilliform rashes), and gastrointestinal
disorders (e.g., nausea, vomiting, gastric retention, intestinal hyper-motility, abdominal pain). At
its most dangerous, a food allergen can trigger a life-threatening episode of anaphylaxis.
Food is one of the most common causes of allergy, and fish is one of the leading causes of food
allergy. The prevalence of fish allergy varies between countries. A Norwegian study followed
3623 children born in the two main maternity clinics in the capital, Oslo. At one year of age, 1.2%
of these children were reported to be allergic to fish but this number increased over time, so that
by the age of two, 3.0% of these children were reported to be fish hypersensitive (Eggesbø and
others 1999). In Spain, a study of 355 food allergic children with a mean age of 5.4 years found
that 30% of these children had fish allergic reactions; another study reported fish to be the third
most common cause of food allergy among Spanish children (Crespo and others 1995; Boyano
and others 1987). Studies from Italy also showed that fish is one of the leading causes of serious
allergic reaction: among 54 episodes of food-induced anaphylaxis in children, 30% were caused
by fish (Novembre and others 1998). Asian countries have the highest rate of fish consumption in
the world, and as the world’s most populous continent, fish allergy affects a larger population there
(Hajab and Selamet 2012). Asian children consume fish for the first time at an earlier age than
anywhere else in the world, sometimes as young as seven months. Connett and others (2012)
reported that in Southeast Asia, the seafood allergy ratios were 2.29%, 0.26%, and 0.29% for 1416 year-old children from the Philippines, Singapore, and Thailand, respectively. Another study
4
conducted in northern Thailand indicated that shrimp, cow’s milk, and fish are the three leading
causes of allergy reactions among preschool children in that country (Lao-araya and
Trakultivakorn 2012). The frequency of fish allergy in Japan was 10.6% in pediatric patients with
a food allergy (Koyama and others 2006). In the U.S, more than one million people (about 0.4%
of the total population) are hypersensitive to fish (Munoz-Furlong and others 2004). An even
higher fish allergy ratio was found in the U.S. by a nationwide random telephone survey, where
2.3% of 5529 households (14,948 individuals) reported having an allergic reactions to fish
(Sicherer and others 2004). The percent of fish allergy among adults in Canada was 0.17%. (BenShoshan and others 2012). Differences in the geographical environment, eating habits, and food
processing methods may also be important risk factors for the development of fish allergy in
different populations.
2.2 Fish Allergens
2.2.1 Fish Parvalbumins
Parvalbumin has been identified to be the major fish allergen. Parvalbumins are a group of
intracellular calcium-binding muscle proteins with low molecule weight (10-13 kDa) that promote
relaxation in the fast-twitch muscle fibers (Rall 1996). They are divided into two different
phylogenic lineages, α and β, according to the composition of their amino acid sequences. β –
parvalbumins have been reported to be more allergenic than α-parvalbumins (Roquet and others
1992) and are responsible for the cross-reactivity among different fish species, including salmon,
whiff, perch, carp, eel, herring, plaice, catfish, grouper, snapper, pollock, wolffish, halibut, and
flounder. It has been found that parvalbumins react with specific IgE in more than 95% of fishallergic individuals, which strongly supports its identification as the major fish allergen
(Griesmeier and others 2010; Chatterjee and others 2006; Poulsen and others 2001; BugajskaSchretter and others 2000). Gad c1, the cod parvalbumin, is the first and most widely studied fish
allergen. It was first identified in Baltic cod (Gadus callarias) and has been well characterized and
sequenced. Another parvalbumin, Salmo salar (Sal s l), was found to be the major allergen in the
white muscle of Atlantic salmon (Lindstrom and others 1996). Parvalbumins identified as the main
allergen in other fish species are listed in Table 1. Parvalbumins share degrees of amino acid
homologies ranging from 60 to 80%, which may explain the clinical cross-reactivity of different
species of fish in fish-allergic patients.
5
The quantity of parvalbumin varies in different types of fish muscle. Generally the content of
parvalbumin is several times lower in dark muscle than in white muscle, which means the dark
muscle is less often implicated in fish allergy than the white muscle. Muscles near the tail have
also been found to contain less parvalbumin than the head and middle area of the fish. Tuna is
reported to exhibit a significant difference in parvalbumin content between the dorsal and ventral
side of their muscles (Coughlin and others 2007). This marked variation in the parvalbumin
concentration in different parts of the fish may be explained by the function of parvalbumin. In
general, fast twitching white muscles that are responsible for rapid movements contain high levels
of parvalbumin, while the dark muscles responsible for continuous swimming contain lower levels
of parvalbumin. Fish species such as tuna and swordfish that contain more dark muscle are
therefore expected to be less allergenic (Kuehn and others 2010).
Parvalbumins are very stable and have been shown to be resistant to heat, chemical denaturation,
and digestive enzymes (Aas and Elsayed 1969, 1971). Unlike most other heat-resistant food
allergens that contain linear epitopes, parvalbumins have conformational epitopes that are
stabilized by the interaction of metal-binding domains. Although the heat resistance of
parvalbumins has been demonstrated, the proteolytic stability of parvalbumins has been questioned
by a number of studies. For example, Untersmayer and others (2007) reported that the proteins of
cod degrade to small fragments after incubation with simulated gastric fluid for as little as 1 min.
Changes to enzyme concentration and pH could therefore have a large effect on the quantity of
parvalbumin and this is one of the factors that stimulated our interest in studying the effects of
vinegar on the allergenicity of finfish.
2.2.2 Other Fish Allergens
Unique IgE-binding bands have been reported at 46 kDa and at 40 kDa in yellow fin tuna (James
and others 1997; Yamada and others 1999) and at 25 kDa in swordfish (Kelso and others 1996).
Das Dores and others (2002) reported a 41 kDa allergen in addition to the 12 kDa parvalbumin in
codfish. This 41 kDa allergen was later identified as aldehyde phosphate dehydrogenase, which is
an enzyme located within the cell that is released from the cytosol after cell death, and this release
increases in non-frozen fish. Collagen (type I) was found in big eye tuna as a high molecular weight
6
allergen, and has also been shown to exist in Japanese eel, alfonsin, mackerel, and skipjack,
indicating that collagen is also a common allergen in several fish species. It was, however,
overlooked in early studies (Hamada and others 2001). Wang and others (2011) identified a 28
kDa protein as a new allergen of mackerel (Scomber japonicus) based on the result of IgEimmunoblotting, and this protein was later identified as triosephosphate isomerase (TPI). TPI was
also found to be an allergen in non-fish species such as lychee, wheat, latex, archaeopotamobius
(Archaeopotamobius sibiriensis), and crangon. A parasite called Anisakis simplex (Moneo and
others 2000) has also been proposed as a fish allergen. This parasite is highly resistant to heat and,
like other fish allergens, can cause severe allergic reactions including anaphylactic shock.
2.3 Effects of Processing on Food Allergens
Generally, in order to improve the quality (e.g., taste, texture, appearance, and preservation time)
and safety of food products, processing steps such as preparation, mechanical processes, separation,
isolation and purification, thermal processes, biochemical processes, high-pressure treatment,
electric field treatment, and irradiation may be involved in the processing of raw material to
produce ready-to-eat foods (Thomas and others 2007). These processes can work exclusively or
together with other factors to influence the allergenicity of food, and can be generally categorized
into two types: thermal and non-thermal processing. Thermal processing methods include boiling,
steaming, frying, grilling, roasting, baking, drying, and pasteurization, while germination,
fermentation, proteolysis, ultrafiltration, enzymatic tissue disintegration, pulping, peeling, and
mashing are classified as non-thermal processing methods (Besler and others 2001; Sathe and
others 2005).
2.3.1 Thermal Processing
The effects of thermal processing on food proteins, especially food allergens, have been
extensively studied. Thermal processing was found to be effective in reducing the IgE-binding
reactivity of several well-known allergens. Food allergens such as Mal d 1 apples (Bohle and others
2006), Api g 1 from celery (Jankiewicz and others 1997), and Cor a 1.04 from hazelnuts (Pastorello
and others 2002) have all been reported to be heat sensitive. After thermal processing, for example
roasting, they may lose some or all of their allergenicity. This reduction in allergenicity can be
7
explained by the alteration of the conformation of heat liable proteins when exposed to heat, and
thus the loss of epitopes.
Many other food allergens have been reported to be heat resistant. For instance, Pru p 1 from
peaches (Brenna and others 2000), and Ara h 1 from peanuts (Nordlee and others 1981) are all
highly resistant to heat. Fish allergens have also been reported to be heat resistant. ParvalbuminLep h 1 is the major allergen in whiff, and Griesmeier and others (2010) reported that an extract
of cooked whiff samples actually has a higher number of IgE reactive bands and exhibits a higher
resistance to pepsinolysis than extracts from raw samples. The exposure of inside epitopes and the
formation of aggregates and polymers by heating may explain this increased allergenicity of
cooked whiff. Other parvalbumins, for example Gad c 1 in cod, have also been reported to be
resistant to heat (Aas and Elsayed 1969). Changes in the food matrix can influence the effect of
heat on allergens, and this effect varies with fish species. Chatterjee and others (2006) reported
that the allergenicity of boiled and fried extracts of mackerel, pomfret, and hilsa were considerably
reduced using IgE based competitive ELISA, while the highest allergenicity of bhetki was found
in the fried extract. The increased allergenicity of fried bhetki may be caused by the exposure of
new epitopes or the Maillard reaction. Like the major allergen in peanuts, Ara h 1 and Ara h 2
bound higher levels of IgE and were more resistant to heat and digestion after they had been
subjected to the Maillard reaction. Roasted peanuts from two different sources bound IgE from
patients with a peanut allergy at approximately 90-fold higher levels than raw peanuts from the
same source (Maleki and others 2000).
2.3.2 Acid Processing
The effects of non-thermal processing on food proteins can vary largely depending on the food
type and processing method. Acid processing has been reported to be effective in reducing the
allergenicity of several different kinds of foods. Perez-Macalalag and others (2007) examined the
effect of vinegar soaking on allergenicity of the major shrimp allergen Sa-II through a skin prick
test (SPT), and reported that mean wheal diameters obtained using shrimp extract that had been
prepared with preliminary vinegar soaking were significantly smaller than mean wheal diameters
obtained using a conventionally prepared extract. Sletten and others (2010) also reported that
pickled herring products (rollmops, Bismarck-herring, brathering and jellied herring) which had
8
been prepared in an acetic acid-salt brine showed decreased IgE binding in 3/5 of the sera by using
IgE based- competitive ELISA. They also found that other types of processing, such as lye
treatment, sugar curing, and salting, may also decrease the allergenicity of fish. A decreased
allergenicity of vinegar-treated peanuts was reported by Kim and others (2012). IgE-binding
intensities for Ara h 1, Ara h 2, and Ara h 3 were significantly reduced after being treated with pH
1.0 acetic acid or commercial vinegar; the local habit of consuming peanuts with vinegar may thus
explain the low prevalence of peanut allergy in Korea. The change in allergenicity can be explained
by an alteration in the allergen structures after treatment with vinegar. A partial loss of structure
of Lep w 1 in whiff was observed at acidic pH (Griesmeier and others 2010) and Neti and Rehbein
(2000) proposed that the proteolytic degradation of parvalbumins by the activity of acidic protease
(cathepsin D) may also explain the disappearance of bands in isoelectric focusing (IEF) gel.
Several reports have indicated that protease treatment might be the most effective way to destroy
the structure of allergens: proteolytic processing has been used to reduce the allergenicity of soy
(Yamanishi and others 1996), wheat flour gluten (Watanabe and others 1995), and milk (Thomas
and others 2007). However, this must be approached with caution, as incomplete hydrolysis may
prevent protease treatments from completely destroying all the epitopes present.
It is possible that acid treatment can alleviate allergic reactions through mechanisms other than
changing the content and structure of the allergens. Armentia and others (2010) considered the
possibility that vinegar could decrease allergenic response in lentils and eggs by decreasing the
gastric pH and thus enhancing the function of digestion and reducing the allergenicity. The
importance of pH and its effect on food allergies has also been indicated by a study conducted by
Pali-Schöll and Jensen-Jarolim (2011), which suggested that gastric acid levels determine the
activation of gastric pepsin and also the release of pancreatic enzymes. When antacid drugs inhibit
or neutralize gastric acid, the likelihood of eliciting allergic reactions via the oral route will be
dramatically increased.
2.4 Detection Methods
2.4.1 Diagnosis of Food Allergies
A diagnosis of a food allergy usually begins with a physical examination and a discussion of the
individual’s medical history. These can determine whether the patient has a food-induced allergic
9
disorder and whether an IgE-mediated or non-IgE-mediated mechanism is most likely to be
responsible. When IgE-mediated allergic reactions are suspected, laboratory evaluations (e.g., a
skin prick test (SPT), radioallergosorbent test (RAST), and/or an enzyme-linked immunosorbent
assay (ELISA)) and oral challenges (e.g., a double-blind placebo-control food challenge
(DBPCFC)) can be used to pinpoint the specific foods causing the allergic reaction.
SPTs are most frequently used to screen patients with food allergies. Glycerinated food extracts
and appropriate positive (histamine) and negative (saline) controls are applied by a pricking
technique. Foods producing wheals at least 3mm larger than wheals induced by a negative control
are considered positive. The predictive accuracy of a positive SPT response is less than 50%
compared with DBPCFC, but the predictive accuracy of a negative SPT response is greater than
95%. Therefore, SPT should be combined with medical history and oral challenges to diagnose
patients who are allergic to specific foodstuffs (Sampson 1999). The DBPCFC, however, remains
the gold standard for food allergy diagnosis. The food challenge is administered in a fasting state,
and the potential allergen is gradually fed to the patient under supervision. It usually begins with
a dose that is unlikely to provoke symptoms (25 to 500 mg of lyophilized food), and the level is
then doubled every 15 to 60 minutes. Once the patient has tolerated 10 g of lyophilized food
blinded in capsules or liquid, clinical reactivity is generally ruled out (Sampson 1999). To confirm
the negative result, an open feeding under observation is needed. SPT and food-specific IgE levels
are used to assess the risks and benefits of conducting a food challenge. However, in the clinical
setting, open oral challenges eliciting no symptoms (negative challenge) or objective symptoms
confirming the history can be considered diagnostic (Sampson 1999).
In vitro assays such as ELISA involve the use of allergen-specific IgE antibodies, which are
principle components in food-allergic reactions. In the most frequently used allergosorbent-type
of assay, after the allergen has been immobilized on a plate, the serum or plasma sample is added
and the plate incubated for a pre-determined period. The immobilized allergen will bind to its
specific IgE antibody, allowing the bound IgE to be detected with an anti-IgE antibody detection
reagent. In the case of ELISA, an enzyme is conjugated to the anti-IgE antibody, but other types
of assay such as sandwich ELISA or latex flow may also be used depending on the application. In
all the commonly used commercial assay systems utilizing immobilized antigens, a standard curve
10
based on purified IgE calibrators is established and used to convert assay signals to mass units of
allergen-specific IgE, given in International Units (IU) per mL of serum or plasma (Asero and
others 2007). When interpreting the results of in vitro ELISA, strong positive results are associated
with clinical sensitivity, while completely negative results are associated with clinical tolerance
(Dorizzi and others 1999). The form of the allergen preparation can have a major effect on the
performance of a test for allergen-specific IgE. Moreover, allergen-specific IgE can be applied to
determine the allergenicity of a food or a food product. Thomas and others (2006) argued that IgE
binding methods are useful for 1) investigating the allergenic potential of proteins; 2) permitting
the comparison of foods before and after processing, and 3) improving the management of
allergenic risk from foods. IgE obtained from food allergic individuals can also be used to detect
allergens in food products. However, the amount of IgE available from sensitized individuals is
usually limited and cross-reactivity to more than one allergenic food may be a problem in human
serum IgE. Therefore, rather than IgE, allergen-specific IgG raised in animals with high specificity
is commonly used in standardized and commercially produced food allergen detection methods
(Besler 2001).
2.4.2 Detection of Allergenic Foods
In order to fulfill the ever increasing demand to confirm the validity of allergen label statements
and assess the risk to food-sensitive consumers, two analytic approaches have become common in
recent years. The first involves immunoassays and DNA-based assays such as ELISA and realtime polymerase chain reactions (RT-PCR), both of which are used for large scale and rapid
screening. The other approach is more detailed and uses multi-analyte methods such as liquid
chromatography–mass spectrometry (LC-MS), which make it possible to carry out full
quantification and confirmation of the analytes of interest.
Immunoassay and DNA-based assays are the most developed and frequently used methods for
detecting allergenic foods and ingredients. Immunoassay’s high precision, simple handling, high
throughput, and good potential for standardization make it a good option for routine food allergen
detection (Monaci and Visconti 2010). The DNA-based methods use primers (short stretches of
DNA) to facilitate the amplification of DNA originating from the offending food, which is
followed by staining the amplified product with a fluorescent dye or using Southern blotting
11
following electrophoresis in an agarose gel to visualize the result. However, using the allergic
protein itself is preferred to using markers that indicate the existence of allergic proteins, so
immunoassays targeting food allergens such as ELISA and lateral flow devices (dipsticks) have
become important tools for detecting food allergens. The first step in developing an immunological
detection method for routine food analysis is the identification and purification of target allergens.
Polyclonal antibodies for the detection of this specific allergenic protein can then be raised in
animals like rabbits, rats, goats, sheep or chickens. Monoclonal antibodies, which are usually IgG
and capable of recognizing only one epitope, are better suited for the recognition of a specific
antigen. ELISA, which is based on a colorimetric reaction following binding with a specific
enzyme-labeled antibody, has been widely used in the detection of allergenic foods and food
allergens since its introduction in the 1980s. Commercial ELISA kits against a great variety of
food allergens with detection limits ranging from 0.05 to 10 mg kg−1, depending on the allergen
and the food matrix, have now been developed (Schubert-Ullrich and others 2009). However, it is
important to note that both thermal and non-thermal food processing may lead to changes in targetprotein structure, and thus significantly affect the result of immunoassays.
Multi-analyte methods, for example mass spectrometry (MS)-based proteomics methods, are
usually used for final confirmation and quantification of the presence of an allergen in different
foodstuffs. In mass spectrometry-based proteomics methods, complete protein sequencing is both
time consuming and difficult to achieve. Specific peptides are used to confirm the presence of
target allergens in food samples. Therefore, prior to submitting samples for MS analysis to identify
intact proteins, pre-fractionation techniques including proteolytic digestion (typically by trypsin)
or advanced liquid chromatography (LC) are performed to separate the peptides. The extract can
then be subjected to MS or tandem-MS (MS2) to obtain a complete sequence of the peptides of
interest. To identify these peptides, spectra are scanned against protein-sequence databases using
search algorithms (Monaci and Visconti 2009). These methods are very sensitive and have been
used to detect the peanut allergen- Ara h 1 in chocolate, with a detection limit as low as 2 ppm
(Shefcheck and others 2006). However, for samples with a high protein content, the results of MSbased proteomics methods may affected by the levels of other proteins in solution and a falsenegative result would be likely if a high content of other proteins masks the peptide signals from
the allergenic protein.
12
CHAPTER THREE
HYPOTHESES AND OBJECTIVES
3.1 Hypotheses
1. If vinegar can influence the detectability of fish proteins, the results of anti-fish monoclonal
antibody (MAb 8F5)-based iELISA and immunoblot against vinegar-treated fish samples will be
different compared with water-treated and non-treated control samples.
2. If vinegar can affect the allergenicity of fish allergens, the results of iELISA using human plasma
(IgE) against vinegar-treated fish samples will be different compared with water-treated and nontreated control samples.
3. If the effects of vinegar on both the detectability and allergenicity of fish are due to the same
chemical reaction, which may consist of acid denaturation, acid proteolysis or acidic precipitation
of antigenic/allergenic protein, their resulting patterns should be similar.
3.2 Objectives
The overall goal of this research was to investigate the effects of vinegar treatment on detectability
by IgG-based immunoassay, and allergenicity by IgE-based immunoassay of whiting, cod and red
grouper.
The specific objectives were to: 1) study the effect of vinegar on the detectability of cooked fish
using anti-fish monoclonal antibody (MAb 8F5)-based iELISA and Western blot; 2) identify the
effect of vinegar on the allergenicity of cooked fish using human plasma (IgE)-based iELISA; and
3) study the chemical action that attribute to the effects of vinegar on fish muscle proteins and fish
allergens.
13
CHAPTER FOUR
MATERIALS AND METHODS
4.1 Materials
White vinegar (Heinz), and fresh whiting, cod, and red grouper were purchased from the Publix
supermarket in Tallahassee, Florida. The fish samples were stored at -20 oC in a freezer until use.
MAb 8F5 was previously developed in our laboratory. Hydrogen peroxide, horseradish peroxidase
conjugated goat anti-mouse IgG (Fc specific), ABTS (2,2′-azino-bis 3-ethylbenzthiazoline- 6sulfonic acid), and β-mercaptoethanol were purchased from Sigma-Aldrich Co., St. Louis, MO.
Bromophenol blue sodium salt was purchased from Allied Chemical Corporation, New York.
Sodium chloride (NaCl), sodium phosphate dibasic anhydrous (Na2HPO4), sodium phosphate
monobasic anhydrous (NaH2PO4), bovine serum albumin (BSA), sodium bicarbonate (NaHCO3),
sodium carbonate (Na2CO3), citric acid monohydrate, sodium dodecyl sulfate, Tween 20 and all
other chemicals, reagents, filters (Whatman No. 1 and No. 4 paper), and 96 well polystyrene
microplates (Costar 9018) were purchased from Fisher Scientific, Fair Lawn, NJ. 0.5 M Tris-HCl
buffer (pH 6.8), 1.5 M tris-HCl (pH 8.8), TEMED (N,N,N,N′-tetra-methyl ethylenediamine),
Precision Plus Protein Kaleidoscope Standards, 30% acrylamide/ bis solution, Tris buffer saline
(TBS), Tris/glycine buffer, 10 ×Tris/glycine/SDS buffer, supported nitrocellulose membrane (0.2
μm), and thick blot paper were purchased from Bio-Rad Laboratories Inc., Hercules, CA. All
solutions were prepared using distilled deionized pure water (DDI water) from the NANO pure
Diamond ultrapure water system (Barnstead International, Dubuque, IA). All chemicals and
reagents were analytical grade. Plasma samples from three fish allergic patients (Table 3) were
purchased from International Plasma Lab, WA. Horseradish peroxidase conjugated mouse antihuman IgE (ε chain specific) secondary antibody was purchased from Southern Biotech
(Birmingham, Al), and alkaline phosphatase conjugated mouse anti-human IgE secondary
antibody was purchased from Thermo Scientific (Pittsburgh, PA).
14
4.2 Methods
4.2.1 Sample Preparation
Fish samples were prepared differently to satisfy the research requirements of the various
experimental objectives.
In order to study the effects of vinegar treatment and treatment time on the detectability and
allergenicity of finfish, 4 g portions of half thawed frozen fish muscle from each species were
weighed into beakers. Vinegar or water was added to the samples in the ratio of 1:2 w/v, and the
samples soaked for <1 min, 15 min, 30 min, and 60 min. A blank control received no processing.
After treatment, the liquid was drained and the samples rinsed with D.I water and patted dry on a
clean paper towel. The beakers containing muscle samples were then covered with aluminum foil,
sealed with adhesive tape, and cooked for 5 min in a boiling water bath.
To study the chemical reactions that caused by vinegar treatment on the detectability and
allergenicity of finfish, three groups of samples were prepared in different ways. In Group A, 4 g
portions of partially-thawed frozen fish muscle were weighed into beakers. Vinegar or water was
added to the samples in the ratio of 1:2 w/v, and the samples soaked for <1 min, 15 min, 30 min,
and 60 min. A blank control received no processing. After treatment, the liquid was drained and
the samples rinsed with D.I water and patted dry on a clean paper towel. The beakers containing
muscle samples were then covered with aluminum foil, sealed with adhesive tape, and cooked
(100°C) for 5 min in a boiling water bath. In Group B, 4 g portions of half-thawed frozen fish
muscle were weighed into beakers. After cooking (100°C at 5 min), vinegar or water was added
to the samples in the ratio of 1:2 w/v and soaked for <1 min, 15 min, 30 min, and 60 min. The
liquid was drained and the samples rinsed with D.I water and patted dry on a clean paper towel.
Control samples were cooked (100°C at 5 min) with no treatment. In Group C, 4 g portions of half
thawed frozen fish muscle were weighed into beakers. Vinegar or water in the ratio of 1:2 w/v was
added to the samples and the beakers covered with aluminum foil, sealed with adhesive tape, and
immediately cooked (100°C) for 5, 15, 30, and 60 min. Control samples were cooked for 5, 15, 30,
60 min with no treatment.
15
4.2.2 Protein Extraction
All prepared samples were mashed into small pieces using a glass rod and scissors after cooling to
room temperature. Extraction buffer (0.15 M NaCl) in the ratio 1:5 w/v was then used to extract
the soluble proteins. Each sample was homogenized at 1000 rpm (Model Ultra-turrax T25 basic,
IKA) for 2 min followed by 1 hour incubation at 4°C. After incubation, samples were centrifuged
(Model 5810R, Eppendorf) at 3220×g at 4°C for 30 minutes. Supernatants obtained after
centrifugation were filtered through Whatman No.4 filtration paper and stored at -20 °C for later
use.
4.2.3 Indirect ELISA
Extracts of the fish samples were tested using iELISA to examine any changes in their
immunoreactivities. As described earlier, MAb 8F5 supernatant and human plasma from fish
allergic individuals were utilized as the source of primary antibodies, while horseradish
peroxidase-conjugated goat anti-mouse IgG-Fc specific antibody (1:3000) and horseradish
peroxidase conjugated mouse anti-human IgE (ε chain specific) antibody (1:1000) were used as
the secondary antibodies for the studies of detectability and allergenicity, respectively. The ELISA
procedure proceeded as follows: 2 μg/100μL of sample protein extract diluted in 0.06 M carbonate
buffer (pH 9.6) was coated onto each well of a 96-well polystyrene microplate and incubated at
37°C for 1 hour. The plate was then washed three times with PBST (0.05% v/v Tween-20 in 10
mM PBS, pH 7.2) and incubated with 200 μL/well blocking solution (1% BSA in 10 mM PBS) at
37°C for 1 hour, followed by another washing step. Properly diluted sample protein extracts in
0.06 M carbonate buffer (pH 9.6) were coated (2 μg/100 μL per well) onto the wells of a 96-well
polystyrene microplate and incubated at 37°C for 1h. After washing three times with PBST, 100
μL of the secondary antibody was added to each well and the plate was incubated at 37°C for 1
hour. After incubation, the plate was washed 5 times and then 100 μl/well ABTS substrate solution
was added to the wells, color was developed at room temperature for 20 min, then 100 μl/well of
0.2 M citric acid was added to stop the reaction. The absorbance was read at 415 nm (Model
MQX200R, BioTek).
16
4.2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and
Western Blot
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were
used to reveal any changes in the antigenic component. SDS-PAGE was performed to resolve the
soluble proteins in different sample extracts according to the method of Laemmli (1970), with
modifications. Briefly, soluble proteins in fixed volumes (15 μl per lane) from the sample extracts
were loaded on 5% stacking gel (pH 6.8) and separated on 12% polyacrylamide separating gel (pH
8.8). The gel was subjected to electrophoresis at 200 V for 45 min using a Mini-Protein 3
electrophoresis cell (Model 161- 3301, Bio-Rad) connected to a power supply (Model 3000, BioRad).
The Western blot assay was carried out according to the method of Towbin and others (1979) with
modifications. After separation of the proteins through SDS-PAGE, protein bands were transferred
electrophoretically (1 hour at 300mA) from the gel to nitrocellulose membranes using a
MiniTrans- Blot unit (Bio-Rad). After completion of the transfer, the membrane was washed with
TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5), and then blocked with 1% BSA in
TBS for 1 hour. After another washing step, the membrane was incubated with the primary
antibody for 1 hour. The membrane was washed with TBST to remove excess antibody and then
incubated for 1 hour at room temperature with the secondary antibody (goat anti-mouse IgGalkaline phosphatase conjugate diluted 1:3000 in antibody buffer). After washing, the membrane
was incubated with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride
(BCIP/NBT) in 0.1 M Tris buffer (pH 9.5) for about 3 minutes to develop the color. D.I water was
applied to stop color development. The appearance of a dark purplish band indicated the antibodybinding site. Prestained broad range protein standards (Precision Plus Protein Kaleidoscope
Standards, Bio-Rad, 161-0375) were used as molecular weight markers for both the SDS-PAGE
and Western blot.
17
CHAPTER FIVE
RESULTS AND DISCUSSION
5.1 Effects of Different Types of Vinegar on the Detectability of Whiting
A preliminary study tested the effects of vinegar, lemon juice and tomato sauce on the
immunoreactivity of cod (Hou and Hsieh 2012). The results showed that among these three acid
ingredients, vinegar has the strongest capacity to reduce immunoreactivity and thus the
detectability of cod by 8F5-based ELISA. Therefore, in this study vinegar was selected as the acid
ingredient used in all the subsequent experiments to study the effect of acid on the detectability of
fish samples. In order to ascertain whether there was any variation between different types of
vinegar, MAb 8F5-based iELISA tests were performed on whiting samples treated by five different
types of vinegar (white, red wine, garlic wine, rice, and apple vinegar). Raw muscle of whiting
was soaked with vinegar in the ratio of 1:2 m/v for 30 min, and then cooked (100C) for 5 min.
The results are shown in Figure 1.
The OD reading decreased dramatically in all the vinegar-treated samples compared with the
water-treated and non-treated control samples. The sample treated with rice vinegar showed the
lowest OD reading (0.45±0.02), while the OD readings of the water-treated and non-treated control
samples were 1.93±0.10 and 1.78±0.00, respectively. There was no considerable difference
amongst the OD readings of the samples treated by different types of vinegar, indicating that these
vinegar products all had a similar effect on reducing the detectability of whiting. The pH of these
vinegars ranged from 2.5 to 3.0. It is plausible that the acid environment caused by adding vinegar
leads to the change in the detectability of whiting. Therefore, the similarity in pH values may
explain the similar effects of the different types of vinegar. Considering that white vinegar is the
most widely consumed vinegar in the U.S., fish samples in the following studies were all treated
with white vinegar.
18
5.2 Effects of Vinegar Treatment Time on the Detectability of Three Common Species of
Finfish
As described earlier, MAb 8F5 is a previously developed fish-specific monoclonal antibody for
fish protein detection. To study the effects of vinegar treatment as well as the treatment time on
the detectability of different fish species (whiting, cod and red grouper), the total soluble protein
concentration of fish samples was determined first. For fish samples treated by vinegar for different
time lengths, less than 1 min treatment was indicated to be most effective in decreasing the total
soluble protein concentration (Table 2). Comparing to water-treated control, decreases of 88.9%,
28.0%, 59.3% in the protein concentrations were observed in vinegar treated (<1 min) whiting,
cod and red grouper sample, respectively, indicating that the amount of antigenic fish muscle
protein present may also have decreased. However, the prolonged vinegar treatment time did not
further intense the decrease trend of crude protein concentrations. In opposite, the total soluble
protein concentrations of vinegar treated samples increased with the increase of treatment time.
The extracts of vinegar treated and control fish samples were examined using the MAb 8F5-based
iELISA. The results are shown in Figure 2. In general, vinegar treatment reduced the
immunoreactivity in the fish samples from all three fish species dramatically (up to 90% of the
OD reading). As shown in Figure 2, the detectability of the vinegar-treated samples decreased
markedly compared with the water-treated and non-treated control samples. After treatment with
vinegar for 60 min, decreases of 81.3%, 76.3%, and 71.3% in the OD readings were observed in
the whiting, cod and red grouper, respectively. These decrease in the OD readings indicated a
lower detectability of the antigenic protein, either due to the decreased amount of the antigen
present or the destruction of the epitope structure of the target antigen. As previously reported
(Chen and Hsieh 2012), the antigenic protein of MAb 8F5 is fish tropomyosin, a 36 kDa
myofibrilar protein. Therefore, vinegar treatment induced changes in tropomyosin, either by
altering its conformation or by lowering its solubility.
Tropomyosin is an essential protein in muscle contraction, both in invertebrates and vertebrates.
Fish tropomyosin is not allergenic, whereas shrimp tropomyosin is the major allergen in shellfish
and is responsible for the cross-reactivity between crustaceans (Zhang and others 2006) and
mollusks (Taylor 2008), as well as insects and mites (Witteman and others 2009; Ree and others
19
1996). Several MAbs against tropomyosin have been documented (Jeounga and others 1997; Lu
and others 2007; Werner and others 2007), but there have been no reports of the effects of acid
ingredients or pH value on the detectability of shellfish using immunochemical assays. However,
in a study to detect mustard in salad dressing, Lee and others (2009) found acidic salad dressing
matrices markedly reduced the detectability of mustard by ELISA. When vinegar was spiked with
mustard flour at pH 3, 3.5, and 4, the detectability of mustard decreased considerably, with the
lowest detectability at pH 3. The authors considered that this reduced detectability was probably
due to the acid precipitation of mustard proteins, which rendered them insoluble and nonextractable.
As shown in Figure 2, the overall immunoreactivity of vinegar-treated whiting, cod, and red
grouper samples was generally much lower than their water-treated and non-treated counterparts.
However, the vinegar treatment time that produced the greatest change in detectability varied
among the three fish species. For whiting, the peak decrease in the OD reading appeared in the
sample treated with vinegar for 15 min (a 90% reduction), whereas whiting samples treated with
vinegar for 30 min or longer showed slight increases in immunoreactivity. Compared with the
whiting sample treated with vinegar for 15 min, which showed a negative iELISA result (OD
reading <0.2), the iELISA result was positive (OD reading= 0.35±0.03) after 60 min treatment.
For cod and red grouper, the OD reading decreased 97% for both samples treated for less than 1
min and an increase in the OD reading can also be seen in the cod and red grouper samples treated
with vinegar for longer times. Long vinegar treatments may expose hidden epitopes, making them
more accessible for antibody binding or the formation of new epitopes, which would both yield
increased immunoreactivity and could explain the increasing trend observed among vinegartreated samples.
5.3 Effects of Vinegar Treatment on Protein Banding Patterns of Finfish
SDS-PAGE was performed to reveal any changes in the protein banding pattern under treatment
with vinegar. In general, the intensity of the bands of the vinegar-treated samples became lighter
than that of the corresponding controls (Figure 3). In the vinegar-treated (for less than 1 min) cod
and red grouper samples, there was no visible band below 150 kDa and in vinegar-treated whiting
20
there were several barely visible bands below 36 kDa. Electrophoretic separation of the fish
extracts showed strongly stained single bands at 36 kDa in water-treated and non-treated control
samples of whiting, cod and red grouper corresponding to fish tropomyosin. Compared to the
control samples, the intensity of the 36 kDa bands in vinegar-treated samples was dramatically
decreased. The fading of the bands indicates a decreased protein content due to the vinegar
treatment. Sletten and others (2009) also reported this reduction in the intensity of the protein
bands in acetic acid-salt brined herring products. Although the overall protein band intensity in
vinegar-treated samples decreased, Figure 3 shows several novel bands appearing between 37 and
100 kDa in the vinegar-treated cod and red grouper samples. These minor novel bands may be
caused by the acid hydrolyzation of larger proteins. Additional protein bands common to whiting
and cod were seen at 10, 12, 17, 19, 24, 36, and 150 kDa, while bands at 12, 36, and 150 kDa were
seen for red grouper. Interesting variations between species were noticed; the banding pattern for
the control samples of cod were more similar to that for whiting, while that of the vinegar-treated
samples of cod more closely resembled the banding pattern for vinegar-treated red grouper. A
major band at 10-13 kDa in these fish samples also faded in vinegar-treated samples when
compared with the control samples. This protein is likely to be fish parvalbumin based on its
molecular weight (Elsayed 1971).
5.4 Effects of Vinegar Treatment on Antigenic Protein of MAb 8F5 in Finfish
Western blot using MAb 8F5 was performed to reveal any changes in the levels of antigenic protein
under the treatment with vinegar. As shown in Figure 4, the monoclonal anti-fish tropomyosin
antibody, MAb 8F5, recognized the 36 kDa tropomyosin bands in all the fish samples. The 72 and
108 kDa bands were recognized by MAb 8F5 in the whiting samples, but not in either the cod or
red grouper samples, and so may be the dimeric and trimeric forms of whiting tropomyosin. As
with the SDS-PAGE results, vinegar-treated samples of the three fish species in Western blot
showed little or no band at 36 kDa, which is also consistent with the decreased iELISA OD reading
in the vinegar-treated fish samples. Furthermore, the changes in the antigenic protein banding
pattern of all three fish species once again agreed with the iELISA results. For whiting, the sample
treated by vinegar for 15 min, which showed the lightest band at 36 kDa, also showed the lowest
OD reading in iELISA. In cod and red grouper, there were no visible bands at all at 36 kDa for
samples treated with vinegar for less than 1 min, and the iELISA results of these two samples were
21
negative (OD reading <0.2). This trend of increased iELISA readings in the samples that received
longer vinegar treatment times supports the findings in Western blot. The combined results of
SDS-PAGE and Western blot provide further evidence that vinegar treatment decreases the IgGbinding activity by either lowering solubility, and thus the amount of antigenic protein in the
extract, or destroying its epitope. In addition, the exposure of hidden epitopes that slightly
increased the IgG-binding could explain the enhanced intensity of bands of the lengthier vinegartreated fish samples.
5.5 Effects of Vinegar Induced Chemical Reactions on the Detectability of Finfish
Three hypotheses were proposed to explain the changes of immunoreactivity of vinegar-treated
fish samples with MAb 8F5. The reduction of immunoreactivity may be caused by 1) proteolysis
of the antigenic protein by acidic proteases in raw fish muscle, which is activated by vinegar; 2)
acid denaturation/hydrolysis of the protein; or 3) the precipitation of antigenic protein at pH around
the pI value of tropomyosin. In order to verify these hypotheses, samples were treated by vinegar
in three different ways according to the detailed sample preparation protocols described in Chapter
Four. In Group A, fish samples were treated with vinegar first, and then cooked (100oC, 5 min).
The iELISA results for Group A (Figure 5) were identical with the data recorded previously:
vinegar treatment dramatically reduced the immunoreactivity in the fish samples of all three fish
species. In Group B, fish samples were cooked to inactivate the proteases in the fish muscle (if
such proteases were present), then the samples were treated with vinegar for different lengths of
time (<1, 15, 30, and 60 min). The results showed that the detectability of all the vinegar-treated
fish samples in Group B also decreased (Figure 6), which implies that the heat inactivation of
proteases had no effect on the action of vinegar on the detectability of these three fish species.
Therefore Hypothesis 1, that the decreased detectability of finfish could be attributed to the acid
activated intrinsic acidic protease, was rejected.
Group C sought to verify Hypothesis 2, that acid denaturation contributes to the decreased
immunoreactivity of finfish. Samples in Group C were cooked with vinegar for gradually
increasing lengths of time (5, 15, 30, and 60 min). The results, shown in Figure 7, revealed that
the iELISA readings of vinegar-treated whiting and cod samples decreased with increasing
treatment time, whereas the OD reading of vinegar-treated red grouper remained fairly stable. The
22
trend of decreased detectability for the fish samples with prolonged vinegar treatment time could
have therefore supported Hypothesis 2, but since the OD readings of the control samples without
vinegar treatment also decreased, and to a similar extent, the dramatically reduced detectability in
vinegar-treated finfish is not likely to be primarily induced by the effect of acid denaturation so
Hypothesis 2 was also rejected.
Subsequently, the pH of fish extracts was determined using a pH meter in an attempt to verify
Hypothesis 3. The pHs of the extracts of the vinegar-treated samples were all around 4.4, while
those of the water-treated and non-treated samples were around 7.1. As the pI value of tropomyosin
is 4.6 (Hamoir 1951), which is very close to the pH of vinegar-treated samples, it is reasonable to
attribute the decreased immunoreactivity of vinegar-treated finfish to the acid precipitation of the
antigenic protein of MAb 8F5 at a pH near its pI, which renders the protein insoluble and nonextractable. The fact that the total soluble protein concentration of the vinegar-treated finfish
samples was lower than that of the control samples also suggests that the decreased
immunoreactivity was indeed mainly caused by the precipitation of antigenic protein, thus
Hypothesis 3 was supported.
In this study, a number of fish species-specific pattern changes were observed in the results. In
Group B, the patterns for vinegar-treated cod and red grouper samples were different to those in
Group A, although both showed dramatically lowered iELISA readings. As shown in Figures 5
and 6, a longer vinegar treatment time was required to reach the lowest reading in Group B
compared to that in Group A. Hatae and others (1990) reported that fish species with a firm texture
had thin muscle fibers with considerable heat-coagulating material between them, while species
with a soft texture had thick muscle fibers with little heat-coagulating material. Since Group B
samples were cooked first, this heat-coagulating material may have obstructed the rapid
penetration of vinegar into the tissue, therefore lengthening the time needed for vinegar to react on
the cooked muscle compared to the raw muscle. For the Group C samples, a slight decrease in
immunoreactivity was observed in both whiting and cod. This finding indicates that although
tropomyosin is thermal-stable, prolonged cooking (60 min) can still denature the protein to a small
extent. Furthermore, as reported by Huang and Ochiai (2005) in their study of thermal stability of
tropomyosin in different fish species, the thermal stability of fish tropomyosins may be species23
specific. They found clear differences in thermal stability among fish tropomyosins of six fish
species, even though the identity of their amino acid sequences was more than 93.3%. This may
explain our finding that unlike the other two fish species, the immunoreactivity of red grouper
remained quite stable during the 60 min cooking.
In conclusion, based on the results of this study, factors such as fish species, the timing of the
vinegar addition and the vinegar treatment time all had an effect on the detectability of vinegartreated finfish. These effect factors and their corresponding iELISA results are summarized in
Table 4. Generally, if the finfish was cooked together with vinegar, the longer the treatment time,
the lower the immunoreactivity. Whether the finfish was treated with vinegar before or after
cooking affected the treatment time that induced the most significant decreased assay signal,
depending on the species of finfish.
5.6 Effects of Vinegar Treatment on the Allergenicity of Finfish
The investigation of altered immunoreactivity thus the detectability in the vinegar-treated finfish
using MAb 8F5-based immunoassays revealed that the total soluble protein concentrations (Table
2) of vinegar-treated samples decreased as a result of the treatment, indicating that the amount of
fish allergens present may also have decreased. Based on the SDS-PAGE gel results, the size of
the protein band at 10-13 kDa found in all three fish species tested corresponded to the amount of
the major fish allergen, parvalbumin, present. The intensity of this band became lighter in vinegartreated samples compared with the corresponding control samples (Figure 3). This finding may
provide evidence for a decrease in the amount of fish allergen in vinegar-treated fish samples,
supporting the findings of studies that have reported that vinegar treatment decreased the
allergenicity of shrimp, herring and peanuts (Perez-Macalalag and others 2007; Sletten and others
2009; Kim and otthers 2012); there have been no previous reports of the effects of vinegar on
allergenicity in finfish. For the current study, the effects of vinegar treatment on the allergenicity
of whiting, cod and red grouper were studied using human plasma (IgE) of three individuals
(subject no. 21290, 18440, and 17427) with a confirmed fish allergy (PlasmaLab International,
Everett, WA). Initially, the IgE-binding reactivity of extracts of raw and cooked salmon and cod
were verified by iELISA. Serological cross-reactivity (OD reading ≥0.2) to different fish samples
was observed in all three individuals. IgE from subject no. 21290 demonstrated the strongest IgE24
binding reactivity, while the IgE-binding reactivity of plasma from subject no. 18440, 17427 are
relatively weaker (Figure 8). The IgE-binding reactivity of these three individuals corresponded to
the information on their specific IgE concentrations (kU/I) provided by the commercial source
(Table 3). The individuals with higher plasma IgE concentrations are known to exhibit stronger
IgE-binding reactivity and Sampson (2001) reported that the quantification of food-specific IgE is
a useful test for diagnosing symptomatic allergies to eggs, milk, peanuts, and fish, and could
eliminate the need to perform double-blind, placebo-controlled food challenges. Therefore, it is
appropriate to use a test measuring alterations in IgE-binding reactivity to predict changes in an
individual’s allergenicity to finfish. All three individuals showed a positive reaction to both raw
and cooked fish samples. Fish allergens were proven to be thermally stable by comparing the
results of iELISA of raw and cooked fish samples.
IgE-based iELISA was performed in order to study the effects of vinegar treatment and treatment
time on the overall allergenicity of whiting, cod and red grouper. A considerable reduction in the
iELISA readings for two of the three plasma samples was observed in vinegar-treated fish samples
of whiting and cod (Figures 9-10). However, vinegar appeared to have little effect on lowering the
OD reading of red grouper (Figure 11). Furthermore, variations were found when comparing the
results of the IgE-iELISA among different individuals. A decrease in OD reading of up to 69.2%
and 83.9% for plasma from subjects no. 18440 and 17427, respectively, indicated that vinegartreated whiting and cod exhibited reduced IgE-binding when compared with water-treated controls.
A decreased reactivity for vinegar-treated whiting and cod samples for plasma from subject no.
21290 was also observed, but the effect was mild. A similar finding of individual variations has
also been reported in other studies examining the allergenicity of various fish products using
human IgE (Sletten and others 2009; Chatterjee and others 2006).
Interestingly, differences between the water-treated and non-treated samples were observed in the
whiting samples (Figure 9). In general, water-treated whiting samples showed higher OD readings
than non-treated ones, which indicates that the presence of water during cooking (100 °C) may
slightly increase the IgE-binding reactivity of whiting. However, this effect was not observed in
either the cod or the red grouper samples. In contrast to our finding, Sletten and others (2009)
reported that increased IgE-binding by dried cod was observed in all 12 sera in their study, as
25
revealed by the decreased IC50 values in competitive ELISA. Studies in peanuts have also indicated
that the involvement of water during cooking reduces IgE-binding reactivity; Mondonlet and
others (2005) found that the IgE-binding reactivity of whole peanut protein extracts prepared from
boiled peanuts was half that of the extracts prepared from raw and roasted peanuts, confirming the
results reported in an earlier study by Beyer and others (2001). However, the effect of dry or wet
heat treatment on the IgE-binding reactivity, which could be used to access the allergenicity of
finfish is unclear, and needs further study.
A similar pattern in the results for IgE-based iELISA (Figures 9 and 10) and MAb 8F5-based
iELISA (Figure 2) was noticeable. Whiting (vinegar-treated for 15 min) and cod (vinegar-treated
for less than 1 min) samples showed the lowest reactivity in both the IgE-based iELISA and MAb
8F5-based iELISA. The increased OD reading with prolonged vinegar treatment time, which may
be caused by either the exposure of hidden epitopes or the formation of new epitopes, was also
observed in IgE-based iELISA for both whiting and cod.
5.7 Effects of Vinegar Induced Chemical Reactions on the Allergenicity of Finfish
Does this similarity in reactivity changes between the IgG and IgE based iELISA results indicate
that vinegar affects both the detectability and allergenicity of finfish through the same chemical
action? As discussed earlier, vinegar decreases the detectability of finfish mainly by precipitating
the antigenic protein, tropomyosin, of the MAb 8F5 at a pH around 4.4, which is close to its pI
value (pI=4.6). The pI value of the major allergen, parvalbumin, varies for different species. The
α-parvalbumins have a pI of 5.0 or higher, while the more allergenic β-parvalbumins contain more
acidic amino acids, resulting in a pI value of 4.5 or lower (Goodman and Pechére 1977). Depending
on the pI range, the decreased allergenicity of finfish could also be induced by the precipitation of
the major fish allergen, parvalbumin. However, previous studies have suggested that other
chemical reactions may also contribute to the reduction of allergenicity. A study by Fink and others
(1994) on the acid denaturation of frog parvalbumin reported that frog parvalbumin unfolds in
solutions with pH 3-4. Since the IgE-binding epitopes on parvalbumin are located on a unique
conformational site in the tertiary structure of the parvalbumin (Perez-Gordo and others 2011; Van
and others 2005), acid induced unfolding may destroy the epitopes, thus decreasing the IgE26
binding reactivity of parvalbumin. Therefore, the decrease of allergenicity in vinegar-treated
samples may also be caused by acid denaturation and the following study of different vinegar
treatments on the allergenicity of finfish was therefore conducted to verify these hypotheses. Fish
samples of whiting, cod and red grouper were treated with vinegar in three ways following the
detailed sample preparation protocol described in Chapter Four. Briefly, samples in Group A were
treated for 15 min, and then cooked (100 C 5 min); samples in Group B were cooked (100 C 5
min) then treated with vinegar for 15 min; and samples in Group C were cooked with vinegar for
different lengths of time (5, 15, 30, and 60 min). Proteins were extracted using 0.15 M NaCl.
Unlike the decrease in OD readings observed in the IgG-based iELISA of vinegar-treated samples
in both Groups A and B (Figures 6 and 7), the decreased OD readings were seen only in the IgEbased iELISA of vinegar-treated samples in Group A, and not those in Group B. As shown in
Figures 12 and 13, the vinegar treatment before cooking (100 °C, 5min) decreased the OD reading
of whiting and cod considerably, whereas implementing the vinegar treatment after cooking had
no effect on the OD reading. As mentioned earlier, the decrease in the OD reading indicates a
reduction in the IgE-binding reactivity, and thus the allergenicity of the allergens. This
demonstrates that vinegar treatment after cooking does not decrease the allergenicity of whiting
and cod. These findings suggest that the chemical reactions that cause the decrease in detectability
and allergenicity are different. The reduction in the allergenicity of finfish as a result of the vinegar
treatment may not be due to the precipitation of fish allergens in an acidic pH environment.
The pattern of results for the Group C samples suggests that rather than allergen precipitation, acid
denaturation may be the chemical action that responsible for the decrease in the allergenicity of
finfish. These results clearly show that 60 min cooking of fish muscle of whiting, cod or red
grouper with vinegar can dramatically decrease their IgE-binding reactivity. Compared with
control samples, an average decrease of 68.9%, 62.0%, and 60.7% of OD reading was observed in
the whiting, cod and red grouper samples, respectively, when tested by IgE from three individuals.
Especially for subjects 18440 and 17427, the iELISA result for whiting and red grouper was
considerably reduced (OD reading <0.2) after the 60 min vinegar treatment, which indicates a
considerable reduction in allergenicity. Moreover, prolonged vinegar cooking time with vinegar
intensified this decreased allergenicity in all three fish species. Comparing the OD reading of the
27
samples cooked with vinegar for 5 min and those for samples cooked with vinegar for 60 min, a
clear decrease is visible (Figures 13-15), indicating that the effect of vinegar on the IgE-binding
reactivity of finfish may be due to acid denaturation -- the combination of heat and acid.
It is noticeable that red grouper is more resistant to acid treatment than either whiting or cod.
Vinegar treatment, either before or after cooking, had no effect on the IgE-binding reactivity of
red grouper. In Group B, vinegar-treated red grouper samples even showed higher IgE-binding
reactivity than control samples when tested by IgE from all three individuals. It is possible that the
15 min vinegar treatment exposed hidden epitopes in the red grouper samples but was unable to
destroy them. However, in Group C, an average decrease of 61.5% in the OD reading was
observed between vinegar-treated (for 60 min) and water-treated (for 60 min) red grouper samples.
Looking at the Group C results (Figures 14-16), iELISA using IgE from subject no. 21290 showed
46.1%, 37.1%, 31.0% decreases in the OD reading for whiting, cod and red grouper samples,
respectively, treated with vinegar for 60 min. This decrease was not observed in samples treated
with vinegar before (Group A) or after cooking (Group B). Therefore, unlike the effects of vinegar
on the detectability of finfish tested by MAb 8F5-based iELISA, it is difficult to determine which
treatment method and treatment time produce the most significant decrease in detectability for
different fish species. Overall, lengthy cooking of fish muscle, together with vinegar treatment, is
the most effective way to reduce the allergenicity of finfish for all fish allergic individuals, despite
the existence of specie and individual variations.
28
CHAPTER SIX
CONCLUSIONS
Changes in immunoreactivity, manifested in terms of the detectability using IgG-based iELISA,
and allergenicity, measured using IgE-based iELISA, as a result of treating whiting, cod and red
grouper samples with vinegar were investigated in this study. The results indicate that vinegar
treatment can decrease both the detectability and allergenicity of whiting, cod and red grouper.
However, the chemical action by which vinegar affects the binding properties between antigenic
tropomyosin-IgG binding and fish allergen-IgE binding are distinctively different. Vinegar
treatment decreased the detectability of finfish to MAb 8F5 by acidic precipitation of the antigenic
protein-tropomyosin, while the allergenicity was reduced mainly by acid denaturation of the
allergenic protein. Therefore, it is not appropriate to use changes in the IgG-binding activity under
the effect of vinegar to predict changes in IgE-binding reactivity, although they do share some
common patterns of immunoreactivity changes. In MAb 8F5-based iELISA, whiting, cod and red
grouper treated for 15 min, <1 min and <1 min, respectively showed negative results (OD reading<
0.2), indicating the most dramatically decreased detectability. To address the difficulty of detecting
fish in acidic pH environments, the production of antibody targeting acid-resistant fish proteins
could be a solution. Moreover, although cooking with vinegar for 60 min decreased the overall
allergenicity of whiting, cod, and red grouper, the variations among individual subjects and
different fish species clearly demonstrates the unpredictable nature of food-allergic responses. The
clinical significance of these findings remains to be established. Although the efficiency of vinegar
treatment in avoiding or alleviating clinical fish allergic reactions is still not clear, vinegar-treated
fish products with decreased allergenicity can be potentially used in oral desensitization therapy
for IgE mediated fish allergic individuals, and it could also be used to lower the risk of fish allergy
in those patients taking anti-acid medications.
29
APPENDIX A
TABLES AND FIGURES
Table 1. Fish allergens
Origin
Allergen
M.W
Protein
(kDa)
Classification
Reference
Baltic cod
(Gadus callarias)
Gad c 1
12.3
β-parvalbumin
Elsayed and Bennich 1975
Atlantic salmon
(Salmo salar)
Sal s 1
11.9
β-parvalbumin
Lindstrom and others 1996
Atlantic cod
(G. morhua)
Gad m 1
11.5
β-parvalbumin
Das Dores and others 2002
Whiff
Lep w 1
12
β-parvalbumin
Griesmeier and others 2010
Mackerel
(Scomber japonis)
Sco j 1
11
β-parvalbumin
Hamada and others 2003
Mackerel
(S. australasicus)
Sco a 1
11
β-parvalbumin
Hamada and others 2003
Mackerel
Sco s 1
11
β-parvalbumin
Hamada and others 2003
(S. scombrus)
Pacific Pilchard
Sar sa
13.1
β-parvalbumin
Beale and others 2009
(Sardinops sagax)
Alaska Pollock
1.0101
The c 1
11.5
β-parvalbumin
Van Do and others 2005
41
aldehyde phosphate
Das Dores and others 2002
(Lepidorhombus
whiffiagonis)
(Theragra chalcogramma)
Cod
(Gadus morhua)
dehydrogenase
Bigeye tuna
(Thunnus obesus)
120
Swordfish
(Xiphias gladius)
25
Mackerel
(Scomber japonicus)
28
Anisakis
Ani s 1
collagen type I
Hamada and others 2001
Kelso and others 1996
triosephosphate
isomerase
21
Wang and others 2010
Moneo and others 2000
(Anisakis simplex)
30
Table 2. Total soluble protein concentration of whiting, cod and red grouper
Soluble Protein Concentration (mg/ml)
Fish
Vinegar treated
Water treated
Non treated
Whiting(< 1 min)
0.06
0.54
0.58
Whiting(15 min)
0.15
0.60
0.53
Whiting(30 min)
0.12
0.57
0.47
Whiting(60 min)
0.17
0.66
0.43
Cod(< 1 min)
0.36
0.50
0.59
Cod(15 min)
0.42
0.50
0.54
Cod(30 min)
0.45
0.57
0.63
Cod(60 min)
0.55
0.52
0.58
Red grouper(< 1 min)
0.77
1.89
2.64
Red grouper(15 min)
1.25
1.99
3.14
Red grouper(30 min)
1.81
1.96
2.86
Red grouper(60 min)
1.95
1.80
3.19
Table 3. Clinical features of patients. (Source: Plasmalab International, Everett, WA)
No.
Age
Sex
Total IgE kU/I
Specific IgE, kU/I (ImmunoCAP)
C
T
S
21290
13
M
2432
84.4
40.4
85.0
18440
32
F
4105
35.8
-
29.7
17427
42
M
398
42.1
-
39.2
C= cod; T= tuna; S= salmon
31
Table 4. Immunoreactivity of MAb 8F5 against vinegar-treated fish samples determined by
iELISA. Samples in group A, B and C were treated with vinegar before, after, and during
cooking, respectively. Absorbance readings at 415 nm: <0.19—“-”, 0.20-0.49—“+”, 0.501.49—“++”, >1.50—“+++”.
Treatment
Group A
Group B
Group C
time(min)
W
C
R
W
C
R
<1
+++
-
-
+++
+++
+++
5
W
C
R
+
++
-
15
+
+
+
+
-
+
+
++
-
30
-
+
+
++
-
-
+
+
-
60
-
+
+
++
+
-
-
+
-
W= Whiting, C= Cod, R= Red grouper.
Figure 1. Effects of different types of vinegar on the immunoreactivity of whiting by MAb
8F5 based-iELISA.
Raw whiting was treated with different types of vinegar for 30 min, after cooking (100 °C, 5 min) the fish protein
was extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5
supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.
32
Figure 2. Effect of vinegar treatment on the immunoreactivity of whiting, cod and red
grouper by MAb 8F5-based iELISA.
Raw whiting, cod and red grouper was treated with vinegar for different time lengths, after cooking (100 °C, 5 min)
the fish protein was extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL.
MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.
33
(a) whiting
(b) cod
(c) red grouper
Figure 3. SDS-PAGE profiles of vinegar-treated, water-treated and control samples of
whiting, cod and red grouper.
Fish extracts of whiting, cod and red grouper diluted 1:1, 1:1, 1:3 v/v, respectively, with sample buffer were used in
the assay. Each lane was loaded with 15 μl of diluted sample extract.
34
(a) whiting
(b) cod
(c) red grouper
Figure 4. Antigenic protein banding patterns of vinegar-treated, water-treated and control
samples of whiting, cod and red grouper.
MAb 8F5 supernatant (1:3) was used as the primary antibody, anti-mouse IgG-AP conjugated antibody (1:1000)
was used as the secondary antibody.
35
Figure 5. Study of the effects of vinegar induced chemical reactions on the
immunoreactivity of whiting, cod and red grouper (Group A).
The samples in Group A were treated with vinegar, water or left untreated for <1, 15, 30, 60 min separately and then
cooked (100 °C) for 5 min. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant
diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.
36
Figure 6. Study of the effects of vinegar induced chemical reactions on the
immunoreactivity of whiting, cod and red grouper (Group B).
The samples in Group B were cooked for 5 min then treated with vinegar, water or left untreated for <1, 15, 30, 60
min separately. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3
was used in the assay. Results were expressed as A415  SD, n =2.
37
Figure 7. Study of the effects of vinegar induced chemical reactions on the
immunoreactivity of whiting, cod and red grouper (Group C).
The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min separately.
Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the
assay. Results were expressed as A415  SD, n =2.
38
Figure 8. Screen for IgE-binding reactivity of raw and cooked salmon and cod. The fish
proteins in raw and cooked (100 °C, 5 min) salmon and cod were extracted with 0.15 M
NaCl.
Plate was coated with samples at the concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:2
v/v were used in the assay. Results were expressed as A415  SD, n =2.
39
Figure 9. Effect of vinegar treatment on the IgE-immunoreactivity of whiting by human
plasma-based iELISA.
Raw whiting was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C, 5
min). The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of
2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay.
Results were expressed as A415  SD, n =2.
40
Figure 10. Effect of vinegar treatment on the IgE-immunoreactivity of cod by human
plasma-based iELISA.
Raw cod was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C, 5 min).
The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100
μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results
were expressed as A415  SD, n =2.
41
Figure 11. Effect of vinegar treatment on the IgE-immunoreactivity of red grouper by
human plasma-based iELISA.
Raw red grouper was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C,
5 min). The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of
2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay.
Results were expressed as A415  SD, n =2.
42
Figure 12. Study of the effects of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group A).
The samples in Group A were treated with vinegar, water or left untreated for 15 min and then cooked (100 °C) for
5 min, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of
2μg/100 μL. Human plasma from three individuals diluted 1:8, 1:4, and 1:4 v/v, respectively were used in the assay.
Results were expressed as A415  SD, n =2.
43
Figure 13. Study of the effects of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group B).
The samples in Group B were cooked (100 °C) for 5 min then treated by vinegar, water or left untreated for 15 min,
the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL.
Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were
expressed as A415  SD, n =2.
44
Figure 14. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of whiting by human plasma-based iELISA (Group C).
The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min, the fish
proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human
plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were
expressed as A415  SD, n =2.
45
Figure 15. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of cod by human plasma-based iELISA (Group C).
The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min separately,
the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL.
Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were
expressed as A415  SD, n =2.
46
Figure 16. Study of the effect of vinegar induced chemical reactions on the IgE-binding
reactivity of red grouper by human plasma-based iELISA (Group C).
The samples in Group C were cooked (100 °C) with vinegar water or left untreated for 5, 15, 30, 60 min separately,
the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL.
Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v respectively were used in the assay. Results were
expressed as A415  SD, n =2.
47
REFERENCES
Aas K, and Elsayed S. 1969. Characterization of a major allergen (cod): effect of enzymic
hydrolysis on the allergenic activity. Journal of Allergy. 44(6): 333-43.
Armentia A, Dueñas-Laita A, Pineda F, Herrero M, Martín B. 2010. Vinegar decreases
allergenic response in lentil and egg food allergy. Allergol. Immunopathol. 38(2):74-7.
Asero R, Ballmer‐Weber BK, Beyer K, Conti A, Dubakiene R, Fernandez‐Rivas M, Vieths S.
2007. IgE-Mediated food allergy diagnosis: Current status and new perspectives. Molecular
Nutrition & Food Research 51(1):135-47.
Beale JE, Jeebhay MF, Lopata AL. 2009. Characterization of purified parvalbumin from five fish
species and nucleotide sequencing of this major allergen from Pacific pilchard. Sardinops sagax.
Mol. Immunol. 46(15):2985-93.
Ben-Shoshan M, Harrington DW, Soller L, Fragapane J, Joseph L, Pierre YS, Clarke AE. 2012.
Demographic predictors of peanut, tree nut, fish, shellfish, and sesame allergy in Canada. J.
Allergy. 2012 (2012):1-6.
Besler M. 2001. Determination of allergens in foods. TrAC Trends in Analytical Chemistry.
20(11): 662-72.
Beyer K, Morrowa E, Li X, Bardina L, Bannon GA, Burks A, Sampson HA. 2001. Effects of
cooking methods on peanut allergenicity. J. Allergy Clin. Immunol. 107(6):1077-81.
Billman GE. 1994. Prevention of ischemia-induced ventricular fibrillation by omega 3 fatty
acids. Proceedings of the National Academy of Sciences - PNAS 91(10):4427.
Boyano MT, Martin Esteban M, Diaz Pena JM, Ojeda JA. 1987. Food allergy in children. I.
Clinical aspects and diagnosis. An. Esp. Pediatr. 26(4):235-40.
Bohle B, Zwölfer B, Heratizadeh A, Jahn-Schmid B, Antonia YD, Alter M, Ebner C
2006. Cooking birch pollen–related food: Divergent consequences for IgE-and T cell–mediated
reactivity in vitro and in vivo. Journal of allergy and clinical immunology, 118(1): 242-9.
Brenna O, Pompei C, Ortolani C, Pravettoni V, Pastorello EA, Farioli L. 2000. Technological
processes to decrease the allergenicity of peach juice and nectar. J. Agric. Food Chem.
48(2):493-7.
Bugajska-Schretter A, Grote M, Vangelista L, Valent P, Sperr WR, Rumpold H, Spitzauer S.
2000. Purification, biochemical, and immunological characterisation of a major food allergen:
different immunoglobulin E recognition of the apo-and calcium-bound forms of carp
parvalbumin. Gut. 46(5), 661-9.
48
Chatterjee U, Mondal G, Chakraborti P, Patra HK, Chatterjee BP. 2006. Changes in the
allergenicity during different preparations of Pomfret, Hilsa, Bhetki and mackerel fish as
illustrated by enzyme-linked immunosorbent assay and immunoblotting. Int. Arch. Allergy
Immunol. 141(1):1.
Chen YT, Hsieh YHP. 2012. Epitope characterization of a fish-specific monoclonal antibody.
Poster presented at the IFT Conference; Las Vegas, USA, June 2012. Chicago, USA: Institute of
Food Technologists.
Connett GJ, Gerez I, Cabrera-Morales EA, Yuenyongviwat A, Ngamphaiboon J, Chatchatee P,
Lee BW. 2012. A population-based study of fish allergy in the Philippines, Singapore and
Thailand. Int. Arch. Allergy Immunol. 159(4):384.
Coughlin DJ, Solomon S, Wilwert JL. 2007. Parvalbumin expression in trout swimming muscle
correlates with relaxation rate. Comparative Biochemistry & Physiology.Part A, Molecular &
Integrative Physiology 147(4):1074-82.
Crespo JF, Pascual C, Burks A, Helm RM, Esteban MM. 1995. Frequency of food allergy in a
pediatric population from Spain. Pediatric Allergy & Immunology 6(1):39-43.
Das Dores S. 2002a. A new oligomeric parvalbumin allergen of Atlantic cod (Gad m 1) encoded
by a gene distinct from that of Gad c 1. Allergy 57(s72):79-83.
Das Dores S, Chopin C, Romano A, Galland‐Irmouli AV, Quaratino D, Pascual C, Guéant JL.
2002b. IgE-binding and cross-reactivity of a new 41 kDa allergen of codfish. Allergy
57(s72):84-7.
Dorizzi RM, Senna G, Caputo M. 1999. Likelihood ratios and Fagan’s nomogram: Valuable but
underrated tools for in vitro latex sensitization assessment. Clinica Chimica Acta 282(1-2):17583.
Eggesbø M, Halvorsen R, Tambs K, Botten G. 1999. Prevalence of parentally perceived adverse
reactions to food in young children. Pediatric Allergy & Immunology 10(2):122-32.
Elsayed S, Aas K. 1971. Characterization of a major allergen (cod): observations on effect of
denaturation on the allergenic activity. Journal of Allergy and Clinical Immunology. 47(5): 28391.
Elsayed S, Bennich H. 1975. The primary structure of allergen M from cod. Scandinavian
journal of immunology. 4(2): 203-8.
FAAN website: resources online [Internet]. Fairfax, VA: Food Allergy and Anaphylaxis
Network; [Accessed 2012 March 28]. Available from: http://www.foodallergy.org/page/fishallergy.
49
Fink AL, Calciano LJ, Goto H, Kurotsu T, Palleros DR. 1994. Classification of acid denaturation
of proteins: intermediates and unfolded states. Biochemistry 33(41):12504-11.
Goodman M, Pechére J. 1977. The evolution of muscular parvalbumins investigated by the
maximum parsimony method. J. Mol. Evol. 9(2):131-58.
Griesmeier U, Vázquez‐Cortés S, Bublin M, Radauer C, Ma Y, Briza P, Breiteneder H. 2010a.
Expression levels of parvalbumins determine allergenicity of fish species. Allergy 65(2):191-8.
Griesmeier U, Bublin M, Radauer C, Vázquez‐Cortés S, Ma Y, Fernández Rivas M,
Breiteneder H. 2010b. Physicochemical properties and thermal stability of Lep w 1, the major
allergen of whiff. Molecular Nutrition & Food Research 54(6):861-9.
Hajeb P, Selamat J. 2012. A contemporary review of seafood allergy. Clin. Rev. Allergy
Immunol. 42(3):365-85.
Hamada Y, Nagashima Y, Shiomi K. 2001. Identification of collargen as a new fish allergen.
Bioscience, Biotechnology and Biochemistry. 65(2): 285-91.
Hamada Y, Tanaka H, Ishizaki S, Ishida M, Nagashima Y, Shiomi K. 2003. Purification,
reactivity with IgE and cDNA cloning of parvalbumin as the major allergen of mackerels. Food
& Chemical Toxicology 41(8):1149-56.
Hamoir G. 1951. Fish tropomyosin and fish nucleotropomyosin. Biochem. J. 48(2):146.
Hatae K, Yoshimatsu F, Matsumoto JJ. 1990. Role of muscle fibers in contributing firmness of
cooked fish. J. Food Sci. 55(3):693-6.
Hou C, Hsieh YHP. 2012. MAb-based competitive ELISA for the detection of commercially
processed fish products. Poster presented at the IFT Conference; Las Vegas, USA, June 2012.
Chicago, USA: Institute of Food Technologist.
Huang M, Ochiai H. 2005. Fish fast skeletal muscle tropomyosins show species-specific thermal
stability. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular
Biology 141(4):461-71.
Jankiewicz A, Baltes W, Bögl KW, Dehne LI, Jamin A, Hoffmann A, Vieths S. 1997. Influence
of food processing on the immunochemical stability of celery allergens. Journal of the Science of
Food and Agriculture. 75(3): 359-70.
James JM, Helm RM, Burks AW, Lehrer SB. 1997. Comparison of pediatric and adult IgE
antibody binding to fish proteins. Annals of Allergy, Asthma, & Immunology 79(2):131-37.
Jeounga B, Reesea G, Hauckb P, Oliverb JB, Daula CB, Lehrera SB. 1997. Quantification of the
major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich
ELISA. J. Allergy Clin. Immunol. 100(2):229-34.
50
Kelso JM, Jones RT, Yunginger JW. 1996. Monospecific allergy to swordfish. Annals of
Allergy, Asthma & Immunology. 77(3): 227-8.
Kim J, Lee, J, Seo WH, Han Y, Ahn K, Lee SI. 2012. Changes in major peanut allergens under
different pH conditions. Allergy, Asthma & Immunology Research 4(3):157-60.
Kleine-Tebbe J, Wangorsch A, Vogel L, Crowell DN, Haustein UF, Vieths S. 2002. Severe oral
allergy syndrome and anaphylactic reactions caused by a Bet v 1–related PR-10 protein in
soybean, SAM22. Journal of allergy and clinical immunology, 110(5), 797-804.
Koyama H, Kakami M, Kawamura M, Tokuda R, Kondo Y, Tsuge I, Urisu A. 2006. Grades of
43 fish species in Japan based on IgE-binding reactivity. Allergol. Int. 55(3):311-6.
Kuehn A, Scheuermann T, Hilger C, Hentges F. 2010. Important variations in parvalbumin
content in common fish species: a factor possibly contributing to variable allergenicity. Int. Arch.
Allergy Immunol. 153(4):359-66.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227(5259):680-5.
Lao-araya M, Trakultivakorn M. 2012. Prevalence of food allergy among preschool children in
northern Thailand: Food allergy in Thai children. Pediatrics International 54(2):238-43.
Lee P, Niemann LM, Lambrecht DM, Nordlee JA, Taylor SL. 2009. Detection of mustard, egg,
milk, and gluten in salad dressing using enzyme-linked immunosorbent assays (ELISAs). J .Food
Sci. 74(5):T46-50.
Lehrer SB. 1996. Why are some proteins allergenic? Implications for biotechnology. Crit. Rev.
Food Sci. Nutr. 36(6):553-64.
Lindstrom C, Van DT, Hordvik I, Endresen C, Elsayed S. 1996. Cloning of two distinct cDNAs
encoding parvalbumin, the major allergen of Atlantic salmon (Salmo salar). Scand. J .Immunol.
44(4):335-44.
Lu H, Ohshima T, Ushio H, Hamada H, Shiomi K. 2007. Immunological characteristics of
monoclonal antibodies against shellfish major allergen tropomyosin. Food Chem. 100(3):1093-9.
Maleki SJ, Chung SY, Champagne ET, Raufman JP. 2000. The effects of roasting on the
allergenic properties of peanut proteins. J. Allergy Clin. Immunol. 106(4):763-8.
Martino M. 1990. Allergy to different fish species in cod-allergic children: In vivo and in vitro
studies. J. Allergy Clin. Immunol. 86(6):909-14.
51
Monaci L, Visconti A. 2010. Immunochemical and DNA-based methods in food allergen
analysis and quality assurance perspectives. Trends in Food Science & Technology. 21(6):27283.
Mondoulet L, Paty E, Drumare MF, Ah-Leung S, Scheinmann P, Willemot RM, Bernard H.
2005. Influence of thermal processing on the allergenicity of peanut proteins. J. Agric. Food
Chem. 53(11):4547-53.
Moneo I, Caballero ML, Gómez F, Ortega E, Alonso MJ. 2000. Isolation and characterization of
a major allergen from the fish parasite Anisakis simplex. J. Allergy Clin. Immunol. 106(1):17782.
Munoz-Furlong A, Sampson HA, Sicherer SH. 2004. Prevalence of self-reported seafood allergy
in the US. 1. J. Allergy Clin. Immunol. 113(2):S100.
Musmand J, Helbling A, Lehrer SB. 1996. Surimi: Something fishy. J. Allergy Clin. Immunol.
98(3):697-9.
Nakamura R. 2013. Evaluation of allergenicity of acid-hydrolyzed wheat protein using an in
vitro elicitation test. Int. Arch. Allergy Immunol. 160(3):259-64.
Neti G, Rehbein H. 2000. Stability of fish muscle parvalbumins at different pH values.
J. Food Biochem. 24(6):493-502.
Nordlee JA, Taylor SL, Jones RT, Yunginger JW. 1981. Allergenicity of various peanut products
as determined by RAST inhibition. J. Allergy Clin. Immunol. 68(5):376-82.
Novembre E, Cianferoni A, Bernardini R, Mugnaini L, Caffarelli C, Cavagni G, Vierucci A.
1998. Anaphylaxis in children: Clinical and allergologic features. Pediatrics 101(4):e8.
Pali-Schöll II, Jensen-Jarolim E. 2011. Anti-acid medication as a risk factor for food allergy.
Allergy 66(4):469-77.
Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, Conti A. 2002.
Identification of hazelnut major allergens in sensitive patients with positive double-blind,
placebo-controlled food challenge results. Journal of allergy and clinical immunology. 109(3):
563-70.
Perez-Gordo M, Lin J, Bardina L, Pastor-Vargas C, Cases B, Vivanco F, Cuesta-Herranz J,
Sampson HA. 2011. Epitope mapping of Atlantic salmon major allergen by peptide microarray
immunoassay. Int. Arch. Allergy Immunol. 157(1):31-40.
Perez-Macalalag E, Sumpaico M, Agbayani B. 2007. Shrimp allergy effect of vinegar soaking on
allergenicity. The World Allergy Organization Journal: S317.
52
Poulsen LK, Hansen TK, Nørgaard A, Vestergaard H, Stahl Skov P, Bindslev‐Jensen C. 2001.
Allergens from fish and egg. Allergy 56(s67):39-42.
Rall JA. 1996. Role of parvalbumin in skeletal muscle relaxation. News in Physiological
Sciences 11(6):249-55.
Ree RV, Antonicelli L, Akkerdaas J, Pajno G, Barberio G, Corbetta L, Ferro G, Zambito M,
Garritani M, Aalberse R. 1996. Asthma after consumption of snails in house‐dust‐mite‐
allergic patients: A case of IgE cross‐reactivity. Allergy 51(6):387-93.
Roquet F, Declercq, JP, Tinant B, Rambaud J, and Parello J. 1992. Crystal structure of the
unique parvalbumin component from muscle of the leopard shark (Triakis semifasciata). J. Mol.
Biol. 223(3):705-20.
Sampson HA. 2001. Utility of food-specific IgE concentrations in predicting symptomatic food
allergy. J. Allergy Clin. Immunol. 107(5):891.
Sampson HA. 1999. Food allergy. Part 2: Diagnosis and management. J. Allergy Clin. Immunol.
103(6):981-9.
Sathe SK, Teuber SS, Roux KH. 2005. Effects of food processing on the stability of food
allergens. Biotechnol. Adv. 23(6):423-9.
Schubert-Ullrich P, Rudolf J, Ansari P, Galler B, Führer M, Molinelli A, Baumgartner S. 2009.
Commercialized rapid immunoanalytical tests for determination of allergenic food proteins: An
overview. Analytical & Bioanalytical Chemistry 395(1):69-81.
Shefcheck KJ, Callahan JH, Musser SM. 2006. Confirmation of peanut protein using peptide
markers in dark chocolate using liquid chromatography−tandem mass spectrometry (LCMS/MS). J. Agric. Food Chem. 54(21):7953-9.
Sicherer SH, Muñoz-Furlong A, Sampson HA. 2004. Prevalence of seafood allergy in the United
States determined by a random telephone survey. J. Allergy Clin. Immunol. 114(1):159-65.
Skerrett PJ. 2003. Consumption of fish and fish oils and decreased risk of stroke. Preventive
Cardiology 6(1):38-41.
Sletten G, Lindvik H, Egaas E, Florvaag E. 2009. Effects of industrial processing on the
immunogenicity of commonly ingested fish species. Int. Arch. Allergy Immunol. 151(3):223-36.
Swoboda II. 2002. Recombinant fish parvalbumins: Candidates for diagnosis and treatment of
fish allergy. Allergy 57(s72):94-6.
Taylor SL, Lehrer SB. 1996. Principles and characteristics of food allergens. Critical Reviews in
Food Science and Nutrition. 36(S1): 91-118.
53
Taylor SL. 2004. Fish allergy: Fish and products thereof. J. Food Sci. 69(8):R175-80.
Taylor SL. 2008. Molluscan shellfish allergy. Adv. Food Nutr. Res. 54139-77.
Thomas K, Herouet-Guicheney C, Ladics G, Bannon G, Cockburn A, Crevel R, Vieths S. 2007.
Evaluating the effect of food processing on the potential human allergenicity of novel proteins:
International workshop report. Food & Chemical Toxicology 45(7):1116-22.
Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of
the National Academy of Sciences - PNAS 76(9):4350-54.
Ueshima H. 2007. Food omega-3 fatty acid intake of individuals (total, linolenic acid, longchain) and their blood pressure: INTERMAP study. Hypertension 50(2):313-9.
Untersmayr E, Vestergaard H, Malling HJ, Jensen LB, Platzer MH, Boltz-Nitulescu G, Poulsen
LK. 2007. Incomplete digestion of codfish represents a risk factor for anaphylaxis in patients
with allergy. J. Allergy Clin. Immunol. 119(3):711-7.
Untersmayr E, Szalai K, Riemer AB. 2006. Mimotopes identify conformational epitopes on
parvalbumin, the major fish allergen. Mol. Immunol. 43(9):1454-61.
Van Do T, Hordvik I, Endresen C, Elsayed S. 2005. Characterization of parvalbumin, the major
allergen in Alaska pollock, and comparison with codfish Allergen M. Mol. Immunol. 42(3):34553.
Wang B, Li Z, Zheng L, Liu Y, Lin H. 2011. Identification and characterization of a new IgEbinding protein in mackerel (Scomber japonicus) by MALDI-TOF-MS. Journal of Ocean
University of China 10(1):93-8.
Watanabe M, Tanabe S, Suzuki T, Ikezawa Z, Arai S. 1995. Primary structure of an allergenic
peptide occurring in the chymotryptic hydrolysate of gluten. Biosci. Biotechnol. Biochem.
59(8):1596-7.
Werner MT, Fæste CK, Egaas E. 2007. Quantitative sandwich ELISA for the determination of
tropomyosin from crustaceans in foods. J. Agric. Food Chem. 55(20):8025-32.
Witteman A, Akkerdaas J, Van Leeuwen J, Van Der Zee J, Aalberse R. 2009.
Identification of a cross-reactive allergen (presumably tropomyosin) in shrimp, mite and insects.
Int. Arch. Allergy Immunol. 105(1):56-61.
Yamada S, Nolte H, Zychlinsky E. 1999. Identification and characterization of allergens in two
species of tuna fish. Annals of Allergy, Asthma, & Immunology 82(4):395-400.
54
Yamanishi R, Tsuji H, Bando N, Yamada H, Nadaoka H, Huang T, Nishikawa K, Emoto S,
Ogawa T. 1996. Reduction of the allergenicity of soybean by treatment with proteases. J. Nutr.
Sci.Vitaminol. 42(6):581-7.
Zhang H, Matsuo H, Morita E. 2006. Cross‐reactivity among shrimp, crab and scallops in a
patient with a seafood allergy. J. Dermatol. 33(3):174-7.
55
BIOGRAPHICAL SKETCH
EDUCATION
The Florida State University
Tallahassee, FL
Master of Science
Major: Nutrition and Food Science
August 2011- present
Thesis: The Effects of Vinegar on Detectability and Allergenicity of Finfish .
Zhejiang Gongshang University
Hangzhou, China
Bachelor of Science in Food Science
Major: Food Safety and Quality
August 2006- June 2010
Thesis: Screening of Biological Preservative Lactic Acid Bacteria and Characterization of Bateriocin .
RESEARCH EXPERIENCE
The Florida State University
January 2012- present
Tallahassee, FL

Effects of acid ingredients on immunoreactivity and allergenicity of finfish.

Food allergens and fish proteins.

Determination of protein concentration, Enzyme-linked immunosorbent assay (ELISA), SDS-PAGE
and Western blot.

Developing research, include paperwork writing, research planning, time arrangement and budget.
Zhejiang Gongshang University
June 2009- November 2009
Hangzhou, China

Screening of biological preservative lactic acid bacteria and characterization of the bateriocin.

Lactic acid bacteria, bacteriocin and their potential to be biological preservative.

Screening bacteriocin-producing lactic acid bacteria, basic microbiology characterization skills and
16S rDNA identification technique.
Zhejiang Gongshang University
January 2009- May 2009

Hangzhou, China
Efficiency of microwave-assisted acid hydrolysis to gas chromatography for monosaccharide
composition analysis of polysaccharide.

Internal standard method, t-test, gas chromatography.

Efficiency of microwave assisted-acid hydrolysis for sample preparation of different test methods.
INTERNSHIP EXPERIENCE
Administration of Quality Supervision, Inspection and Quarantine in Zhejiang, Research Assistant
Hangzhou, China
Nov. 2009- May 2010
56

Determination of contents of illegal food additives (e.g. melamine) in animal products using highperformance liquid chromatography (HPLC).

Hepatitis E virus infection in swine in Zhejiang province using reverse transcription polymerase
chain reaction (RT-PCR).

Quality management and techniques for illegal food additives in China.

Record management and classification.
PROFESSIONAL EXPERIENCE
Training Experience

HACCP Certificate Registered, International HACCP Alliance
August 2012
Experience: training of HACCP background knowledge (seven principles HACCP, physical, chemical,
biological hazards and hazard control), HACCP plans writing and implement HACCP in food
processing, distribution and preparation environment.

Hazardous Waste Awareness Refresher Training, The Florida State University
May 2012
Experience: training of hazardous waste determination and definitions, hazardous waste generator
status, accumulation limits, container management, spill control etc.
Teaching Experience

Instructor of English Reading course, Nasi Education
January- June 2011
Responsibilities: Instruct knowledge about how to improve English reading ability and skills;
Develop lectures;
Make PPT and video tutorials to trains students,;
Prepare and graded exams.
Computer Skills and Certificates

MS Office (Word, Excel, PowerPoint), SPSS, Origin 7.0, Auto CAD

International HACCP Alliance HACCP Certification (USA, 2012)

Food Quality Inspector Certification (China2010)

National Compute Level II certification (China, 2008)
Leadership Activities

Investigation of Hygienic Production Conditions in Small Workshops, China
July 2007
Responsibilities: event planning and organization, communication with local government food
safety department and media, etc. The team won the award of Provincial Excellent Team of Summer
Social Practice .

Popularizing Food and Drug Safety Knowledge in Impecunious County, China
Responsibilities: event planning and organization

Student Union in College of Food and Biology Engineering, Zhejiang Gongshang University-Minister
of Secretariat
May 2007- May 2008
Responsibilities: organization and communication between different departments of College Student
Union, document management, assets management.
57
July 2008
PUBLICATION

Junli Zhu, Ye Wang, Jianrong Li. Screening of biological preservative Lactic acid bacteria and
characterization of the bacteriocin[J], China Chewing, 2010, 5: 42-45
AWARDS AND HORNORS

Research Assistantship, Florida State University, 2013

Florida-China linkage scholarship, Florida, 2012-2013

7th Annual Experience Asia Festival, Tallahassee- Finger language performer, 2011

Excellent Undergraduate Thesis Paper, Zhejiang Gongshang University, 2010

Social Practice Special Scholarship, Zhejiang Gongshang University, 2009

College Comprehensive Scholarship, Zhejiang Gongshang University, 2007, 2008

Provincial Excellent Team of Summer Social Practice award, Zhejiang, China, 2007

Excellent Student Award, Zhejiang Gongshang University, 2007

Outstanding Member in Summer Internship, Zhejiang Gongshang University, 2007
AFFILIATION

Student Membership of Institute of Food Technologists
58