The document discusses antigen-antibody techniques used for disease diagnosis. It defines antigens and antibodies, and describes various agglutination tests like bacterial agglutination and hemagglutination which detect antibodies through visible clumping. Precipitation tests detect insoluble antigen-antibody complexes. ELISA and immunoelectrophoresis are also summarized, with ELISA being a sensitive and specific immunoassay and immunoelectrophoresis separating antigens based on charge and mobility. Monoclonal versus polyclonal antibodies are compared.

1. ANTIGEN-ANTIBODY
DISEASEDIAGNOSIS
TECHNIQUES
PRESENTED BY-
JAYSHRI SHELKE
B.F.SC 3RD YEAR
ENROLL NO- F/15/050

2. What is antigen ?
Antigens are any substance that stimulates the immune system to
produce antibodies. Antigens can be bacteria, viruses, or fungi
that cause infection and disease.

3. What is antibody?
Antibodies, also called immunoglobulins, Y-shaped
molecules are proteins manufactured by the body that help
fight against foreign substances called antigens.

6. Principle of Agglutination
 The reaction between a particulate antigen and an antibody results in visible clumping
called agglutination, Antibodies that produce such reactions are known as agglutinins.
 The principle of Agglutination reactions are similar to precipitation reactions; they depend on the cross
linking of polyvalent antigens.
 Agglutination is the process that occurs if an antigen is mixed with its corresponding antibody called
isoagglutinin. This term is commonly used in blood grouping. This occurs in biology in three main
examples: The clumping of cells such as bacteria or red blood cells in the presence of an antibody or
complement.

7.  Types of Agglutination-
 Identification of an organism with known antibodies
 Identification of serum antibodies with known antigens.
 Bacterial agglutination test
 Measure the antibody produced by the host against bacterial agglutinins
 Best performance when used in sterile physiologic saline
 Uses:
 Disease diagnosis: bacterial agent is difficult to cultivate in vitro. Some examples of such diseases are:
 Tetanus: Clostridium tetani
 Yersiniosis: Yersinia pestis
 Leptospirosis: Leptospira spp.
 Brucellosis: Brucella spp
 Tularemia: Francisellatularensis

8. Slide Agglutination test
Use of Antisera (Ab)
to identify Salmonella and Shigella by causing agglutination of the organisms
Diagnostic purpose (Hospital laboratories): Antisera directed against the cell wall O antigens of
Salmonella and Shigella
Epidemiologic purpose (Public health laboratories): Antisera against flagellar H antigen and
capsular Vi antigen of salmonella
Particle agglutination test
Agglutination of an artificial carrier particle with specific antigen bound to its surface
Size of the carrier enhances the visibility of agglutination.
Examples include
Latex particles: Latex agglutination test
Treated Red blood cells i.e. Hemagglutination
Whole bacterial cells.
Reaction is dependent on
Amount and avidity of antigen bound to carrier
Time of the incubation with specimen
Environment of interaction (pH, Protein concentration etc)

9. Hemagglutination
Treated animal RBC is used as a carrier of antigen
Passive hemagglutination: Ag that are being bound by Ab are not the Ag
of RBC but are passively bound Ag.
Examples
Microhemagglutination test for Syphilis (MHA-TP)
Hemagglutinationtreponemal test for Syphilis (HATTS)
Passive hemagglutination tests for antibody to extracellular antigen
of Streptococci
Rubella indirect hemagglutination test
Hemagglutination Inhibition Test (HAI) for Avian Influenza
Quantitative Micro Hemagglutination Test (HA)
Latex agglutination test for Ag detection
Latex beads coated with specific antibody are agglutinated in the
presence of homologous antigen (bacteria).
Used to determine the presence of capsular antigen of
H. influenzae
N. meningitidis: rapid detection of meningococcal capsular
polysaccharides in CSF.

10. Latex agglutination test for Ab detection-
Latex particles coated with specific antigen
Commercially available for the accurate and sensitive detection of antibody to
Cytomegalovirus
Rubella virus
Varicella-zoster virus
Heterophile antibody of infectious mononucleosis

11. Coagglutination (COAG)
Specific antibody is bound to the
surface protein A of staphylococci
(Cowan type 1 strain of Staphylococcus
aureus). Soluble microbial antigen in
the specimen is mixed with the COAG
reagent, resulting in the agglutination
of the staphylococcal cells.
Antibody COAG Reagent + Antigen in
specimen = Staphylococcal cells
(Agglutinated)
Coagglutination
Test

12. precipitation test
Precipitation reactions are based on the interaction of
antibodies and antigens. They are based on two soluble
reactants that come together to make one insoluble
product, the precipitate. These reactions depend on the
formation of lattices (cross-links) when antigen and
antibody exist in optimal proportions.
Precipation test is use for
medical Definition of precipitin test. : a
serological test using a precipitin reaction to detect the
presence of a specific antigen; specifically : a test used in
criminology for determining the human or other source of
a bloodstain.

13. Precipitation:

14. Difference between Agglutination& Precipitation
Both reactions are highly specific because they depend on the specific antibody and
antigen pair. The main difference between these two reactions is the size of antigens.
For precipitation, antigens are soluble molecules, and for agglutination, antigens are
large, easily sedimented particles.

15. Immunoelectrophoresis: -Technique based on the principles of
electrophoresis of antigens and immunodiffusion of the electrophoresed
antigens with a specific antiserum to form precipitin bands. -It is used to
detect the presence of antibodies. - Used mainly to determine the blood
levels of three major immunoglobulins: immunoglobulin M (IgM),
immunoglobulin G (IgG), and immunoglobulin A (IgA)
During electrophoresis -, molecules placed in an electric field acquire a
charge and move towards appropriate electrode. Mobility of the molecule is
dependent on a number of factors: -Size of molecules to be separated. -
concentration of agarose gel. -Voltage applied. -The buffer used for
electrophoresis.

16. Applications:
-Immunoelectrophoresis is used in clinical laboratories to detect the
presence or absence of proteins in the serum.
This technique is useful in determining whether a patient produces
abnormally low amounts of one or more isotypes of Ig , characteristic of
certain immunodeficiency diseases.
It can also show whether a patient overproduces some serum protein, such
as albumin, immunoglobulin, or transferrin

17. Monoclonal Antibody Polyclonal Antibody
Consists of one antibody
class/subclass which is selective
for a single epitope on the antigen
antigen Contains a mixture of
antibodies (mainly IgGs), often
recognizing multiple epitopes on
the antigen
Because of their specificity, they
are less likely to cross-react with
other proteins, giving lower
background than polyclonal
antibodies
May contain non-specific
antibodies resulting in
background staining

18. What is ELISA?

19. What are the types of ELISA tests?
There are four types or kinds of ELISA tests:
1)Direct ELISA: attachment of an antigen to a polystyrene plate followed by an
enzyme-labeled antibody that can react with the antigen and a substrate that
can be measured
2)Indirect ELISA: attachment of an antigen to a polystyrene plate followed by an
unlabeled or primary antibody followed by an enzyme-labeled antibody that can
react with both the primary antibody and substrate
3)Sandwich ELISA: A capture antibody is attached to the polystyrene plate, then
antigen is added that specifically attaches or captures the antigen. A second
antibody, also specific for the antigen but not the same as the capture antibody
is added and "sandwiches" the antigen. This second antibody is then followed by
an enzyme-labeled antibody specific for the second antibody that can react with
a substrate that can be measured
4)Competitive ELISA: This test is like the sandwich ELISA but involves the
addition of competing antibodies or proteins when the second antibody is
added. This results in a decrease in the substrate signal that is generated. This
test is considered to give good, highly specific results.

20. What are the advantages of ELISA testing?
ELISA tests are generally good and accurate tests. They are considered highly
sensitive and specific (accurate) and compare favorably with other methods used for
the detection of substances in the body. The ELISA testing method is more
straightforward and easier to perform than older laboratory techniques, which often
required radioactive materials.