Ag, Ab reaction

Antigen Antibody Reaction

Dr. Mejbah Uddin Ahmed

Antigen Antibody Reaction

Serum: Fluid that remains after blood

has clotted and cells have been removed.

Antiserum: Serum containing

antibodies to a specific antigen(s).

Antigen Antibody Reaction

Immunological test: Detection of

antibodies or antigens in any body fluids

including serum or tissues.

Serological test: Detection of

antibodies or antigens in serum.

Antigen Antibody Reaction

Titer: The term titer refers to the highest

dilution of serum at which agglutination still

takes place and beyond which no

agglutination occurs.

Serology: Study of serum in the

context of antigen or antibody.

Antigen Antibody Reaction

Uses of serologic test:A. Diagnosis of infectious diseases:

When organism can not be cultured,e.g, Syphilis.

When culture is dangerous: rickettsial disease. When culture technique is not readily available:

HIV.

Antigen Antibody Reaction

When culture require too long time:

Mycobacteria.

B. Diagnosis of Autoimmune

disease: Ab against normal body

components.

C. Determination of blood

grouping and HLA typing.

Antigen Antibody Reaction Ag-Ab tests are broadly classified into following types:

1. Agglutination test

2. Precipitation test

3. Complement fixation test (CFT)

4. Immunoassay using labeled reagents

5. Immunofluorescence (IF)

     6. Enzyme linked assay

     7. Radio immunoassay (RIA)

Antigen Antibody Reaction

Involve particulate antigens and antibodies.

Antigens may be:

On a cell or attached to latex particles.

Figure 18.4

Agglutination test

Antigen Antibody Reaction

Different types of agglutination tests:

Direct: When particulate Ag directly reacts with the Ab.

A. Slide agglutination e.g. Blood grouping, Serotyping of bacteria

B. Tube agglutination e.g. Widal test, Weil-Felix test.

Agglutination Reactions

Indirect or passive: Agglutination

of an antigen, which is not particulate by

itself but is coated in carrier particles to

make it particulate: RBC, Latex, Gelatin

and Protein-A of Staph. Aureus.

Agglutination Reactions

Accordingly the tests are

named as follows :A.Hemagglutination e.g. Pregnancy test, TPHA,

Anti-HBbsAg test,

B.Latex agglutination e.g. HBsAg test, ASO titer.

C.Particle agglutination e.g. Anti-HIVAb.

D.Co-agglutination e.g. Detection of antigen

from CSF in case of bacterial meningitis

Coombs test: Also known as anti-

immunoglobulin test because it employs

antibodies against immunoglobulin.

A.Direct: To detect Rh antibody already bound

to the surface Rh antigen of fetal RBC.

B.Indirect: To detect circulating Rh Ab in the

serum of Rh-negative mother bearing the Rh-

positive baby.

Coombs test

Coombs test

•Direct Coombs Test– Detects antibodies on erythrocytes

+

Patient’s RBCs Coombs Reagent(Antiglobulin)

Coombs test

Indirect Coombs Test Detects anti-erythrocyte antibodies in

serum

Patient’s Serum

TargetRBCs

+ Step 1

+

Coombs Reagent(Antiglobulin)

Step 2

Precipitation Reactions

Involve soluble antigens with antibodies

Figure 18.3

Precipitation

A. Single diffusion.

B. Double diffusion.

Precipitation in electrophoresis :

A. Immune electrophoresis: used for the diagnosis of paraproteinemia, immune deficiency etc.

B.Counter current Immunoelectrophoresis: used for the detection of cryptococcus antigen in CSF, carcinoembryonic antigen etc.

Precipitation in gel:

Precipitation

Interpretation: Diameter of ring is proportional to the concentration

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

Immunoelectrophoresis

Method

Ags are separated by electrophoresis

Ab is placed in trough cut in the agar

Ag

-+

Ag

Ab

Ag

Ab

Countercurrent electrophoresis

Method Ag and Ab migrate toward each

other by electrophoresis Used only when Ag and Ab have

opposite charges

Ag Ab

- +

Complement fixation Test

Steps of CFT: Ag & Ab( one is known) are mixed. Measured amount of complement is

added. If Ag & Ab match , complements will be

used. Sensitized RBC( RBC + anti RBC antibody)

is added.

Ag

Patient’sserum

Ag

Complement fixation Test

Ag No Ag

Complement fixation Test

Interpretation:

Presence of hemolysis indicates that

antigen and antibody did not form a complex,

i.e, test is negative.

Absence of hemolysis indicates Ag-Ab

complex complex was formed and utilized

most or all of the complement i.e. test is

positive.

Complement Fixation

Figure 18.9.1

Complement fixation Test

Complement Fixation

Figure 18.9.2

Complement fixation Test

Neutralization Reactions

Figure 18.8b

Eliminate the harmful effect of a virus or exotoxin

Immunoflourescence

Immunoflourescence: Is highly sensitive

but requires a special and costly instrument

not affordable by small laboratories.

Enzyme-Linked Immunosorbent Assay

ELISA: Is very sensitive,

technically demanding and time

consuming but without radiation

hazards. Once it was very popular

and still used for diagnosis of HIV,

HBV and HCV etc.

Radioimmunoassay

RIA: Is highly sensitive, but technically

demanding, with possible radiation

hazards, costly and time consuming. It

is used for detection of very small

amounts of antigens like hormones,

drugs etc.

Commonly Used Immunological Test

Diagnosis of bacterial diseases: ASO titer-Post streptococcal disease. Widal test- Enteric fever. VDRL- Syphilis. TPHA-Syphilis. Weil-Feix test.

Commonly Used Immunological Test

Diagnosis of bacterial diseases: HBsAg Ati-HBsAb Anti-HBcAb HBeAg Anti-HBeAb Anti-HCVAb

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