King Abdulaziz University
Faculty of Science
Department of Biochemistry
Girls Section
Immunochemistry Lab
BIOC 446
Organized by lecturers:
Omima Niazy and Wedam Alghazzawi
Contents
Week #
Experiment
Page
1
Immunochemistry history
Immunological tests
2
Human blood groups
7
3
Standard Curve of Immunoglobulin
10
2,3
4,5
Quantitative perception test
Immunochemical assays
12,14
6
Determination of fibrinogen
18
Purification of immunoglobulin by ion exchange
chromatography
21
7,8
9
10,11
hCG Pregnancy test and Human Immunodeficiency Virus
test
28,29
Enzyme-Linked Immunosorbent Assay
30
12
Ouchterlony double diffusion for antigen and antibody
patterns
33
10
References
35
11
Further readings
36
1
Definition:
Immunochemistry (chemoimmunology) is studying the chemical reaction between
antigens and antibodies that occur in immune system.
The structure of an antibody is a large Y-shape glycoprotein produced by plasma
cells that is used by the immune system to identify, and foreign objects called
an antigen. Each tip of the "Y" of an antibody contains a paratope (similarly to a lock)
that is specific for one particular epitope (similarly to a key) on an antigen, allowing
these two structures to bind together.
2
It is involve the measurement of one or other of the components of the antibodyantigen system, namely, antibody, antigen and the immune complex. There are a
very large number of tests possible with a given combination of antibody and antigen
and here you can see the following examples:
These Tests include:
1. Precipitation
2. Agglutination
3. Immunofluorescence
4. Radioimmunoassay (RIA)
5. Enzyme-Linked Immuno sorbent Assay (ELISA)
6. Western Blotting
The major differences between the assays are the sensitivities and the time taken to
carry them out. As expected, the most sensitive methods are usually the most
difficult technically and therefore the ones that can give the greatest error.
Immunochemical are now used extensively in the biological sciences and some
examples of those currently in use are given in the following series of experiments.
3
1. Precipitation
Soluble antigen interacts with polyclonal antibody to form a lattice (a visible
precipitate). It is called precipitins (equivalence).
No precipitate is formed
Precipitate is formed
1. Precipitation reactions (Immunology/Serology)
I- Precipitation in Solution

Bottom Precipitate

Ring Precipitate (Ring Test)
II- Simple Immunodiffusion (ID)

Double ID (Ouchterlony)

Single Radial ID (RID) (Mancini)
III- Electro-Immnodiffusion
I- Precipitation in Solution
Bottom Precipitate
Ring Precipitate
4
II- Simple Immunodiffusion (ID)
Double ID
Single Radial ID
5
2. Agglutination
It will be discussed in blood group lab.
6. Western blotting
3. Immunofluorescence
4. Radioimmunoassay
5. Enzyme-Linked Immunosorbent Assay
It will be discussed in ELISA lab.
6
Principle:
Blood consists of different types of cells floating in plasma. The types of cells are the
red blood cells contain hemoglobin binding with oxygen; the white blood cells
defending the body against infection; the platelets help the blood clotting and the
plasma contains dissolved protein and salt.
The differences in human blood type or blood group are based on the presence or
absence of specific protein molecules called antigens and antibodies on red blood
cells. Antigens are located on the surface of the red blood cells and the antibodies
exist in the blood plasma. The blood groups in persons depend on what the persons
inherited from their parents.
In blood transfusions, the process of receiving blood from another type leads to the
antibodies bind to the foreign antigens, causing dangerous clumping or agglutination
of the blood. Therefore, the successful transfusion depends on matching antigens on
the surface of the red blood cells between two persons.
Rhesus factor (Rh factor). It was first discovered in the blood of Rhesus monkeys.
The persons who have the antigen in their blood are called Rh positive and the
persons who lack that protein are called Rh negative. For example, the Rh factor is an
important in mother whose is Rh negative and her fetus whose blood is Rh positive
that lead to the mother blood attack the fetus blood.
AB0 blood grouping system
There are four different kinds of blood groups: A, B, AB or 0 (null).
7
Blood group A
The person has A antigens on the surface of red blood cells and B
antibodies generated in blood plasma.
Receive from A and O blood groups
Give to A and AB blood groups
Blood group B
The person has B antigens on the surface of red blood cells and A
antibodies generated in blood plasma.
Receive from B and O blood groups
Give to B and AB blood groups
Blood group AB
The person has A and B antigens on the surface of red blood cells
and no A or B antibodies in blood plasma.
Receive from all blood groups
Give to AB blood group
Blood group 0
The person does not have A and B antigens on the surface of red
blood cells and A and B antibodies generated in blood plasma.
Receive from O blood groups
Give to all blood groups
Rh+ blood
The person has Rh+ antigens on the surface of red blood cells
and no Rh- antibodies generated in blood plasma.
Receive from Rh+ and Rh- blood groups
Give to Rh+ blood group
Rh- blood does not have antigens on the surface of red blood
cells but it has Rh+ antibodies generated in blood plasma.
Receive from Rh- blood group
Give to Rh+ and Rh- blood groups
Material:

Clean microscopic slide

Blood drops

Marker

Ani-A, Anti B and Anti-D antibodies reagents
Methods:
1. Mark one end for A, other end for B, and the last end for D on the slide.
2. Add one drop of blood on each position of the marked.
8
3. Add one drop of Ani-A, Anti B and Anti-D antibodies reagents on each side
respectively.
4. Mix each side with a toothpick and spread each mixture slightly.
5. Observe if any agglutination for red blood cells.
9
Principle:
Immunoglobulin (Ig) is a membrane-bound form of glycoprotein that is attached to
the surface of a B cell and is known as the B cell receptor while antibody is a soluble
form that is secreted from the plasma cell into the blood and tissue fluids. The five
major types of Immunoglobulins are IgA, IgG, IgM, IgE, and IgD. Gamma globulin
(IgG) is a type of immunoglobulin can be isolated from human blood and injected for
passive immunization against some diseases like hepatitis. Here in this experiment,
we will quantify the protein by Biuret reagent. Increasing quantities of γ-globulin are
assayed by measuring the extinction at 540 nm.
Materials (Preparation for 100 students):

Isolated γ-globulin.

D.W.

Biuret reagent

Spectrophotometer

Vortex

Incubator

Five test tubes

Pipette (1ml)
Procedure:
Tube
γ-globulin (ml)
H2O (ml)
Blank
1
2
3
4
-
0.25
0.5
0.75
1
0.25
1.5
1.5
1
0.75
0.5
1.5
1.5
1.5
Biuret (ml)
Vortex all tubes and incubate at 37°C for 15 min.
Read the absorbance of each tube at 540 nm.
10
Result Sheet
Lab partners: ------------------------------Standard concentration: _____________
A. Calculate the different concentrations of proteins and read the absorbance at 540
nm for each one.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
B. Fill the below table
Tube #
Concentration
(mg/ml)
Absorbance
C. Plot a standard curve of the absorbance against the concentration of γ-globulin.
11
Functional assays:
Biological fluids contain a complex mixture of proteins. There are many assay
systems for assaying a specific protein by reacting with that protein such as
antibodies that react with specific protein so that immunochemical techniques are
accurate and highly specific.
Currently there are a number of assay systems depend on the formation of an immune
complex between the antibody and the antigen.
All antigen-antibody reaction is reversible and a typical 'titration curve' refers to
increasing amount of antigen added to a fixed concentration of antibody as below
figures. Be clearly to identify the antigen excess region if the results are not to be
misinterpreted.
Figure 1: A precipitation curve for a system of one antigen and its antibodies.
12
Figure 2: A typical antigen-antibody precipitation curve for an antigen titrated against a
fixed concentration of antibody.
13
Principle:
Gamma globulin (γ-globulin) fraction will isolate from the human serum by salt
precipitation used as the antigen in this experiment. Increasing quantities of antigen
are mixed with a constant amount of antiserum and the protein precipitated as the
immune complex assayed by measuring the extinction at 280 nm.
Maximum interaction between the antibody and the antigen is only obtained if the
incubation at 4°C is carried out for several days but with class experiments this is not
very convenient and quite reasonable results can be obtained after 1 hour.
Materials: (Preparation for 100 students):

Fresh human serum (100 ml).

Saturated Ammonium Sulphate (NH4)2SO4: Add 155 g of Ammonium Sulphate to
200 ml of water.

NaOH (0.1mol/liter) 2 liters.

Antiserum (rabbit anti-human whole serum).

UV spectrophotometers

Bench centrifuges

Autoclave

Red-top blood container

Centrifuge tubes

Pipette (1ml & 2ml)

Micropipette (0.5-10 μl) ,(10-200 μl), (100-5000 μl)
Procedure:
1. Pipette 2 ml of human serum into a test tube and slowly add 2 ml of saturated
ammonium sulphate solution; leave on ice for about 30 min with occasional
stirring then centrifuge at 2500 g for 15 min.
2. Remove the supernatant and resuspend the pellet in a known volume of the
buffered saline and suitably diluted, write the volume taken.
14
3. Measure the extinction at 280 nm and calculate the amount of protein present in
the diluted sample by assuming that 1 mg/ml has an extinction of 1.40 at 280 nm.
4. In a series of tubes, prepare different concentrations of γ-globulin in μg (0, 10, 20,
30, 50, 100, 200, 300, 400, 500, and 1000) by calculating the added volume of γglobulin from step 3.
5. Add buffered saline in tubes to bring the final volume to the volume in the last
tube.
6. Add 50 μl of antiserum to each tube, mix thoroughly then incubate at 37°C for 1
h, then 4°C for 1 h or overnight.
7. Centrifuge the tubes at 25000g for 5 min and remove the supernatant.
8. Wash the precipitate twice with 1ml ice-cold buffered saline, centrifuge, and
decant washing.
9. Finally dissolve completely the final pellet in 0.l mol/liter NaOH.
10. Read the extinction of each tube at 280 nm.
11. Plot a graph of the extinction against the amount of antigen present.
15
Result Sheet
Lab partners: -------------------------------
Data table:
Tube no.
Concetration
(μg/ml)
1(blank)
0
2
10
3
20
4
30
5
50
6
100
7
200
8
300
9
400
10
500
11
1000
Volume of
imunoglobulin (μl(
Calculations:
16
Volume of buffer
saline (μl(
Absorbance
at 280 nm
c. Plot a graph of the extinction against the amount of protein present.
17
Principle:
Fibrinogen is a plasma protein and it is important in blood clotting. It synthesized by
liver. A number of simple methods for determining fibrinogen are available. Some of
which are useful, and for example, when a quick result is required. They include
several precipitation tests and the tyrosine method (Lempert).
Determination of plasma proteins:
While Kjeldahl methods properly carried out have usually been considered the
most reliable, they are more time consuming than colorimetric methods and need
more care in execution. The colorimetric methods may not be so sound theoretically
but can give sufficiently accurate results. If a colorimetric method is used it should be
compared with a Kjeldahl method.
Materials:

Calcium Chloride solution (2.5%)

Sodium hydroxide (N/4)

Sulphuric acid (1N)

Sodium Carbonate (20%)

Tyrosine solution (containing 20 mg of tyrosine per 100 ml of 0.1 N hydrochloric
acid).

Folin-Ciocalteu reagent (the undiluted reagent), dangerous.

D.W.

Bench centrifuge.

Spectrophotometer.

pH meter.

Water bath.

Centrifuge tubes.

Pipettes & Glass rod & dropper.

Universal paper 0-14 & 5-8

Measuring cylinder
18
Procedure:
1. Collect 5 ml blood into Sodium Citrate tube (blue cap), (0.5ml citrate/5ml blood),
and then centrifuge for 10 min at 3000 rpm.
2. Mix 0.5 ml. of plasma with 14 ml of distilled water and 0.5 ml of calcium chloride
solution in a small measuring cylinder,
3. Place a fine glass rod (clean capillary tube) in the liquid and allow standing in an
incubator at 37° C until a clot is formed. Thirty minutes is sufficient but it may be
need to leave even overnight.
4. After incubator collect the clot by rotating the rod on it.
5. Press the rod against the side of the beaker to squeeze out any solution and to
compress the clot.
6. Take care to pick up on the rod any small pieces of clot which may have become
detached.
7. Dry by pressing carefully against a piece of filter paper.
8. Transfer the clot to the tube in which the digestion is to be carried out.
9. Collect the clot on a glass rod wash in several portions of distilled water; dissolve
in 5 ml of N/4 sodium hydroxide (in a boiling water bath).
10. Neutralize with sulphuric acid.
11. Transfer the contents into volumetric flask.
12. Add 0.5 ml of the Folin-Ciocalteu reagent.
13. Add 3 ml of Sodium Carbonate.
14. Make up to 25 ml of D.W. and place in an incubator at 37°C for an hour (not
more).
15. As standard use 0.5 ml of a tyrosine and treat in the same way as the test (steps
12, 13, and 14).
16. Read absorption at 680 nm by colorimeter with using water as blank.
17. Since 1 mg of tyrosine is equivalent to 16.4 mg fibrinogen.
18. Mg fibrinogen per 100 ml. = Abs. of unknown x 100 X 0.1 X 16.4
Abs. of standard
0.5
19
Result Sheet
Lab partners: -------------------------------
Mention the quantity of fibrinogen per 100 ml of plasma.
20
Principle:
Ion exchange chromatography is a process in which ions are exchanged between a
solution and an insoluble solid (usually resinous and open permeable molecular
structure so ion and solvent ion can move freely in and out).
Several side-chain groups of the amino acid residues in proteins are ionizable,
Therefore proteins are charged molecules. This characteristic can be used to separate
different proteins by ion-exchange chromatography.
When human serum is added to an ion exchange column such as diethylaminoethylSephadex (DEAE-Sephadex), most of the serum proteins become bound to the
column. However, by using a buffer with a pH greater than 6.5, IgG is not absorbed
and is eluted in the first fractions. The other proteins are then removed from the
column by a stepwise elution with increasing concentrations of salt. The isolated
fractions
are
then
analyzed
by
cellulose
acetate
electrophoresis
immunoelectrophoresis.
Figure 1: Ion exchange chromatography separation & analyzing.
21
and
There are two types of ion exchange chromatography: Cation exchange
chromatography and Anion exchange chromatography.
Component of ion exchange chromatography are mixture of positive and negative
proteins molecules, stationary phase (DEAE-Sephadex), and mobile phase (Tris-HCl
buffer and then buffered saline solutions).
See the below figure for separation of particles by AEX chromatography.
22
Column
Peristaltic pump
Eluants
Protein
Figure 2: Ion exchange chromatography system.
Peristaltic pump
Fraction collector
Figure 3: Ion exchange chromatography system.
23
Figure 3: Filtration under vacuum.
Materials:
1. DEAE-Sephadex A50-120
2g
2. Tris-HCl buffer (10 mmol/litre, pH 8.0)
3 liters
3. NaOH (1 mol/liter)
1 liter
4. HC1(1 mol/liter)
1 liter
5. Human serum (5 ml)
6. Buffered saline solutions. (Prepare the following concentrations of 250 ml saline
in the tris-HCl buffer)
a) 20 mmol/liter
b) 50 mmol/liter
c) 100 mmol/liter
d) 150 mmol/liter
e) 200 mmol/liter
f) 300 mmol/liter
7. Solid polyethyleneglycol (500 g)
8. UV spectrophotometers
9. Column monitoring equipment
10. Buchner filtration apparatus
11. Disposable syringes (10 ml)
12. Glass wool or plastic
13. Peristaltic pumps that can pump less than 60 ml/h
24
Procedure:
1. Preparation the stationary phase of the column:
a) Add 2 g of DEAE-Sephadex A50-120 to 70 ml of water and leave until swollen.
b) Degas the suspension under vacuum and leave to equilibrate to room
temperature.
c) Wash the column with 1 mol/litre NaOH then repeatedly with water until the pH
falls to about 7.
d) Wash with saline then 1 mol/litre HC1 and finally with the tris-HCl buffer until
the eluate registers pH 8.0. Alternatively the material can be washed on a
Buchner funnel.
e) Place some glass wool in the bottom of a 10 ml disposable syringe as a support
and prepare a column of the ion exchange material.
2. Separation of immunoglobulins:
a) Load 0.5-1.0 ml of your own serum on to the DEAE-Sephadex.
b) Pumping the tris-HCl buffer (about 2.5 ml) through the column.
c) Collect five fractions about 2.5 ml for each one.
d) In the meantime prepare 25 ml of a series of fluids containing sodium
chloride at the following concentrations in the buffer: 20 mmol/litre, 50
mmol/litre, 100 mmol/litre, 150 mmol/litre, 200 mmol/litre, 300 mmol/litre.
e) Pump these solutions through the column in turn starting with the lowest salt
concentration and collect fractions until all the protein has been eluted with
that solution, then move on to a higher concentration of salt.
f) Carefully monitor the proteins eluted by measuring the absorbance of each
fraction at 280 nm and plot the extinction against fraction number.
g) Concentrate each fraction between 5 and 10 times by dialysing against solid
polyethyleneglycol for about 3 h and examine the protein concentrates by
cellulose acetate electrophoresis, immunodiffusion and immunoelectrophoresis
25
Result Sheet
Lab partner: -------------------------------
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
26
1
2
3
4
5
6
7
8
9
10
Abs.
Tube no.
(300 mmol/l)
Abs.
Tube no.
(200 mmol/l)
Abs.
Tube no.
(150 mmol/l)
Abs.
Tube no.
(100 mmol/l)
Abs.
Tube no.
(50 mmol/l)
Abs.
Tube no.
(20 mmol/l)
Tube no.
(Tris-HCl)
Buffered saline solutions
1
2
3
4
5
6
7
8
9
10
Abs.
Plot a graph of the extinction against fraction number and paste the photo here.
27
Principle:
“Human chorionic gonadotropin (hCG). It is a glycoprotein hormone secreted by
viable placental tissue during pregnancy, is excreted in urine and serum
approximately 20 days after the last menstrual period. hCG levels rise rapidly,
reaching peak levels after 60-80 days. The appearance of hCG in urine and serum
soon after conception and its rapid rise in concentration makes it an ideal marker for
the early detection and confirmation of pregnancy. However, elevated hCG levels are
frequently associated with trophoblastic and non-trophoblastic neoplasms and hence
these conditions should be considered before a diagnosis of pregnancy can be made”.
Source: http://www.btnx.com/Content/Products/hCG%20Combo%20Cassette.pdf
Interpretation of results:

Positive results:
1. The test strip is dipped in a urine sample containing hCG hormone (as
antigen), the hCG is drawn by capillary action up the test strip to the reaction
zone, where it binds with mouse monoclonal anti-hCG antibody-enzyme
conjugate.
2. The hCG bound antibody-enzyme conjugates are dissolved and carried along
in the capillary flow to arrive at the test zone. Other epitopes on the hCG
molecules bind to the immobilized polyclonal hCG antibodies.
3. In the control zone, the unbound anti-hCG antibody-enzyme conjugate binds
to immobilized anti-mouse antibodies.
4. The fixed enzymes are able to activate the dye substrate in both the test and
control zones.

Negative results:
1. The test strip is dipped in a urine sample not containing hCG hormone, the
urine sample is drawn by capillary action up to the reaction zone, where it
helps the mouse monoclonal anti-hCG antibody-enzyme conjugate move to
the control zone, where it binds with immobilized anti-mouse antibodies.
2. The fixed enzyme is able to activate the dye substrate in control zone.
28
Principle:
Human Immunodeficiency Virus (HIV) that causes acquired immunodeficiency
syndrome (AIDS) which progressive failure of the immune system.
Method:
Interpretation of results:

Positive results:
1. The test strip is dipped in a urine sample containing HIV antibody, the HIV
antibody is drawn by capillary action up the test strip to the reaction zone,
and where it binds to HIV antigen coated gold particles to form the complex.
2. The complex moves to the test zone by capillary action, where it binds to
recombinant HIV antigen (coated on the test membrane).
3. In the control zone, the unbound HIV antigen coated gold particles binds to
immobilized anti-HIV antibodies.
4. The fixed enzymes are able to activate the dye substrate in both the test and
control zones

Negative results
1. The test strip is dipped in a urine sample not containing HIV antibody; the
urine sample is drawn by capillary action up to the reaction zone, where it
helps the HIV antigen coated gold particles move to the control zone , where
it binds with immobilized anti-HIV antibodies.
2. The fixed enzyme is able to activate the dye substrate in control zone.
29
Introduction:
Enzyme-Linked Immunosorbent Assay (ELISA) is the most commonly used
immunological assay to detect the presence of an antibody or an antigen in a sample.
ELISA may be run in a qualitative or quantitative format.

Qualitative results mean a positive or negative result for a sample. The cutoff between positive and negative is determined statistical.

Quantitative results means the amount of colored product is direct
proportional to the amount of enzyme-linked antibody or antigen. A standard
curve can be plotted each standard optical density against the standard
concentrations, then concentration the unknown antigen or antibody can be
determined from the standard curve.
ELISA results are read it as a number using a spectrophotometer (micro
reader plate) or spectrofluorometer ... etc.
ELISAs are commonly performed in 96-well (or 384-well) usually
polystyrene microtiter plates, which fix with antibodies or antigens.
96 - well microtiter plate
Advantages of ELISA:

Safest and not need to radioactive material such as RIA.

Allows for easy visualization of results with high level of accuracy.

Sensitive and specific.

Large numbers of tests can be done.

Require minimal reagents.

Qualitative & Quantitative assays
- Qualitative  eg. HIV testing
- Quantitative assays  eg. Therapeutic drug monitoring
30

Wells can be coated with antigens or antibodies
Requirements to do the assay

Sample (test).

Purified antigen (if you want to detect or quantify antibody).

Purified antibody (if you want to detect or quantify antigen).

Standard solutions (positive and negative controls).

Microtiter plates: plastic trays with small wells in which the assay is done.

Wash fluid (buffer).

Enzyme-labeled antibody and enzyme substrate.

ELISA reader (spectrophotometer) for quantitative measurements.
Types of ELISA assay
Variation of different types of ELISA assay depends upon the labeling and signal
detection methodology. The basic approaches stay the same: fixing either antigen or
antibody and detecting antibody-antigen complex.
ELISA
Direct
Indirect
31
Sandwich
Competitive
Direct ELISA
In this ELISA method, directly enzyme linked antibodies added to
react with target antigens (fixed to microtiter plate). The color or the
signal produced as a result of addition of substrate is proportional to
antibodies in the sample.
Indirect ELISA
This method differs than direct ELISA in that one more labeled
secondary antibody is added in the reaction. The primary antibody
added to the fixed antigen. Then labeled secondary antibody added
that recognizes the primary antibody. The color or the signal
produced as a result of addition of substrate is proportional to
antibodies in the sample.
Sandwich ELISA
It measures the amount of antigen between two layers of
antibodies - the capture antibody and the detection antibody, just
like a sandwich. The antigens to be measured must contain at least
two antigenic sites, since at least two antibodies bind to antigen.
The color or the signal produced as a result of addition of substrate
is proportional to antigen. For example, measuring the HIV
antibody.
Competitive ELISA
It measures the amount of antigen in a sample. In this type of ELISA, the antigen is
labeled instead of the antibody. Unlabeled antigen and the labeled antigen compete
for binding to the capture antibody. The color or the signal produced as a result of
addition of substrate is inversely proportional to antigens in the sample. For example,
the absence of the antigen in the sample will result in a dark color, whereas the
presence of the antigen will result in a light color or no color as the concentration of
the antigen increases.
32
Principle:
Ouchterlony double diffusion is used to detect, identify, and quantify antibody and
antigen. Antigens from different species are loaded into two wells and the known
antibody is loaded into a third well and antigens and antibody are diffused into the gel
to form different geometrical patterns depending on the similarity between the
antigens.
During incubation, the antibody and antigen react after diffusing through the gel and
form a precipitate where they meet. The immune complex is soluble in excess antigen
and so the equivalence point is marked by a sharp line. The position of this line
depends on:

The relative concentration of antibody and antigen in the agar which in turn
depends on the concentration in the well.

The molecular weights.

The diffusion coefficients of the constituents.
If the antibody and antigen contain several reactive species then multiple lines of
precipitation will be seen as below figure.
A
B
C
Ab
Ab
Ab
Ag
Ag
Ag
Pattern A: Both antigens react with antibody to form smooth line of perception and
antibody cannot distinguish between two antigens and that means the two antigens are
immunologically identity.
Pattern B: Antibody reacts only with one antigen.
Pattern C: It means both two antigens are not identity and then the antibody cannot
react with any antigen.
33
Material:

Saline (0.9 per cent w/vNaCl; 0.1 per cent w/v Na azide)toxic 1litre

Agar (1.5 per cent w/v in saline; 3.Human serum

cow serum &human serum
50 ml

Rabbit anticow serum
50 ml

Rabbit antihuman serum 50ml

Stain

Destain

Tannic acid (1 per cent w/v)

Incubators at 37*C

Gel punch pattern or pasture pipette (for cutting wells).
Method:
1. Heat the agar solution to 90°C until all the clumps of agar have dissolved. Cool to
55°C then pour into plastic Petri dishes to a depth of 6 mm) or pour 1.3 ml onto
microscopic slide cover with heated pipette.
2. Allow the plates to cool.
3. Cut out two wells with dis.3mm between them
4. Remove the gel plugs by gentle suction and
5. Place your own serum or the cow serum in the in the well and antiserum in the
other wells until they are just full.
6. Put the slid cover in the Petri dishes which has some drops of water.
7. Cover.
8. Incubate at 37°C in a moist chamber overnight
9. Dye for 15 min
10. Remove dye 3 times each 4 min.
Treat with 1 per cent w/v tannic acid to aid visualization
11. Examine the patterns obtained and interpret the results.
34
References:
Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.
Blood Groups, Blood Typing and Blood Transfusions". Nobelprize.org. 29 Dec 2012
http://www.nobelprize.org/educational/medicine/landsteiner/readmore.html
http://www.immunochemistry.com/what-history-immunoassays
http://www.ihcworld.com/_books/Dako_Handbook.pdf
http://train-srv.manipalu.com/wpress/?p=123867
Harold Varley, Practical Clinical Biochemistry. Edition, 4. Publisher, Heinemann
Medical, 1967
http://www.flowinjection.com/sichrom/sichrom.aspx
http://upload.wikimedia.org/wikipedia/commons/d/da/Vacuum-filtration-diagram.png
http://en.wikipedia.org/wiki/Ion_exchange
Chromatographic methods for protein purification (by M. Hedhammar, A.E.
Karlström, S. Hober, Royal Institute of Technology)
Protein purification (by J. Doudna, University of California)
http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E836FE09D8403
http://immunoassays.blogspot.com/
http://exploreable.wordpress.com/2011/05/25/elisa-enzyme-linked-immunosorbentassay/
http://www.labome.com/method/Protein-Purification-through-ColumnChromatography.html
http://www.analyticalventura.com/iex-hplc.shtml
http://water.me.vccs.edu/turbidometer.html
https://www.score95.com/blog/blog/immunology-nephelometry/
http://tneraj.blogspot.com/2008/10/precipitation-tests.html
Agglutination reactions. From Applegate, 2000.
http://cnx.org/content/m44823/latest/
http://biosiva.50webs.org/compft.htm
Direct immunofluorescence. In direct immunofluorescence the object is visualized
using a fluorescein-tagged antibody. From Hart and Shears, 1997.
http://www.immunoassay.org/radioimmunoassay-ria/
http://drugline.org/
Corley RB. (2005). A Guide to Methods in the Biomedical Sciences. Springer.
p. 11.ISBN 978-0-387-22844-0
http://classes.midlandstech.com/carterp/Courses/bio225/chap18/ss2.htm
http://bbs.pranovo.com/basic-theories-f11/antigen-antibody-interactions-principlesand-applications-t739-10.html
http://dc352.4shared.com/doc/BuvY7zIw/preview.html
http://immuneweb.xxmu.edu.cn/monoclonal/fda-fig13-2.html
http://quizlet.com/
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Ag_Ab.html
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Further readings:
 Animated blood types
 Introduction to techniques in immunology
 Salting out
 Enzyme purification by salt (Ammonium Sulfate) precipitation
 Analysis of protein
 Immunological tests
 Antibody-Ag
 Immunoassays tests
 Precipitin reaction (Molecular Biology)
 Precipitation reaction heavy chain
 Antigen Antibody Reactions in the Laboratory
 Human fibrinogen antigen
 Fibrinogen antigen assay by Radial Immunodiffusion (RID)
 Tests for fibrinogen
 Plasma fibrinogen determination
 Pregnancy test animation
 Animation Quiz 1 - ELISA Enzyme-Linked Immunosorbent Assay
 Enzyme-Linked Immunosorbent Assay (ELISA) animation
 Protein purification
 Blood bank antigens and antibodies
 Antibody
 Human Immunodeficiency Virus Rapid Test Device
 Immunology
 Ion exchange chromatography
 Ion exchange chromatography: Principals and Methods
 Principles of ion exchange chromatography
 http://www.youtube.com/watch?v=hmK7yYr2T54
 http://water.me.vccs.edu/turbidometer.html
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