ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 1519
FOOD COMPOSITION AND ADDITIVES
Enzyme-Linked Immunosorbent Assay Kit for
Beta-Lactoglobulin Determination: Interlaboratory Study
FRANTIÒEK ÒTUMR
SEDIUM RD, s.r.o., ðelezni…ního pluku 1361, 530 02 Pardubice, Czech Republic
DANA GABROVSK;, JANA RYSOVÁ, and PETR HAN;K
Food Research Institute Prague, Radiov< 7, 102 31 Praha 10, Czech Republic
JAN PLICKA
IMMUNOTECH, a Beckman Coulter Co., Radiov< 1, 102 27 Praha 10, Czech Republic
KVŒTA TOMKOV;
SEDIUM RD, s.r.o., ðelezni…ního pluku 1361, 530 02 Pardubice, Czech Republic
PETR CUHRA, MARTIN KUBQK, SOÀA BARÒOV;, and LENKA KARÒULQNOV;
State Agriculture and Food Inspection Authority, Za Opravnou 300/6, 150 06 Praha 5, Czech Republic
HANA BULAWOV; and JOSEF BRYCHTA
State Veterinary Institute, RantíÍovsk< 93, 586 05 Jihlava, Czech Republic
An interlaboratory study was performed in six
laboratories to prove the validation of the ELISA
method developed for quantitative determination of
beta-lactoglobulin (BLG) in foods. The ELISA kit
used for this study is based on rabbit polyclonal
antibody. In-house validation of the kit did not
produce false-positive results or cross-reactivity in
a broad range of food matrixes containing no milk
proteins. All participants obtained the BLG kit with
a standard operational procedure, the list of the
samples, samples, and a protocol for recording
test results. The study included 14 food samples
(extruded breakfast cereals, bread, two soy
desserts, butter, chicken ham, chicken meat, wheat
flour, long grain rice, jelly, two whey drinks,
crackers, and bitter chocolate) and six spiked
samples (two rice, two wheat flour, and two
chicken meat). Nine samples of food matrixes
containing no milk proteins showed BLG content
lower than the first standard (0.15 mg/kg). Two
samples of food matrixes with no milk proteins
revealed BLG content higher than standard 3
(1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three
food samples containing milk were tested as
positive, and all spiked samples were evaluated as
positive. The statistical tests (Cochran, Dixon, and
Mandel) and analysis of variance were used to
evaluate the interlaboratory study results.
Repeatability and reproducibility limits, as well as
LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg),
for the kit were calculated.
Received August 28, 2008. Accepted by SG January 12, 2009.
Corresponding author’s e-mail: [email protected]
C
ow’s milk allergy is the most frequent cause of food
allergy in infants (1). Hypersensitivity to cow milk
proteins may persist through adulthood and can be
very severe. Different clinical symptoms of milk allergy have
been established (2). Data on the prevalence of milk allergy
vary according to the various countries, and results of studies
on about 1% of the general population of adults and 2–3% of
children can be considered approximate (3).
Cow’s milk contains 30–35 g proteins/L. Its acidification
to pH 4.6 causes formation of two fractions: whey (20% of all
proteins content) and casein curd (80% of all proteins).
The whey contains mainly beta-lactoglobulin (BLG),
alpha-lactalbumine (ALA), bovine serum albumin (BSA),
lactoferrin (LF), and immunoglobulins (Igs). The curd
consists of casein (CAS), occurring in four isozymes named
Alphas1, Alphas2, Beta, and Kappa. The curd consists of CAS,
comprising four proteins coded by different genes carried on
the same chromosome (Alphas1, Alphas2, Beta, and Kappa).
BLG is the major allergen of cow’s milk, and occurs
naturally in the form of a 36 kDa dimer. No homologue of
BLG is present in human milk. Two disulfide bridges span the
molecule, and another cystein is present as a free residue. This
structure is responsible for the main physicochemical
properties and for interaction with CAS during heat treatment.
The relative resistance of BLG to acid and enzymatic
hydrolysis allows the protein to be absorbed intact through the
intestinal mucosa. BLG belongs to the lipocalin family, a
group of proteins with high allergenic potential (3, 4).
There is no unambiguous relation between cow’s milk
allergenicity and its heat processing. Boiling of milk for 2, 5,
or 10 min results in either no difference or in a reduction of
about 50–66% of the positive reactions compared to raw
milk (3).
BLG is thermolabile, but it may be protected through
interaction with casein. The loss of organized protein
1520 ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009
Table 1. Samples included in the study
Sample
No.
Content
1
Bread (sliced)
2
Crackers with herbs de Provence
3
Bitter chocolate
4
Jelly Sour fish
5
Soy dessert apricot
6
Soy dessert caramel
7
Chicken ham
8
Rice long grain
9
Rice long grain spiked with 200 mL of 100´ diluted milk/1 g
10
Rice long grain spiked with 660 mL of 100´ diluted milk/1 g
11
Wheat flour
12
Wheat flour spiked with 200 mL of 100´ diluted milk/1 g
13
Wheat flour spiked with 660 mL of 100´ diluted milk/1 g
14
Chicken meat
15
Chicken meat spiked with 200 mL of 100´ diluted milk/1 g
16
Chicken meat spiked with 660 mL of 100´ diluted milk/1 g
17
Extruded breakfast cereals with cinnamon
18
Butter with plant oil
19
Whey fruit drink
20
Whey drink for sportsmen
CS 1
Negative sample from the kit
CS 2
Positive sample from the kit
structures upon heat treatment does not necessarily result in a
decreased allergenic potential. The resulting allergenicity of
BLG depends mainly on temperature, heating time, and
possible interactions within the food matrix.
Determination of the allergenicity of a low molecular
fraction of whey protein and sodium caseinate hydrolysates
was studied. Alcalase, pepsin, and lactozyme were used in the
multistep hydrolysis. Allergenicity was examined with the
ELISA method by applying sera from eight patients. The
results did not reveal significant differences compared to
ultrahigh temperature (UHT) milk (5). On the other hand, the
formation of aggregates during heating may increase the
allergenicity of the product (3, 6, 7). Increasing antigenicity
was detected in skim milk and sweet whey at temperatures
ranging from 50 to 100°C. The highest antigenicity was
detected at 80 and 90°C. Above 100°C, the BLG antigenicity
of skim milk and sweet whey decreased with increasing
temperature and holding time (8). The effect of high-pressure
treatment on antigenic response of bovine BLG was also
studied (9). Whey protein isolate solution, sweet whey, and
raw skim milk were pressurized, and the antigenic response
was determined by means of an indirect competitive ELISA.
The solutions containing BLG were pressurized at 200, 400,
and 600 MPa for 0, 10, and 30 min at temperatures between 30
Figure 1. Calibration curve.
and 68°C. The antigenicity of BLG increased with increasing
pressure and with holding time in all solutions (9).
Few data are available on threshold levels of BLG
available in clinical studies using single-, double-blind,
placebo-controlled food challenge (SBPCFC and DBPCFC).
The lowest observed adverse effect level (LOAEL) has been
observed at <0.1 mL of milk (10). Indirect indications on
possible threshold doses have been obtained from reports of
severe adverse reactions that occurred after ingestion of a
minute amount of dairy product. Food allergens, including
BLG and other milk proteins, are absorbed and excreted
through breast milk, which may be responsible for adverse
reactions in breast-fed infants. In reports where breast-fed
babies experienced severe reactions, the concentration of
cow’s milk proteins were as low as a few ng/mL of milk. They
ranged from 0.5 to 50 ng/mL, and reactions often reportedly
occurred at about 5 ng/mL (3). For the detection and
identification of cow’s milk proteins, various analytical
methods have been used (ELISA, chromatographic
methods, electrophoretic methods, immunoblotting, MS, and
ion-exchange chromatography (11–13). Several commercial
ELISA kits for BLG determination are available. The
influence of heat treatment on BLG immunoreactivity and
assay applicability was also tested (14). A newly published
method uses an optical biosensor chip and gold nanoparticles
based on the resonance-enhanced absorption effect (15).
Tracing of allergens in food have become quite important
in recent times, as food allergens are recognized as the cause
of many symptoms of atopy in predisposed consumers.
According to the European Union (EU) and Czech national
legislation, all selected substances with allergenic effect are
ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 1521
Table 2. Content of beta-lactoglobulin (BLG) in the
samples (average from two replicates)
BLG, mg/kg
Sample
Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
1
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
2
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
3
10.0
12.9
7.74
13.5
14.8
15.7
4
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
5
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
6
2.01
1.02
2.18
2.01
3.31
2.04
7
<0.15
<0.15
0.264
<0.15
<0.15
<0.15
8
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
9
1.89
2.29
1.90
0.85
1.43
2.53
10
5.72
4.65
8.39
7.66
4.70
9.14
11
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
12
0.18
0.46
0.29
<0.15
0.21
0.20
13
4.80
5.67
4.36
2.17
4.19
1.06
14
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
15
0.80
1.53
1.40
0.97
1.14
2.01
16
5.83
3.89
3.42
3.81
3.82
6.77
17
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
18
8.58
6.10
8.19
7.36
4.07
8.80
19
10.7
33.2
9.33
18.6
22.1
20
11.1
31.1
12.8
5.96
9.37
15.0
13.9
CS 1
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
CS 2
0.51
0.17
0.52
0.33
0.42
0.74
crackers, jelly, soy desserts, chicken ham and chicken meat,
wheat flour, rice, and extruded breakfast cereals) were
purchased in a supermarket. Bread, crackers, bitter chocolate,
chicken ham and chicken meat, rice, and extruded breakfast
cereals were milled. A rotor-speed mill IKA® was used to mill
the samples. The spiked samples were prepared individually
by addition of 200 or 660 mL UHT milk diluted 100 times in
the extraction buffer per 1 g sample containing no milk
proteins. The amount of UHT milk addition was selected to
achieve absorbance of spiked samples in the linear part of the
calibration curve. The samples of foods were distributed
together with the ELISA kit for interlaboratory study. The
samples are listed in Table 1.
ELISA Kit Chemicals
(a) The BLG ELISA kit.—Cat. No. FA 00107 (SEDIUM
RD, s.r.o., Czech Republic). The format of the newly
developed ELISA kit for BLG determination in food and raw
materials is a two-step sandwich assay based on polyclonal
bovine BLG antibody produced in a rabbit. This antibody
reacts specifically with bovine BLG and may cross-react with
BLG from other species.
(b) Antibody.—Cat. No. A10-125A (Bethyl Laboratories,
Inc., Mongomery, TX). This antibody was used for
solid-phase coating and signal conjugate with horseradish
peroxidase (Cat. No. 10121606, Roche Diagnostics GmbH,
Mannheim, Germany).
(c) BLG.—Cat. No. 9045-23-2 (Sigma-Aldrich, St. Louis,
MO) was used for standard solutions preparation.
(d) 3,3¢,5,5¢-Tetramethyl-benzidine (TMB).—Cat. No.
T5513 (Sigma-Aldrich), TMB Liquid Substrate for ELISA.
The substrate, supplied as a one component ready-to-use
solution, was used as TMB substrate.
ELISA Kit Composition
subject to labeling rules for common foods and raw materials.
This means that all food producers must provide appropriate
information to consumers who suffer from a food allergy. This
EU food legislation would result in better food quality and
labeling control by state inspection authorities. New validated
analytical methods are needed in this respect (3, 16–18).
Some commercial kits for the detection of food allergens
are already available. However, the spectrum of food
allergens is not covered in full; confirmation of detected
quantities is possible only with a system based on more than
one kit. Also, in any particular food, a number of substances
can compose its allergenicity. Therefore, it is advantageous to
detect more than one allergen present in particular food. The
ELISA kit used in this study fulfills the need for detection of
one cow’s milk allergen: BLG. Validation of the kit by
collaborative study throughout its testing on a broad scale of
food matrixes increases its value as a versatile detection tool.
Experimental
Samples
The selected foods containing milk proteins (butter, whey
drinks) or foods with zero content of milk proteins (bread,
(a) Microtiter plate (MTP) with lid.—96 wells (arranged
in 12 strips, each containing eight wells), inner walls of which
are coated with the specific antibody.
(b) Buffer solution for the first incubation.—
Phosphate-based buffer solution, pH 7.2, colored blue. One
bottle containing 24 mL solution (ready-to-use).
(c) Standards (ready-to-use).—Six vials, each containing
0.5 mL standard solutions. The vials contain BLG in
concentrations of 0, 0.15, 0.50, 1.50, 5.00, and 15.00 mg/kg,
colored yellow.
Table 3. Classification of samples into categories
Category
Samples
1a
1, 2, 4, 5, 7, 8, 11, 14, 17, CS 1
1b
3, 19, 20
2a
6, 18
2b
9, 10, 12, 13, 15, 16, CS 2
1522 ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009
Table 4. Contingency table of false-positive and
false-negative statements
Sample
True
False
F/T ratio
Positive sample
TP 108
FN 2
0.018
Negative sample
TN 118
FP 2
0.017
(d) Control samples (CS 1-negative: 0 mg BLG/kg; CS
2-positive: 0.5 mg BLG/kg).—Two vials, each containing
0.5 mL, yellow colored (ready-to-use).
(e) Concentrate of extraction buffer (5´).—Yellowcolored Tris buffer, pH 8.5. One bottle, containing 90 mL
concentrate.
(f) Concentrate of conjugate (80´).—One vial, containing
0.4 mL concentrated conjugate of the specific antibody with
peroxidase in phosphate buffer solution containing stabilizers.
(g) Buffer solution for dilution of conjugate.—Phosphate
buffer solution, pH 7.2, colored pink. One bottle containing
24 mL (ready-to-use).
(h) Concentrate of wash solution (20´).—One bottle,
phosphate buffer with detergents, pH 7.2. One bottle
containing 50 mL concentrate.
(i) TMB substrate.—One bottle containing 22 mL
(ready-to-use).
(j) Stop solution.—One vial containing 5.5 mL (ready-touse); 2 M HCl solution.
(k) Vial for conjugate dilution.—One piece.
Assay Parameters
In-house validation of the kit was performed to determine
the following assay parameters.
Analytical sensitivity.—First standard (0.15 mg/kg) was
diluted with zero standard twice and four times to
obtain solutions with concentration of 0, 0.0375, 0.075, and
0.15 mg/kg. These solutions were measured 10´ (zero
standard 20´), and analytical sensitivity, called LOD, was
calculated from in-house validation (LODIH). The lowest
concentration at which absorbance was detected represents
the analytical sensitivity. The value determined by this
procedure is 0.06 mg BLG/kg.
Functional sensitivity.—Defined addition of BLG to three
naturally milk-free matrixes increased its content to levels of
0.02, 0.05, 0.10, and 0.15 mg/kg. The value 0.1 mg BLG/kg
was determined as a reliable value that can be established (as
LOQ from in-house validation (LOQIH).
Repeatability.—Intra-assay variance of the kit was tested
for extracts of three samples in 25 multiplicates. Maximum
CV was 9.98%. Interassay variance of the kit was determined
for three samples in 10 runs, and maximum CV was 14.2%.
Extraction effect.—Defined amount of BLG was added to
four naturally milk-free samples to obtain two levels of BLG
content. The extraction of each sample and determination of
BLG content were performed in two runs. The differences
between runs were <25%.
Dilution effect.—Sets of diluted samples using dilution
buffer from extracts of two real samples with natural content
of BLG 4.5 and 2.3 mg/kg were prepared. All samples were
then analyzed in one run. Recovery was in the range of
89.5–117%.
Recovery.—Defined addition of BLG to two naturally
milk-free matrixes (meat and rice) increased its content to
levels of 0.5, 1.0, and 2.0 mg/kg. These extracts of the samples
were analyzed, and recovery was in the range of 91–118%.
Zero matrixes.—Analyses included raw materials for
foodstuffs, the nature of which should justify the expectation
of their zero content of milk proteins: wheat flour, fish meat,
chicken meat, beef; pork, rice, soy, fruit juice, vegetable oil,
and eggs. The analyses detected no BLG in these matrixes.
Goat milk was also tested, and a very high positive result
was found.
Interlaboratory Study
Six laboratories with daily routine ELISA experiences
were included in this study. The aim was that each laboratory
would perform a complete evaluation of BLG content in the
samples provided. The participants received the BLG kit with
instructions and all samples. They were required to perform
complete duplicate analyses of the samples, including
calibration. The calibration data, absorbance values, and
remarks on technical conditions (washing procedure, ELISA
reader, centrifugation) from participants were required.
Sample Treatment
Laboratories received 1 g of each food sample and spiked
samples. All samples were prepared for direct extraction; only
10 mL of extraction buffer had to be added. The samples were
shaken for 30 min and immediately centrifuged at 1800 ´ g for
10 min. The supernatant was used for the assay directly
without dilution.
Determination
A 200 mL volume of buffer for the first incubation was
added into each well, and then 20 mL of standards, or control
samples or samples was added into corresponding wells. The
plate was covered and incubated for 1 h at room temperature
without shaking. The content of all wells was washed four
times with 300 mL wash solution using the washer or repeating
dispenser. Conjugate working solution (200 mL) was
immediately dispensed into all the wells.
The plate was covered and incubated for 1 h at room
temperature without shaking. The content of all wells was
washed four times with 300 mL wash solution using the
washer or repeating dispenser. TMB substrate (200 mL) was
immediately dispensed into all the wells. The plate was
incubated for 20 min in the dark without shaking. A 50 mL
volume of stop solution was added and shaken briefly, and
within 15 min the absorbance reading was made at 450 nm.
ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 1523
Table 5.
Repeatability and reproducibility data from the interlaboratory study
Sample No.
Performance characteristic
6
18
9
12
Mean value (y, mg/kg)
2.10
7.84
1.67
0.24
No. of laboratories (after exclusion of outliers; n)
6
6
5
6
No. of outliers (N)
0
0
1
0
Repeatability standard deviation (sr)
0.28
0.42
0.14
0.022
Relative standard deviation of repeatability (RSDr, %)
Repeatability limit (r)
Reproducibility standard deviation (sR)
Relative standard deviation of reproducibility (RSDR, %)
13.3
0.78
0.76
36.1
5.82
8.51
8.92
1.17
0.4
0.061
1.84
25.6
0.56
33.6
0.12
48.7
Reproducibility limit (R)
2.12
5.15
1.57
0.33
r/R ratio (r/R)
0.37
0.23
0.25
0.18
RSDR from Horwitz equation [CV(R)%]
HorRat [RSDR/CV(R)]
14.3
2.52
Calculations
A calibration graph was constructed as the dependence of
measured absorbance of corresponding calibration solutions
subtracted by the absorbance of zero standard (vertical
axis–logarithmic scale) versus BLG concentration in the
solution (horizontal axis–logarithmic scale). Logarithmic
equation was used for graph construction. An example of a
calibration curve is shown in Figure 1.
Results and Discussion
Sample absorbance values obtained from participating
laboratories were evaluated by Excel® software. The
calculated results of all samples in the collaborative study are
presented in Table 2. Nine samples of food matrix with zero
content of milk proteins (samples 1, 2, 4, 5, 7, 8, 11, 14, and
17) revealed BLG content lower than the first standard
(0.15 mg/kg). Sample 3 (bitter chocolate) revealed BLG
content higher than standard 4 (standard 5 resp.). The label of
this product stated that it could contain traces of milk proteins.
Sample 6 (soy dessert caramel) had BLG content higher than
standard 3 (standard 2 resp.), and no remark concerning milk
as an ingredient was mentioned on the label. This means the
product is probably contaminated by milk and is not suitable
for people with milk allergy. The samples with milk or whey
proteins content in the list of ingredients (samples 18–20)
revealed positive results, in two cases even above the highest
calibration point.
Statistical Evaluation
Samples included in the test could be classified in two types:
(1) Samples out of scope of the kit: (a) Negative
samples.—Samples without BLG content and lower than the
lowest point of the calibration curve (in the majority of
11. 9
2.15
14.8
2.27
19.8
2.46
laboratories). These samples were not assessed in quantitative
way; concentration values are only roughly informative.
(b) Highly positive samples.—Samples with BLG content
higher than the highest point of the calibration curve (in the
majority of laboratories). These samples were not assessed in
quantitative way; concentration values are only roughly
informative.
(2) Samples with quantification accomplished, i.e.,
findings between the lowest and highest calibration point:
(a) naturally positive samples; and, (b) samples with known
intentional addition of milk. Further statistical evaluation
included only results within the calibration range, i.e., samples
from categories 2a and 2b. Table 3 presents the classification
of samples into categories.
The following parameters were determined within the
collaborative study: repeatability, reproducibility; LOD;
LOQ; and ratios of false-negative (FN/TP) and false-positive
results (FP/TN).
LOD and LOQ
The LOQ (sometimes called lowest observable quantity)
of the kit was judged as 10 times the value of average standard
deviation of reproducibility (LOQ = 10´sR) on levels close to
the LOQ, and the value of 0.22 mg/kg was found. The LOD of
the kit was judged as three times the value of the average
standard deviation of reproducibility (LOD = 3´sR) with a
value of 0.07 mg/kg. The LOQ is slightly higher than the
lowest point in the calibration curve (0.15 mg/kg), and reflects
concentration above which the presence of BLG is beyond
reasonable doubt at the probability level of 95%.
False-Positive/False-Negative
Based on the assumption that samples in category 1a are
truly negative, one can count the number of false statements
(i.e., statements that proclaim these negative samples positive;
1524 ÒTUMR ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009
Table 6. Repeatability and reproducibility data from the interlaboratory study
Sample No.
Performance characteristic
15
10
13
16
CS 2
Mean value (y, mg/kg)
1.41
6.22
3.71
3.73
0.5
No. of laboratories (after exclusion of outliers; n)
5
5
6
4
5
No. of outliers (N)
1
1
0
2
1
Repeatability standard deviation (sr)
0.22
0.34
0.46
0.81
0.061
Relative standard deviation of repeatability (RSDr, %)
Repeatability limit (r)
Reproducibility standard deviation (sR)
15.7
0.62
0.43
5.51
12.5
0.96
1.3
1.74
1.77
0.81
0.17
0.16
Reproducibility value (R)
1.20
4.86
4.96
2.27
0.44
r/R ratio (r/R)
0.52
0.20
0.26
1.00
0.39
15.2
2.00
therefore, FP). Similarly, in category 2, the fraction of FN
statements can be determined as the number of false
statements, i.e., those when positive result was proclaimed
negative. Table 4 presents an overview of numbers of the
above-mentioned statements.
F/T = ratio of number of false statements to number of true
statements. As presented in Table 4, FN/TP was 0.018, while
FP/TN was 0.017. Both ratios (approximately 1.7%, resp.
1.8%, of false statements from total number of all statements)
are generally considered as low, and acceptable for the
intended use of the kit. These ratios characterize reliability
and selectivity of the kit. All four false results can be
considered as the fault of the laboratory, because the measured
concentrations were on levels near the lowest calibration
points. All false results were considered as outliers within
statistical evaluation.
Repeatability and Reproducibility
Values of repeatability and reproducibility were calculated
from nine samples that were appropriate for quantification
(category 2). Results are summarized in Tables 5 and 6.
Although repeatability expressed as RSDr, % is <16%
except in one case, and in four cases even <10%, values of
RSD of reproducibility (RSDR, %) are higher than could be
expected from the Horwitz equation (19; see also line Horwitz
CVR %), but still acceptable. However, in all cases but one, the
determined RSDR value grouped around 2 times the value of
Horwitz CV(R). Such values are still generally considered as
an acceptable level of precision (20). Cochran, Grubbs, and
Mandel tests according to ISO 5725 were used for detection
of outliers (21). Probability level a = 1% was used for the
exclusion of a laboratory from further statistical evaluation.
12.2
2.29
13.1
3.63
21.7
12.2
30.4
RSDR from Horwitz equation [CV(R)%]
47.7
2.27
Relative standard deviation of reproducibility (RSDR, %)
HorRat [RSDR/CV(R)]
27.9
21.7
13.1
1.66
31.5
17.7
1.77
Conclusions
The newly developed sandwich ELISA kit for quantification
of BLG was developed and validated. Analytical sensitivity,
functional sensitivity, recovery, accuracy, and other parameters
were determined by in-house validation.
The published collaborative study confirmed that the
developed ELISA kit is suitable for BLG content
determination in food products and raw material. All negative
samples were determined as negative in all laboratories, as
were all positive samples. LOD and LOQ were determined
within the collaborative study. All spiked samples revealed
BLG content higher than the first standard (samples spiked by
200 mL UHT milk) and higher than standard 3 or 4 (samples
spiked by 660 mL UHT milk). Performance characteristics are
acceptable for routine use in food control laboratories.
Acknowledgments
The work was supported by the Czech Ministry of
Education, Youth and Sports, Research Project No. 2B06138.
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