1. Antigens, Antibodies and Their Interactions
SEROLOGIC
REACTIONS
Antigens, Antibodies and Their Interactions

2. in-vitro Ag-Ab reactions.
Serologic reactions:

3. Antigens:
Any foreign substances which when introduced into an animal, can stimulate a specific immune response, in the form of production of antibodies and specific reactive T-lymphycytes.
Antigens have the ability to combine specifically with the antibodies produced or sensitized T-cells induced.

4. Antibodies:
Glycoproteins that bind specifically to the antigen that induced their formation.

5. Ab Ag Ag Ab Ag Ag Ag Ab Ab

6. Diagnostic applications of serologic reactions :
1- Diagnosis of infectious diseases:
known antigen preparations are used to detect circulating antibodies in patient's serum as evidence of a current or previous infection with that agent OR
known antibodies are used to detect antigens associated with an infectious agent directly in body fluids.
2- Identification of unknown cultures:
known antibodies are used to detect their homologous antigens in cultures.

7. Methods for Detecting
an Ag-Ab Reaction

8. Precipitation reactions (Ag is soluble) precipitation.
Agglutination reactions (Ag is particles) clumping.
Complement fixation reactions.
Labelling methods:
a-Immuno-fluorescence reactions.
b- ELISA.

9. Precipitation
Reactions

10. Precipitation Reactions
This is an Ag-Ab reaction in which the Ag is soluble (eg: Protein ; Bacterial toxin).
When antigens and antibody mixed in the proper proportion, they form large macromolecular complexes called precipitates
One of the easiest of serologic tests

11. Precipitation Reactions

12. Example of Precipitation Reactions
Agar Gel diffusion method:
1- Double diffusion:
a- Elek’s Toxigenicity Test:
b- Ouchterlony method:
2- Single radial immunodiffusion.

13. Example of Precipitation Reactions
Elek’s Toxigenicity Test:
To determined the toxigenic strain of C. diphtheriae
Principle:
Toxin production by C. diphtheriae can be demonstrated by a precipitation reaction between exotoxin and diphtheria antitoxin.
Procedure:
1. Place a strip of filter paper saturated with diphtheria antitoxin on a serum agar plate.
2. Streak the test organism across the plate at right angle to the filter paper.
3. Incubate the plate at 35oC for 24 hrs.

14. Example of Precipitation Reactions
Results:
Positive test: formation of four radiating lines resulting from the precipitation reaction between exotoxin and diphtheria antitoxin.

15. Example of Precipitation Reactions
Ouchterlony method:
Wells are punched in the agar. The antigen in one well and the antibody is placed in another.
Both will diffuse in the agar, and precipitation bands are formed where they meet at optimal proportions.

16. Example of Precipitation Reactions
Ouchterlony method: 2 1 C 3 Ag 2 1
Incubate for
1h at 37°c Ag C 3

18. Example of Precipitation Reactions
Single radial immunodiffusion:
The antibody is mixed with the agar before pouring it in the plate, while the antigen is placed in a well punched in the agar.
the Ag diffuses in all directions, and where its concentration is optimal in relation to the antibody, a precipitation ring will form around the well. The diameter of the well depends on the Ag concentration.
e.g., quantitation of various Ig classes in human serum samples.

19. Agglutination
Reactions

20. Agglutination Reactions:
When Ag is in form of particles, it will become clump if react with specific Ab.

21. Agglutination Reactions:
Example of Slide agglutination:
Blood grouping (ABO grouping)
There are 4 blood groups
depending on the presence
or absence of either or both
two types of antigens A and B
on the surface of RBCs.

22. Blood grouping Blood group A B AB O RBC surface Ag A B A & B None
Serum Ab
Anti-B
Anti-A
None
Anti-A & Anti-B
Universal donor
Universal Acceptor

24. Agglutination in 2 only gp B Agglutination in 1 and 2 gp AB
Anti-A
Anti-B
Drop of blood 1 2
Agglutination in 1 only gp A
Agglutination in 2 only gp B
Agglutination in 1 and gp AB
No Agglutination in 1 or gp O

25. Rhesus blood group Rh or D is clinically and medically important.
According to presence or absence of Rh antigen on the RBCs surface, the individuals classify to Rh+ve (if present) 0r Rh-ve ( if absent).
Anti-Rh (anti-D)
If agglutination occures Rh+ve
If No agglutination Rh-ve
Drop of blood

29. Complement Fixation
Reactions

30. Complement Fixation Reactions
The complement is a group o heat-labile proteins normally found in blood and tissue fluids (except urine & CSF).
CF is an Ag-Ab reaction that occurs in the presence of the complement.
The Ag unites with its specific Ab and the resulting complex fixes (consumes) the complement

31. Complement Fixation Reactions
Two systems are used in CFT:
1- Test system:
The serum sample (heated to 56°)
Measured amount of Ag.
Complement (Guinea pig serum).
if the serum contains the specific Ab→ Ag-Ab complexes→ will fix all the complement.
Ag + Ab + C fixation
Ag + C No fixation (free complement)
2- Indicator system e.g; sensitized sheep RBCs.
(Sheep RBCs coated with their specific Abs).

32. Result:
+ve reaction No Lysis
-ve reaction Haemolysis

34. Labeling
methods

35. Immuno-fluorescence
These are Ag-Ab reactions in which Ab is labelled with fluorescein.
Fluorescein is a dye which emits greenish fluorescence under UV light.
There are two ways for this test;
Direct immunofluorescence,
Indirect immunofluorescence.

36. Direct immunofluorescence
In this test a fluorescein-labelled Ab is added to detect the presence of Ag in tissue section fixed on a microscopic slide.
A drop of the labelled Ab is placed on the section and left to react for some min.
the excess unattached Ab is washed
Examine under UV rays.
If Ag is present fluorescence
If Not No fluorescence
Disadvantage: expensive method (for each Ag we need specific labelled Ab)

37. Indirect immunofluorescence
The test is used to detect Ab in patients’ sera.
Fluorescein labelled anti-human Ig is used .
Known Ag is fixed on a slide
Add the patient's serum & allow to react for some time
the excess is washed,
add Fluorescein labelled anti-human Ig (attach to the Fc portion of the human Ig if present).
examine under UV.

38.                                                                 
                                       
positive test for rabies
 negative test for rabies

39. Enzyme-Linked Immuno Sorbant Assay
ELISA
Enzyme-Linked Immuno Sorbant Assay
This technique is ;
Very sensitive
Does not require specialized equipment
Avoid the hazards of radioactivity.
The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is added as a quantitative measure of enzyme activity.

40. ELISA Direct ELISA (double Ab technique): used for detection of Ags.
Known specific Ab is immobilized by adsorption onto a plastic surface.
Clinical sample is added (if Ag present it will bind to the immobilized Ab)
enzyme-labelled specific Ab is addad
(attach to the fixed Ag if present)
wash the excess
add the substrate

41. If Ab specific to Ag change the color
If Not specific No color change
Dark yellow highly +ve
Yellow moderate +ve
Color less ve
Disadvantages: expensive method (for each Ag we need specific Ab labelled)

42. wash +
Antigens
Antibody labelled
with enzyme +
substrate

43. ELISA
Indirect ELISA:
In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera.
Known Ag is fixed by adsorption onto a plastic surface.
The serum sample is added ( if specific Ab is present, it will bind the fixed Ag).
Wash
Add the enzyme-labelled antihuman Ig
wash the excess
add the substrate, then quantitatively measure for the degree of color change.

44. wash + =
Antigens
Antibody = + = +
substrate
Ig enzyme labelled

46. Thank You