NAME: ARISHA SAIF

SEAT NO: H1831009

CLASS: 2ND YEAR, 4TH SEMESTER

COURSE:
INTRODUCTION TO IMMUNOLOGY (LAB)- 404
COURSE INCHARGE:
DR. MUSTAFA KAMAL & DR. MARIAM SIDDIQA
 CONTENTS
INTRODUCTION

PRECIPITATION TEST

AGGLUTINATION TEST

COMPLEMENT FIXATION TEST

IMMUNOELECTROPHORESIS TEST

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

TEST

IMMUNOBLOTTING ASSAY/ WESTERN BLOT TEST

IMMUNOFLUORESCENCE ASSAY TEST

FLOW CYTOMETRY TEST

NEUTRALIZATION TEST
IMMUNOLOGICAL
TECHNIQUES
 INTRODUCTION
Immunological techniques involve the study of immune system through both, the exploratory
methods and use of immunological reagents as experimental apparatuses.
Immunological techniques are the wide combinations of procedures and specific preliminary shows
detailed by immunologists for provoking, assessing, and depicting safe responses. They grant the
immunologists to adjust the safe system through cell, sub-nuclear and inherited control. These
methodologies are not bound to the field of immunology , anyway, are by and large applied by
fundamental specialists in various other characteristic orders and by clinicians in human and
veterinary prescription.
The most prevailed immunological techniques include the production and utilization of antibodies in
order to suspect certain proteins present in a biological sample. Antibodies are used to recognize
pathogens and also offers to detect the time of exposure of particular substances in a biological
sample.
Immunological techniques are based on the principle of antibody-antigen test. This test uses one the
two substances to detect the presence of another one. Antigen used to distinguish antibodies to a
microbe in patient’s sample whereas antibody is used to detect the presence of antigen of the
microbes in patient’s sample. These both molecules play the key role to produce accurate result.
Following are the Immunological techniques that are commonly used:
1. Precipitation Test
2. Agglutination Test
3. Complement Fixation Test
4. Immunoelectrophoresis Test
5. Enzyme Linked Immunosorbent Assay (Elisa) Test
6. Immunoblotting Assay/ Western Blot Test
7. Immunofluorescence Assay Test
8. Flow Cytometry Test
9. Neutralization Test

IMMUNOASSAY: It is a technique which is basically used for the detection and measurement of
specific protein as antigen and antibodies. A test that utilizes the binding of antibodies to antigens for
the detection and measurement of certain substances. Immunoassays is a biochemical test, basically
used for the detection of diseases. From the above techniques, few techniques come under
immunoassay technique i.e., Enzyme Linked Immunosorbent Assay (Elisa) Test,
Radioimmunoassay(RIA), Immunoblotting Assay/ Western Blot Test, Immunofluorescence Assay Test.
 1. PRECIPITATION TEST
Precipitation test estimates the presence of certain antigens which when comes in contact with
homologous antibodies, shows visible precipitation due to antigen- antibody complex in a solution.
There are four types of precipitation tests but are limited for their use due to low sensitivity, since
large amounts of antibodies are required.

TYPES OF PRECIPITATION TEST

1. SIMPLE PRECIPITATION TEST:

a. FLOCCULATION TEST:

Flocculation or slide precipitation test is basically performed to detect antibody and the interaction
between antigens and antibodies. They produce precipitate that can be seen with naked eye. This
test is done on glass slide. A drop of reagent (antigen or antibody) is set on a slide. At that point one
drop of serum test is added to it. Then slide is pivoted to blend the serum and reagent. If precipitate
occur, it is framed as positive test.

b. TUBE PRECIPITATION TEST:

In this test a narrow test tube is taken in which antibody solution is placed which is then layered
slowly with the solution containing antibodies, which results in the appearance of a ring at the
interface of the two liquids.

2. IMMUNO-DIFFUSION TEST (GEL DIFFUSION)

a. SINGLE DIFFUSION IN ONE DIMENSION (OUDIN ):

Antibody is incorporated in the agar to which antigen solution is added which then diffuses down
through the agar gel and precipitation line is formed. The number of precipitation bands appeared
gives the types of antigens.
b. DOUBLE DIFFUSION IN ONE DIMENSION (OAKLEY- FULTHROPE):
This method also known as Oakley–Fulthrope includes the joining of antibody in agar gel in a test
tube, above which a layer of plain agar is put. The antigen is layered on top of this plain agar. During
bring forth, the antigen and antibody push toward each other through the interceding layer of plain
agar. In this zone of plain agar, both antigen and antibody respond with one another to outline a
band of precipitation at their ideal concentration.
c. SINGLE DIFFUSION IN TWO DIMENSIONS (RADIAL IMMUNODIFFUSION):
Antibodies are mixed with the agar solution; this solution is then used to make a layer over the top of
a glass slide. Cuts are made through this layer over which the antigen solution is poured. The resulting
reaction is the formation ring shaped precipitation band due to the diffusion of antigen solution
through the antibody- agar layer. The diameter of the band gives the concentration of antigens in the
solution. This test is used to identify antibodies of influenza virus and to quantify IgG, IgM and IgA in
sera.
d. DOUBLE DIFFUSION IN TWO DIMENSIONS (OUCHTERLONY):
This test is usually done for the toxicity test of C. Diphtheria. in this test a petri dish is taken over
which a layer of agar gel is formed. This layer is then carved into wells in the center of which antibody
solution is added which is surrounded by antigen solution on the walls. As a result, different patterns
of are observed.
i. Precipitation line will be fused if the adjacent antigens are related.
ii. Precipitation line will be crossed if the adjacent antigens are not related.
iii. Spur formation will be observed if the adjacent antigens are partially related.
 2. AGGLUTINATION TEST
It is a blood test that is used to identify unknown antigens in the sample with the help of known
antibodies. Agglutination may or may not occur identifies the antigens and is used for tissue matching
blood grouping and diagnosis of certain infections.

TYPES OF AGGLUTINATION TEST

1. Active/ Direct Agglutination Test
2. Passive Agglutination Test
3. Hemagglutination Test

1. ACTIVE/ DIRECT AGGLUTINATION TEST:

When antigens are present naturally on a particle surface, it is known as active or direct
agglutination. In active agglutination, the antigens react directly with the antibodies that is produced
by a host body attacked by a pathogen.

Direct agglutination is of different types:

 Slide/ Tile Agglutination Test:
This test is performed on slide and its final result is indicated by clumps.
 Tube Agglutination Test:
This test is performed in tube and is used to determine antibody titre.
 Heterophile agglutination Test:
It is used to demonstrate heterophilic antibodies in serum of infections.
 Antiglobulin (Coombs) Test:
They are of two types direct and indirect. The direct test detects the RBCS that are attached to the
surface of antibodies whereas indirect test detects the antibodies that are floating in the blood.
2. PASSIVE AGGLUTINATION TEST:
This is a method in which either the antibody or the antigen is coated on the surface of an inert
carrier particle which on the exposure of corresponding antibody or antigen causes agglutination.
Most commonly used inert carriers are latex particles, carbon particles and bentonite etc. if the
antigen is adsorbed on the carrier particle it is the passive agglutination but if the antibody is attached
at the carrier surface it is the reverse passive agglutination.

 Latex Agglutination:
In this type of agglutination, the antigen
and antibody are adsorbed on the surface
of latex beads, which eventually increases
the number of antigen binding sites. If the
corresponding antigen or antibody is
present in the sample, resulting product
will be the formation of crosslinked
aggregates.

3. HEMAGGLUTINATION TEST:
In this test RBCs are used as carrier particle. Human, sheep and chick RBCs are commonly used in
this test. In Indirect hemagglutination test, to detect antibodies , RBC are coated with antigen in
the serum whereas in Reverse passive hemagglutination, RBCs are attached with antibodies for
the detection of microbial antigen. Following are the types of this test:
 Viral hemagglutination:
In Viral hemagglutination, viruses () have the ability to agglutinate RBCs without antigen-antibody
reaction. Inhibition of this hemagglutination by antibody against virus is known as
hemagglutination inhibition.
 Coagglutination test:
The test is done using Staphylococcus aureus strain Cowan I (i.e., it contains Protein A) that is
coated with anti-goat pox serum to detect presence of goat pox antigen of affected goat skin or
kid kidney cell culture. This is the rapid test to detect the presence of goat pox.
 3. COMPLEMENT FIXATION TEST
Complement fixation is a technique that evaluates the presence of an antibody in the blood serum of
a patient. In this method fungal antigens and positive controls are used to detect the antibody in the
patient’s blood sample.

PRINCIPLE OF COMPLEMENT FIXATION:

The antibodies present in the patient’s blood serum sample when reacts with the corresponding
antigen, it fixes to the component of the serum. These fixed components are then analyzed by using
the sheep red blood cells (SRBC) that is sensitized with anti- SRC (hemolysin). As a result, if the SRBC
would not be lysed, it serves as the indicator that the components are fixed.
Complement fixation generally involves two basic phenomena:
1. The relative concentration of antigen or antibody is determined by the degree of irreversible
fixation of the components to their corresponding antigen- antibody complexes.
2. The relative concentration of non- fixed (unbound) complements may be determined by the
breakdown (lysis) of the SRBC due to their sensitization through the hemolysin.
Since many of the fungal infections have similar symptoms and their antigens may show similar
antigenicity, patient’s blood sera should be exposed to each of the antigen. The higher complement
fixation rate refers to the presence of the same antigen that serves as the causative agent for the
infection.

4. IMMUNOELECTROPHORESIS TEST
This is basically a test that occurs when precipitation in agar occur under special electric field. This
process mainly comprises of two reactions i.e., immuno-diffusion and electrophoresis. In first step
antigen is separated into simpler form from the mixture through electrophoresis and then in the next
step it is tested through immuno-diffusion. Firstly, agar is layered on a glass slide, then a well is
formed on its surface and antigen solution is added on the well. After 1 hour electrophoresis is
performed. For the migration of antigen, a trough is cut in a rectangular shape and antibody solution
is added into the trough and it is left for 18-24 hours for diffusions. This results in the formation of
precipitation band on each separate compound. This technique is basically used to detect different
antigen that is present in human serum as well as abnormal and normal serum protein is detected
through this technique.

TYPES OF IMMUNOELECTROPHORESIS TEST

1. COUNTER IMMUNOELECTROPHORESIS:
It is a technique that is used to assess the binding of antibody with its antigen. This technique is same
as immuno-diffusion with a slight expansion of an applied electrical field across diffusion process. In
this rapid migration of antibody and antigen is seen towards is well, forming precipitation line,
indicating binding. This test is basically based on principle of movement of antibody and antigen in
opposite direction.

2. ROCKET IMMUNOELECTROPHORESIS:
It is developed by Laurell. Basically, it is a modification of radial immuno-diffusion, also known as
electro-immunoassay or electro-immunodiffusion. It is called rocket immunoelectrophoreisis
because of the shape of precipitation band as it has cone-like appearance. In this case, the migration
of antigen occurs in a layer of agarose gel that contain antibody in an electric field. This migration of
antigen gives the rocket shaped patterns of precipitation.

3. RADIOIMMUNOASSAY (RIA):
This technique was first discovered by Berson and Yallow. A radioimmunoassay is an immunoassay
that utilizes radiolabeled particles to form immune complexes. Substance concentrations and antigen
concentrations (e.g.: hormone level in blood) is made by using these technique as it is sensitive in
vitro assay technique. The main principle of this technique is competitive binding of antigen between
radio-active antigen fights with non-radio-active antigen. There are two methods of
radioimmunoassay:
i. Double-antibody Radioimmunoassay
 ii. Coated tube Radioimmunoassay
5. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) TEST
ELISA is a sensitive and simple technique that is used to identify antigen or antibody and is based on
an enzyme that works by acting on its substrate and showing color representing positive result.
Depending on the principle, ELISA is of different types. This technique is mostly prevailed to detect
HIV antibodies, Hepatitis B and mycobacterium antibodies in the sera and rotavirus & E.coli in feces.

TYPES OF ELISA TEST

1. DIRECT ELISA:
In this method the antigen sample is placed on a plate and the antibody used for the detection is
added over it which then binds to the target antigen. Substrate is then added which shows the
amount of analyte in the sample. This method is advantageous since its fast and simple but is less
specific as it only detects one antigen.
2. INDIRECT ELISA:
It is similar to the direct ELISA as the antigen gets immobilized on the plate but there is one more
step. Initially, the primary antibody is used that binds with the desired antigen, over it a conjugated
antibody is added for detection. At the end of the procedure, substrate produces a signal in order to
detect the quantity of bound antigen.

3. SANDWICH ELISA:
These are the most commonly used type of ELISA and are highly specific, as they involve the binding
of two antibodies on the epitope of a single antigen. Here the term ‘matched antibody pairs’ is used
since an antigen is sandwiched between two specific antibodies. The antibody is coated on a
microplate over which the sample is poured, and protein of interest gets immobilized over the plate.
A conjugated detection antibody binds to another site on the target protein. Substrate added then
gives a signal that quantifies the amount of antigen present in the sample.

4. COMPETITIVE ELISA:
When the protein of interest is too small that it gets
difficult to sandwich between two antibodies,
competitive ELISA is brought into consideration, as it
is commonly used for small particles. Here the captured antibody is covered on a microplate and
instead of a conjugated antibody, a conjugated detection antigen is used to complete the process of
binding of antigen to the antibody. The more the antibody present in the sample, lesser would be the
binding of conjugated antigen to the captured antibody. Similarly, the substrate is added at the end
that indicates the number of antigen present in the sample.
 6. IMMUNOBLOTTING ASSAY/ WESTERN BLOT TEST
Immunoblotting(western blotting) is a rapid technique that is used for the identification of
antimicrobial antibodies in the sample of patient’s blood by exposing them to the specific antigens
that has already been immobilized by blotting on the surface of a membrane. This technique involves
the phenomena of gel electrophoresis which can easily distinguish between different types of protein
on the basis of molecular weight. Each protein produces a band when the results are transferred on a
membrane which is then reacted with specific antibodies desired for the protein of interest. This
method involves three major tasks:
 Separation of protein based on sizes.
 Transfer of protein to a solid support (microplate).
 Exposure of target protein (antigen) to the primary and secondary antibodies for the purpose of
visualization.

TYPES OF IMMUNOBLOTTING TEST

1. NORTHERN BLOTTING:
This type of immunoblotting is used for the detection of certain RNA molecules amongst mixture of
RNA. The RNA analyzed in a tissue, or a cell is used for the detection of particular expression genes.
2. SOUTHERN BLOTTING:
This is a laboratory procedure that is used for the detection of particular DNA sequence that is
present in a blood or tissue sample. DNA is cut fragments through restriction enzymes and is then
separated through the use of gel electrophoresis which is then transferred to the surface of the
membrane that is out of the gel. Afterwards the membrane is brought in contact with the DNA probe,
which if binds together indicates that probe sequence is present in the sample.
 7. IMMUNOFLUORESCENCE ASSAY TEST
Immunofluorescence assay (IFA) is microscopic technique that involves the antigen- antibody reaction
in order to visualize viral proteins present on a cell. Firstly, the infected cells are layered on cover
glass and then reacted with paraformaldehyde for the detection of antigen. Specific antibody is then
brought in contact with cell surface in order to detect the viral antigen. Then a secondary antibody is
applied which is attached to a fluorescence dye by the help of which antigen can be visualized under
a fluorescence microscope. Through this technique, two or three antigens can be identified by using
relatively equal numbers of distinct antibodies.

TYPES OF IMMUNOFLUORESCENCE TEST

1. DIRECT IMMUNOFLUORESCENCE:
This laboratory method is also called ‘direct immune fluorescent’ or ‘primary immunofluorescence’
which is used for the detection of diseases that is related tom skin, kidney and other organs of the
body.
2. INDIRECT IMMUNOFLUORESCENCE:
This laboratory method is also called ‘secondary immunofluorescence’ which is used for the detection
of autoantibodies that are circulating in the blood sera of the patient. It is also used for the detection
of autoimmune blistering diseases.
 8. FLOW CYTOMETERY TEST
Flow cytometry is a technique that involves rapid analysis of single cells in a solution with multiple
parameters. This is a laboratory process that is used for the detection, identification and
quantification of particular cells. It also helps in the identification of specific compounds that are
present in the cells, as this information relies on physical traits and/ or indicators called antigens that
are present on the surface or within the cells.
This method is also used for the identification of different types of cells (including cancer cells) that
are present in the sample of blood and bone marrow. It distinguishes different kind of disease cells
that depends on either presence or absence of certain protein markers (antigens) on cell’s surface.
This cell is significant in diagnosing CLL. It utilizes a machine that searches for specific substances
(markers) on or cells for the identification of their cell type. This test can be utilized to check whether
the lymphocytes blood sample have CLL cells or not.
 9. NEUTRALIZATION TEST
Neutralization test is a method in which equal proportions of virus and blood sera are mixed together
and inoculated in the animal tissue. Unknown viruses are identified using known antibody or
measurement of virus in serum samples against a known infection. Basically, Neutralization of
infections by their particular antibodies are called virus neutralization tests. They are difficult to
perform but are specific and sensitive test.

TYPES OF NEUTRALIZATION TEST

 Viral Hemagglutination Inhibition Test: This test is used for the detection of viral
infection (i.e., measles,mumps and influenza). If antibody against specific virus is present in
patient’s serum and it have the property of agglutinating the RBCs, the virus will combine with
antibodies and will inhibit agglutination of RBCs.
 Toxin Neutralization Test: This test neutralizes microbial toxins of particular antibody
which is join in a way that the active part of toxin is blocked. This reaction can obstruct the
impact of many microbial toxins.