IMMUNOLOGY LAB MANUAL

Third year students

BIOL 349

Dr. Salam Nasreddine

Dr Salam Nasreddine
The Blood Group Antigens

Dr Salam Nasreddine
Plasma: Serum + coagulant factor.

Serum : plasma –coagulant factor

Dr. Salam Nasreddine

Plasma: non cellular, intercellular substance of blood + coagulate factor of the blood,
containing the antibodies.

Serum: plasma free of the coagulate factor, containing the antibodies.

Dr. Salam Nasreddine

Blood group (Erythrocyte):

Ø Blood group antigens are surface markers on the red blood cell
membrane.

ØThe two most important ones are:

* ABO system
Incompatible transfused red cells react with the patient's
own anti A or anti B antibodies and cause an acute severe
clinical reaction
* Rhesus, RH system
The immunogenicity of its antigens leads frequently
Hemolytic Disease of the Newborn (HDN)

Dr Salam Nasreddine
1.1. Blood group (Erythrocyte):

The most important blood-group system in

human-blood transfusion

1- ABO system 2- Rhesus, RH system

Characteristics of blood groups

• A, B, AB , O •The commonly-used terms Rh factor, Rh

positive (+ ) and Rh negative (-) refer to the D
• ABO blood groups are defined by the antigen only
presence of antigens on erythrocytes • RH system groups are defined by the
surface and regular antibody in plasma presence of antigens on erythrocytes surface

Dr Salam Nasreddine
ABO System

Anti-A and Anti-B are natural antibodies (regular antibodies) : have been defined
as antibodies that are produced without any previous infection, vaccination
Are IgM type (do not cross the placenta).
Antibodies anti A, B and A+B are active at 37°C.
Dr Salam Nasreddine
 3. Determination of blood groups

3.1. Blood samples

- Labeling is required (name, date of birth, address)

3.2. Determination of ABO group

- It must necessarily involve the simultaneous search of globular antigens

and serum antibodies

Reaction of Beth Vincent Reaction of SIMONIN

(search of globular antigens) (search of serum antibodies)
- The patient's red blood cells incubated - Blood groups (A, B, AB, O)
with antihuman globulin (anti-A, anti-B, incubated with the patient’s serum
anti A+B)

Dr Salam Nasreddine
Ø The production of these antibodies do not exist at birth
Ø These natural antibodies appear in children 6 months
and peak at 5 to 10 years

à So reaction of SIMONIN before 6 months can cause false negative results

Dr Salam Nasreddine
Reaction of Beth Vincent Reaction of SIMONIN

Dr Salam Nasreddine
The difference between coagulation, agglutination and precipitation

???
9.3. Difficulties in determining the ABO blood types:

SIMONIN false negative:

Absence of anti-A, Anti-B and anti-AB agglutinin:

- Newborn (antibodies are not produced)
- Hypogammaglobulinemia is a low level of immunoglobulins (antibodies) in blood
that is caused by a lack of B-lymphocytes
- Presence of hemolysis, which masks the agglutination
- Inbition of Anti-A by a soluble substance

-SIMONIN false positive:

- Infected blood clot or from some types of bacterial

- Presence of fibrinogen (soluble plasma glycoprotein) in the blood

Dr Salam Nasreddine
 Red blood cell compatibility :
Blood transfusions – who can receive
blood from whom?

Ø Blood group O are called "universal donors" .

Ø Blood group AB are called "universal receivers.

Dr Salam Nasreddine
 8. Plasma transfusions

- Plasma extracted from type AB blood can be

transfused to individuals of any blood group "universal
donors"
- Individuals of blood group O can receive plasma
from any blood group; and type O plasma can be used
only by type O recipients "universal receivers”

Dr Salam Nasreddine
 Rh antigen
• Rh antigens are transmembrane proteins exposed at the surface of red blood cells.
• RBCs that are "Rh positive" express the antigen designated D (RH1).
• Some of us have it, some of us don't.
• If it is present, the blood is RhD positive, if not it's RhD negative.
• Unlike the ABO system, Anti-Rh antibodies of the system are not normally
present in the plasma, but can be produced if an individual with
Rh - is exposed to Rh +

Ø Rh antibodies are post-immune antibodies (irregular antibodies)

- There are formed by exposing to foreign erythrocytes, by transfusion or

pregnancy à RH antibodies are called irregular antibodies

- D antigen has intense immunogenicity

- Most of them are IgG (IgG do not cause spontaneous
agglutination à are called incomplete antibodies)
- IgG cross the placenta

- IgM is more effective than IgG in agglutinating red blood cells

Dr Salam Nasreddine
1.2- ABO distribution by country

World Distribution

Global
Blood Group
distribution
O+ 38 %
A+ 34 %
B+ 9%
O- 7%
A- 6%
AB + 3%
B- 2%
AB - 1%

Dr Salam Nasreddine
Blood transfusions

• A person with Rh+ blood can receive blood from a person with
Rh- blood without any problems.

• A person with Rh- blood can not receive blood from a person
with Rh+ blood

Dr Salam Nasreddine
RH incompatibility:

Hemolytic Disease of the Newborn (HDN)

HDN is the most common problem with Rh incompatability.

Dr Salam Nasreddine
 Development of HDN of the
Newborn
• A small quantity of fetal blood leaks across the placenta
into the maternal blood stream.

• If the mother is Rh- and the baby is Rh+, the mother’s

immune system begins to produce anti-Rh antibodies.

• The mother’s antibodies cross the placenta during the

subsequent pregnancy into the fetal blood.

• If the second fetus is Rh+, the antigen-antibody reaction

causes hemolysis of fetal RBCs and it results in HDN

Dr Salam Nasreddine
Symptoms of hemolytic disease of the newborn:
Ø Each baby may experience symptoms differently

Ø During pregnancy symptoms may include:

- At birth and during the newborn period, symptoms include a mild anemia
and jaundice, both of which may resolve without treatment

- The newborn may have an enlarged liver and spleen

- Neurological damage

- Newborn death

Dr Salam Nasreddine
 Treatment of HDN
• If a woman has Rh- and gives birth to a child, or if she
has a miscarriage or abortion, she is given an injection of
anti-Rh antibodies called anti-Rh gamma globulin or
RhoGAM to prevent HDN

• Intravenous injection of RhoGAM

• The Rhogam should be given within 48 to 72 hours

The mode of action

• The antibodies bind to the fetal Rh antigens and

inactivates them if they crossed the placenta during birth,
and the mother’s immune system does not respond by
producing antibodies Dr Salam Nasreddine
Important :

Ø Unlike the Rh system, ABO incompatibility between mother

and fetus is not dangerous

à Anti-Rh-antibodies are mainly IgG which is capable of

crossing the placenta and entering the fetal circulation. The
natural antibodies to A and B blood group substances, however,
are mostly of the IgM class and therefore do not cross the placenta

Dr Salam Nasreddine
A- Direct Combos Test (DAT): To detect the incomplte antibodies
bound to RBC surface

Ø The Direct Coombs Test is used in the hemolytic disease of the Newborn
(HDN), in transfusion reaction, during anemia.

Dr Salam Nasreddine
ABO BLOOD GROUP

Dr Salam Nasreddine
Slide test for determination of ABO group of RBCs.

Dr Salam Nasreddine
•If the Rh test is negative, add a second drop of anti-IgG and then
read again.

Dr Salam Nasreddine
Tube test for determination of ABO group of RBCs

1 - Wash the blood two or three times (1/4) by physiological salt solution (9 g NaCl in 1 L
of water D): 1ml + 3 ml of blood
2 - Centrifuge 3000 rpm / 5 min
3 - Remove the supernatant (with the aid of a Pasteur pipette) and collect the red blood
cells
4 - Preparation of either 5 to 10% red cell suspension (50μl of blood + 950 μl of
physiological salt solution)
- 5 to 10 % red cell suspension is critical to any agglutination test.

5 - Shake gently to re-suspend completely (by the pipette)

5 - Add 50 μl of the diluted blood in 8 test tubes
6 – Add to: the first 2 tubes: 50 μl of Anti-A
the tube 3 and 4: 50 μl of Anti-B
the tube 5 and 6: 50 μl of Anti-D
7 - Mix gently
8 - Observe whether agglutination occurs with the naked eye (1 minute, 3 minutes)

Dr Salam Nasreddine
 50 μl

50 μl of blood
+ 950 μl
physiological
salt solution 50 μl 50 μl 50 μl
Anti-A Anti-B Anti-D

Anti-D
Anti-D
Dr Salam Nasreddine
Certain factors may cause false results:

1 - Contamination of blood samples, reagents or materials.

2 - Blood samples too old, which may give weaker reactions than fresh.
3 - Suspensions very diluted or very concentrated.
4 – Inadequate incubation times or temperatures.
5 – Incorrect centrifugation:
6 - Shake vigorously can cause incorrect agglutination.

Dr Salam Nasreddine
Isolation of peripheral blood mononuclear cells (PBMC)
Introduction

The mixture of lymphocytes includes T cells, B cell and natural killer (NK) cells.
-Lymphocytes and monocytes: together are referred to as peripheral blood mononuclear cells
(PBMC).
- Blood also contains neutrophils, basophils and eosinophils which are referred to as
polymorphonuclear cells (or granulocytes).

Mast cells
 Isolation of peripheral blood mononuclear cells (PBMC)

Principle
- Peripheral blood is a primary source of lymphoid cells for the investigation of the
human immune system.

- Its use is facilitated by Ficoll-Hypaque density gradient centrifugation, a simple and

rapid method of isolating peripheral blood mononuclear cells (PBMC) that takes
advantage of the density differences between mononuclear cells and other elements found
in the blood sample.
Ficoll:
- The Ficoll Isopaque is a liquid with density (d = 1.077), containing of a mixture of
polysaccharide polymer (polysucrose), and sodium metrizoate.
- Ficoll is used to separate blood to its components (erythrocytes, leukocytes etc.)
- Ficoll-Paque is normally placed at the bottom of a conical tube, and blood is then slowly
layered above Ficoll-Paque.
- After being centrifuged, the following layers will be visible in the conical tube, from top to
bottom:
- plasma and other constituents
- a layer of mono-nuclear cells called buffy coat (PBMC/MNC),
- Ficoll-Paque
- Erythrocytes & granulocytes which should be present in pellet form.
-Mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer because they
have a lower density: in contrast, red blood cells and granulocytes have a higher density
than Ficoll-Hypaque and collect at the bottom of the layer.

- Ethylene diamine tetra-acetate (EDTA) and heparin are commonly used in

conjunction with Ficoll-Paque to prevent clotting.
 Size and density of blood cells
Sedimentation
rate
Size (µm) Density +
RBC 7 (6,5-7,5) 1,098 (1,089-1,105)
Eosinophils 12 (12-15) 1,091 (1,087-1,096)
Neutrophils 12 (12-15) 1,088 (1,082-1,097)
Basophils 9,5 (9-10) 1,078 (1,074-1,082)
Monocytes 15 (15-20) 1,071 (1,065-1,075)
Lymphocytes 9 (8-10) 1,063 (1,057-1,067)
Platelets 2-4 1,040 -

Ø Monocytes and lymphocytes are agranulocytes..

Ø Monocytes are the largest leukocytes with a proportion 2 -10%
Ø Lymphocytes are the smaller leukocytes with a proportion 28 -33%
Procedure
1.Obtain blood into heparinized vacutainer tubes (green top).
2. Blood Dilution 2X: in a 15ml Falcon tube, mix 1 ml PBS and 1 ml of blood

3. Mix by inverting the tube 3-4 times gently.

4. Underlay this carefully with 1 mL of FICOLL using a pipette Pasteur. (Another method
consists of putting the FICOLL at the bottom of the tube and gently overlaying with the
blood-PBS mix).
5. Spin at 1700rpm for 15-30 minutes at 18 et 20 0 C or 3000 for 25 minutes at room
temperature , brake off (since you have a gradient i.e. cells are in layers).

6. Resultant layers are approximately from top to bottom:

Plasma – platelets -- PBMC – Ficoll – red blood cells (with granulocytes).

Heparinized vacutainer tubes

 BIOL 349
TP-2

• Immunoprecepitation (Ouchterlony )
• The principle of serial dilutions
• Equivalence zone.

Dr Salam Nasreddine
Dr Salam Nasreddine
A- Immunoprecipitation:

Ø Immunoprecipitation (IP) is the technique of precipitating a protein antigen

of solution using an antibody that specifically binds to that particular protein.

Ø Immunoprecipitation (l’immunodiffusion) is used:

- to study the specificity of a Ac against different Ags (or vise versa)

Dr Salam Nasreddine
Equivalence zone a variable ratio of antigen and antibody which results
in precipitation in which there is no unbound antibody or antigen.
Ouchterlony Immunodiffusion (double diffusion)

- Double diffusion
- Qualitative test
- It is based upon the simultaneous application of Ag
and Ab in separate but adjacent wells of an agar plate.
-There are three basic patterns of precipitation as
shown in the figure below.

Dr Salam Nasreddine
 Ouchterlony - A reaction of identity

•A reaction of identity occurs

between an Ab and Ag containing
identical antigenic determinants,
and produces smoothly fused
precipitin band. The two antigens
are immunologically identical.
- If the Ag A (patient) is the same as the Ag A
(control), the reaction with the Ab will be the
same and the result is a solid, continuous,
smooth line of identity between the Ag wells
and the Ab well.
à this type of reaction is termed a band of
identity.

Dr Salam Nasreddine
 Ouchterlony – Non-Identity

•A reaction of non-identity occurs when the

antiserum contains antibodies to both antigens
but the two antigens do not share a common
determinant. The two-precipitin lines are
formed independently with different antibody
molecules and cross without interaction. The
two antigens are immunologically unrelated as
far as that antiserum is concerned.

- If Ag A (patient) is different from Ag B

(control), and both react with the Abs to A & B,
the precipitin lines cross and a double spur is
formed à this is a line of nonidentity.

Dr Salam Nasreddine
 Ouchterlony – Partial Identity
•A reaction of partial identity occurs when two antigens have at least
one common determinant, but where the antiserum contains
antibodies to a determinant in one antigen that is absent from the
other.

- If Ag A (patient) and Ag A1 (control) share a

common element but are not exactly the same
(Abs to A), a single spur is formed. à This is
the line of partial identity.
Dr Salam Nasreddine
 Ouchterlony-Interpret
• Determine which interpretation fits for samples 1, 2 and 3.

Dr Salam Nasreddine
 Experimental procedure

1. Coating petri dishes with agarose

•Weight 1.5 g of agarose
•Add 100ml of PBS (0.1M PH 7.2) or distilled water in a beaker.

- The agarose is dissolved by heating (100°C) while stirring à then cooled to 55°C.
- The warm 1.5% agarose solution is then pipetted on the petri dishes of 5cm diameter,
adding 5ml in each dish.
- The dish is air dried completely.

Dr Salam Nasreddine
2. Preparing the plates

Punch holes of 0.5cm in the agarose dish (6 holes surrounding a central hole)

Label on the plate cover the name of the

sample and mark a line on both the dish
and cover.

Dr Salam Nasreddine
3. Deposit solution

- The antibody (anti human IgG: 1mg/ml) is placed in the centre well and the antigens
(human IgG) are placed in the surrounding 6 wells.

Ø Prepare 5 solutions containing different concentrations of human IgG (Ag)

- [IgG]i = 20 mg/ml.
- Dilution with PBS

Add 20 μl diluted sample

to designated sample wells (peripheral well).

Ag
Ag Ag
Ac
Ø Add 20μl anti-human IgG in the central well (0.5 ou 1 Ag Ag
mg/ml) Dr Salam Nasreddine PBS
Place the slide in a humid chamber and incubate overnight at room temperature.
A humid chamber is prepared by covering the bottom of a petri plate with moist paper
towels with PBS or water.
- To prevent excessive drying of the agarose

Staining of Gel:
The dried slides are stained to help distinguish
precipitin lines, to allow the slide to be stored
indefinitely and to make photography easier.
The slide is stained with the protein dye,
Coomassie blue.

Typical Petri plate showing double diffusion

Dr Salam Nasreddine
 Dilution

C1V1=C2V2 à Dilution=C2/C1=V1/V2

Dilution = C final / C initial (dilution * C initial = C obtained)

Or
Dilution = Original volume taken/Final volume achieves (Dilution * total
volume= the volume taken from solution)

Dilution F= Df=F= 1/diluton

V buffer= v total-v taken

Dilution factor=F=
Cinitialx Dilution = C final Cinitial / final
Ex: dilution ½
Dr Salam Nasreddine à Df= 2 =1/dilution
 BIOL 349

- Experience: Dot blot

- Interpretation of the result ( detecting

the equivalence zone and the title).

Dr Salam Nasreddine
 Lap Biol 394
TP-6

- Western Blot
- Dot Blot

Dr Salam Nasreddine
TP- DOT BLOT

Dr Salam Nasreddine
DOT BLOT
Semi-Quantitative Measurement of Proteins by Dot Blotting

Principle:

†A technique for detecting, analyzing, and identifying proteins/RNA/DNA:

• Similar to the western blot technique but differing in that protein samples are not separated
electrophoretically but are spotted through circular templates directly onto nitrocellulose
membrane.

• Concentration of proteins can be estimated semi-quantitatively (qualitative) by using

specific antibody against it.

Dr Salam Nasreddine
Procédure

Procedure:
1. Have nitrocellulose membrane ready (10 cm x 0.5 cm). Draw 8 grids (-1.5 cm apart)
by pencil to indicate the regions you are going to blot (see below).

1 2 3 4 5 6 7 8

2. Prepare in the PBS buffer (0.1 M, pH: 7.2) a solution of the human IgG:
Make 7 serial dilutions (1/2) from the original tube with 1 mg/ml IgG, such that the final
volume will be 50 µL.

Dr Salam Nasreddine
 0,015 0,008 mg/ml

Dot 1 2 3 4 5 6 7 8

3. Spot 2 µL of samples onto the nitrocellulose membrane at the center of the grid.

N.B:
Place the drops: from the less concentrated solution to the more concentrated.
Dr Salam Nasreddine
4. Dry the strips by putting them in the oven for 5 minutes at 37° C.

5. Use a (pincer) to place the strips in the plate.

6. Wash: to remove the unbound IgG (Tween-20).

- Wash the membranes 3 times with washing buffer (PBS-Tween 20).

8. Add 1.5 mL/well (by adding 750 µL, two times): of the anti-IgG (labeled with HRP:
peroxidase) solution diluted at 1/500 in PBS-Gelatin.

9. Incubate during 30 minutes at room temperature: to allow IgG and anti-IgG recognition.

10. Remove the solution from the wells.

11. Wash 4 times : to remove the unbound anti-IgG

- 3 times: with washing buffer (PBS-Tween 20).
- The last wash will be done with PBS buffer without Tween 20.

Dr Salam Nasreddine
12. Revelation (Colorimetric Detection):
- Add 750µL (2 times) / well of the revelation solution (chromogenic substrate: 4-
chloro-1-naphtol) + H2O2.
- Incubate for 5 to 10 minutes in dark.
- Pour out the solution.

13. Stop the reaction: with distilled water

14. Dry the strips in the oven for 5 minutes / 37°C

15. Read the results

Dr Salam Nasreddine
Colorimetric HRP systems
- 4CN (4-chloro-1-naphtol) are commonly used chromogenic substrates for HRP.
In the presence of H2O2, HRP catalyzes the oxidation of the substrate into a product
that is visible on a blot.

4-chloro-1-naphtol

4-chloro-1-naphtone

Dr Salam Nasreddine
 TP-4

• TP : ELISA (different types of ELISAs)

Dr Salam Nasreddine
Dr Salam Nasreddine
The E.L.I.S.A. test is an immunoassay.

Dr Salam Nasreddine
ELISA
Enzyme-linked immunosorbent assay (ELISA) depends on an enzyme.
An enzyme conjugated with an antibody or with an antigen reacts with a colorless substrate to
generate a colored reaction product.

Dr Salam Nasreddine
Ø There are many different types of ELISAs:

- Heterogeneous (solid phase):

- Direct ELISA
- Indirect ELISA (the most common, classic ELISA)
- Sandwich ELISA (also called direct)
- Double Sandwich ELISA (also called indirect)

Dr Salam Nasreddine
 Heterogeneous ELISA
Direct ELISA
Enzyme conjugated antibody Enzyme conjugated antigen

Indirect ELISA

Dr Salam Nasreddine Research antibody into plasma

Heterogeneous ELISA
Sandwich ELISA

Dr Salam Nasreddine
Preparation of immunosorbent (solid phase):
- There are many carriers capable of binding antigen or antibodies.
- This attachment can be made either by simple passive
absorption or by non-covalent bonding.
- The polystyrene plates are most commonly used for this type of non-
covalent binding.
- For good attachment of proteins on polystyrene, you have to play with
four factors:
- The incubation time,
- The concentration of the protein
- The carbonate-bicarbonate buffer (0.1M, pH 9.6).
- Temperature
The polystyrene plates

Dr Salam Nasreddine
Direct ELISA
- An antigen coated to a multiwell plate is detected by an antibody that has been directly
conjugated to an enzyme.
-This can also be reversed, with an antibody coated to the plate and a labeled antigen used
for detection, but the second option is less common.

- This type of ELISA has two main advantages:

It is faster, since fewer steps are required
It is less prone to error, since there are fewer steps and reagents

Dr Salam Nasreddine
Indirect ELISA
- Antigen coated to a polystyrene multiwell plate is detected in two stages or layers.
-First an unlabeled primary antibody, which is specific for the antigen, is applied.
- Next, an enzyme-labeled secondary antibody is bound to the first antibody.
- The secondary antibody is usually an anti-species antibody and is often polyclonal.
- This method has several advantages:
- Increased sensitivity, since more than one labeled antibody is bound per primary
antibody
- Flexibility, since different primary detection antibodies can be used with a single
labeled secondary antibody
- Cost savings, since fewer labeled antibodies are required

Dr Salam Nasreddine
Sandwich ELISA
- Sandwich ELISAs typically require the use of matched antibody pairs, where each
antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule.
- The first antibody, termed the capture antibody, is coated to the polystyrene plate.
- Next, the sample solution is added to the well.
- A second antibody layer, the detection antibody, follows this step in order to measure the
concentration of the sample.
- If the detection antibody is conjugated to an enzyme, then the assay is called
a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection
antibody will be needed resulting in an indirect sandwich ELISA.

Dr Salam Nasreddine
 Sandwich ELISA

This type of assay has several advantages:

- High specificity, since two antibodies are used the antigen/analyte is specifically
captured and detected
- Flexibility and sensitivity, since both direct and indirect detection methods can be used

Dr Salam Nasreddine
ELISA Results
The ELISA assay yields three different types of data output:
1) Qualitative:
ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is
present in a sample, as compared to a blank well containing no antigen or an unrelated control
antigen.

2) Semi-quantitative:
ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity
of signal will vary directly with antigen concentration.

3) Quantitative:
ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known,
purified antigen) in order to precisely calculate the concentrations of antigen in various samples.

Dr Salam Nasreddine
.

- Alternatively, a standard curve based on known concentrations of antibody or antigen is

prepared, from which the unknown concentration of a sample can be determined.`

Standard curve

Dr Salam Nasreddine
Color changes from blue to yellow
Application

- Determination of proteins at low concentration.

- ELISA performed to aid in the diagnosis or detection of disease (by
detection of Ag or Ab).

Sensitivity

- ELISAs are one of the most sensitive immunoassays available.

- The typical detection range for an 0.01 ng to 0.1 ng.

Dr Salam Nasreddine
 TP-5

ELISA

Dr Salam Nasreddine

Dr Salam Nasreddine
Steps:
1- Antigen (human Ig or serum) is passively attached to a plastic solid phase by period
of incubation.
- Ig is the 20mg/ml concentration
- Group: initial concentration = 100 µg / ml
- Group: initial concentration = 200 µg / ml
-7 serial dilution of serum-1/5 (in a solution of carbonate and bicarbonate) and one
control tube (without antigen).

- Add 100 µl/well of each dilution.

2. Incubate for 30 min at 37 oC. 150 µl

Each student
receives two
strips of ELISA
plate.
100 µg /ml Ig

600 µl of carbonate and bicarbonate

Total volume =750 µl
Dr Salam Nasreddine
 Etud: 1 2 3 4 5 6

[ Ac ] Dilution
d’AC
100 microg/ml
-

20 1/5

4 1/25

0.8 1/125

0.16 1/625
0.032 1/3125
0.0064 1/15625

- Ss Ac

Dr Salam Nasreddine
3. Wash the membranes 3 times with washing buffer (PBS-Tween 20)
- Wash: to remove the unbound IgG (Tween-20 is a detergent that helps to remove
the unbound proteins).

4. Add 100 µl/well of an anti-IgG (Ab) that is linked covalently to an enzyme peroxidase, in
order to detect Ag.
- Anti-IgG (labeled with HRP: peroxidase) solution diluted at 1/1000 in PBS-
Gelatin.

5. Incubate during 30 minutes at 37° C : to allow IgG and anti-IgG recognition

- Gelatin (bloking reagents) to prevent the

nonspecific binding of the antibodies (prevent
Dr Salam Nasreddine
false positive results).
6. Remove the solution from the wells.

7. Wash 4 times : to remove the unbound anti-IgG

- 3 times: with washing buffer (PBS-Tween 20).
- The last wash will be done with PBS buffer without Tween 20.
- Because tween can stop the enzymatic reaction

8. Revelation (Colorimetric Detection):

Addition 100 µl/well of a substrate (OPD: ortho-phenylenediamine dihydrochloride
with H2O2) whereby enzyme activity produces a color change.

9. Incubate for 5 to 10 minutes at room Temperature in dark.

10. Stop the reaction with sulfuric acid H2SO4: 50µl/ well.

11. Read the optical density of the color at 490 nm by spectrophotometry.

To determine the catalytic activity of the enzyme (enzyme activity).

A color change indicates that the sample Colorimetric Detection

contains antibodies that react against the
original antigen.

Dr Salam Nasreddine
11. Plot absorbance vs antiserum dilution using the mean for each set.
12. Estimate the inflection point (calculate the concentration of Abs from standard
curve).
13. Interpolate the titer by drawing a line down to the x-axis

For example, the graph shows titer at 1/100

Titre

Concentration of serum= DO
at the inflection point (detect
concentration from standard Concentration
curve ) * reverse dilution.

Dr Salam Nasreddine
Ø Out DO> 3

Ø Negative control <0.09

Dr Salam Nasreddine
- ELISA data is typically graphed with optical density vs log
concentration to produce a sigmoidal curve as shown in figure below.
- Known concentrations of antigen are used to produce a standard curve and then this data is
used to measure the concentration of unknown samples by comparison to the linear portion of
the standard curve. This can be done directly on the graph or with curve fitting software which
is typically found on ELISA plate readers.

Sensitivity

- ELISAs are one of the most sensitive immunoassays available.

Dr Salam Nasreddine
- The typical detection range for an 0.01 ng to 0.1 ng.
 Standard curve

Dr Salam Nasreddine
.

- Alternatively, a standard curve based on known concentrations of antibody or antigen is

prepared, from which the unknown concentration of a sample can be determined.`

Standard curve

Dr Salam Nasreddine
Color changes from blue to yellow
Inflection point: A point where the concavity
changes (from up to down or down to up).

Dr Salam Nasreddine