This document describes various methods for detecting antigen-antibody reactions, including primary reactions like precipitation and agglutination that are visible to the naked eye, as well as secondary reactions that require labels like enzymes, radioisotopes, or fluorescent substances and can be seen using techniques like immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blotting, and flow cytometry. It provides details on the principles, procedures, applications, and examples of each method.

1. By
Eman A. Abd
Alrahman
Antigen Antibody
Reactions

2. Methods of Detection of Antigen –
Antibody Reactions
I-Primary reactions
AgAb complexes visible
by naked eye or by
microscope.
1. Precipitation
2. Agglutination
3. Complement fixation
4. Neutralization
II-Secondary reactions
AgAb complexes visible
by using labels e.g. enzyme,
radioisotopes ,
fluorescentsubstance.
1. IF
2. ELISA
3. RIA
4. mmunoblotting
5. Flow cytometry

3. I-Primary reactions
4. Neutralization
• In vivo Toxin anti toxin
neutralization
• In vetro Toxin anti
toxin neutralization
• Viral neutralizationb)Viral
neutralization
a)Toxin anti toxin
neutralization

4. A)Toxin anti toxin neutralization
In vivo Toxin anti toxin neutralization
e.g.
• Chick test: test susceptibility to diphtheria.
• Dick test: test susceptibility to scarlet fever.
• Schuiltz –Charlton test: diagnosis of
scarlet fever.

5. In vitro Toxin anti toxin neutralization e.g:
Anti streptolysin (O) test
•Used for: diagnosis of post streptococcal diseases.
•Procedure : serial dilution of patient serum is mixed with
streptolysin (O) toxin and incubate, then RBCs
suspension added and incubated, In presence of
neutralizing anti toxin in sera no hemolysis will occur.
1/50 C -ve1/16001/8001/4001/100 C
+ve

6. •Titer : the reciprocal of the highest dilution of
sera showing no hemolysis(>200 Todd unit is
diagnostic)

7. B) Virus neutralization :
Depends on the neutralization of cytopathic
effect of unknown virus on tissue culture by
suspected antibodies in patient serum.
Used for :diagnosis of viral infection by detection
of antibody in the patient serum using known
virus.

8. II- Secondary reactions
A)Immunoflourscence (IF)
Antigen antibody reaction in Ab is labelled with
flourscenct dye seen by flourscenct microscope
1)Direct IF:(used for detection of unknown Ag)
2)Indirect IF:(used for detection of unknown Ab)

9. 1)Direct IF
•Used to detect presence of Ag in
tissue fixed on a microscopic slide
• Specific fluorescein labelled Ab
is used .
•if the Ag is present Ab will bind to
it and appear as green stain on the
sample when examined under U/V
rays.
•e.g diagnosis of rabies in brain of
rabid animals

10. 2)Indirect IF
•Used to detect presence of Abs in
patient serum using known Ag
fixed on a microscopic slide.
•Followed by washing then
fluorescein labelled anti-
-globulins added and examined
for the presence of fluorescent
spot in +ve case .
•e.g. diagnosis of syphilis.

11. Enzyme-linked Immunosorbant Assay
(ELISA)
Principle
•Use of enzyme-labelled immunoglobulin to
detect antigens or antibodies.
• Signals are developed by the action of
hydrolyzing enzyme on chromogenic substrate
optical density measured by micro-plate
reader.
Examples
Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)

12. Types of ELISA used in the detection of
antigens and antibodies
1)Non-competitive
Indirect ELISA (Ab)
Sandwich ELISA(Ag)

13. 2)Competitive
In this test, antibody is first incubated in solution with a
sample containing antigen. The antigen-antibody mixture is
then added to the microtitre well which is coated with
antigen.
The more the antigen present in the sample, the less free
antibody will be available to bind to the antigen-coated well.

14. •After the well is washed, enzyme conjugated
secondary antibody specific for isotype of the
primary antibody is added to determine the
amount of primary antibody bound to the well.
The higher the concentration of antigen in the
sample, the lower the absorbance.

15. ELISA
Micro-plate reader
96-well micro-plate
Positive result

16. Application of ELISA
Presence of antigen or the presence of antibody in a
sample can be evaluated.
Determination of serum antibody concentrations in a
viral infections.
Used in food industry when detecting potential food
allergens.
Applied in disease outbreaks- tracking the spread of
disease e.g. HIV, bird flu, common, colds, cholera,
STD etc.

18. Procedure:
o A known quantity of an antigen is made radioactive, by
labeling it with gamma-radioactive isotopes of iodine.
o This radiolabeled antigen is then mixed with a known
amount of antibody for that antigen, and as a result, the
two specifically bind to one another.
3)Radioimmunoassay (RIA)
Is a very sensitive in vitro assay technique used to
measure concentrations of antigens (for example,
hormone levels in blood).

19. oThen, a sample of serum from a patient containing an
unknown quantity of that same antigen is added. This
causes the unlabeled antigen from the serum to compete
with the radiolabeled antigen for antibody binding sites.
oAs the concentration of "cold" antigen is increased, more
of it binds to the antibody, displacing the radiolabeled
variant, and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled antigen

20. The bound antigens are then separated from the
unbound ones, and the radioactivity of the bound antigen
remaining in the supernatant is measured using a gamma
counter.

24. WESTERN BLOTTING
 Used for detection of unknown is Ab rather than Ag
 Steps:
Separation of complex antigenic material (eg., viral
proteins) by electrophoresis
◦ Separated components transferred from gel to
nitrocellulose paper by “blotting”
◦ Unknown (or control) sera (which may have Abs)
incubated with paper strips; Ag - Ab complexes ppt. at site
of transfer
◦ Strips washed; staining reveals complexes

25. WESTERN BLOTTING

26. Applications
The confirmatory HIV test employs a western blot to
detect anti-HIV antibody in a human serum sample.
Proteins from known HIV-infected cells are separated and
blotted on a membrane as above. Then, the serum to be
tested is applied in the primary antibody incubation step;
free antibody is washed away, and a secondary anti-human
antibody linked to an enzyme signal is added. The stained
bands then indicate the proteins to which the patient's
serum contains antibody.

27. A western blot is also used as the definitive test for
bovine spongiform encephalopathy (BSE, commonly
referred to as 'mad cow disease').Some forms of Lyme
disease testing employ western blotting.
A western blot can also be used as a confirmatory test for
Hepatitis B infection and HSV-2 (Herpes Type 2)
infection.

28. Flow cytometry
Flow cytometry is a technology that is used to analyze the
physical and chemical characteristics of particles in a fluid
as it passes through at least one laser. Cell components are
fluorescently labelled and then excited by the laser to emit
light at varying wavelengths.

29. It has broad application in medicine (especially in
transplantation, hematology, tumor immunology and
chemotherapy, prenatal diagnosis, genetics and sperm
sorting for sex preselection). Also, it is extensively used in
research for the detection of DNA damage, and apoptosis.
Applications
Flow cytometry is used to perform several procedures
including:
 Cell counting
Cell sorting
Detection of biomarkers
Protein engineering