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)
 Reaction
Agglutination
test
 Involve
particulate
antigens and
antibodies.
 Antigens may
be:
 On a cell or
attached to
latex particles.
Figure 18.4
 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
 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

•Direct Coombs Test

– Detects antibodies on erythrocytes

+ 

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Coombs
test
 Indirect Coombs Test
 Detects anti-erythrocyte antibodies in serum

Step 1
+ 
Patient’s Target
Serum RBCs

Step 2

+ 
Coombs Reagent
(Antiglobulin)
 Reactions

 Involve
soluble
antigens
with
antibodies

Figure 18.3
 tion
Precipitation
in gel:
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.
 tion
Ab in gel

Ag Ag Ag Ag
• Method
– Ab in gel
– Ag in a well
 Interpretation:
Diameter of ring is
Diameter2

proportional to the
concentration

Ag Concentration
 Immunoelectroph
oresis
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.
Complement fixation Test

Ag No Ag

Ag
Patient’s
serum
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 Test

Complement Fixation

Figure 18.9.1
Complement fixation Test
Complement Fixation

Figure 18.9.2
 Reactions
Eliminate the harmful effect of a virus or
exotoxin

Figure 18.8b
 Immunofloures
cence

 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.
 Radioimmuno
assay

 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
Thank You