Serologic reactions

in-vitro Ag-Ab reactions.

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.


Antibodies (also known as immunoglobulins Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

Ag
Ab
Ag
Ab
Ag
Ab
Ag
Ab
Ag



Features of Ag-Ab reactions.The reaction is highly specific.There is no denaturation of Ag or Ab during the reaction.The combination is firm but reversible


A\ Unlabelled tests Precipitation reactions (Ag is soluble) precipitation. Agglutination reactions (Ag is particles) clumping. Complement fixation reactions. Neutralization B\ Labelled tests 1-Immuno-fluorescence reactions. 2 - ELISA. 3-Radioimmuno electrophoresis


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 in the presence of electrolytes at the suitable temperature and pH.

Agar Gel diffusion method: a- Elek’s Toxigenicity Test: b- Ouchterlony method:

Elek’s Toxigenicity Test: Principle:
To determined the toxigenic strain of C. diphtheriae
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.
3. Incubate the plate at 35oC for 24 hrs.
2. Streak the test organism across the plate at right angle to the filter paper.



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

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.


2
1
C
3
Ag
2
1
C
3
Ag

Ouchterlony method:

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

Examples of agglutination reactions


Slide agglutination Tube agglutinationHaemagglutination testA:Direct coomb’s testB:Indirect coomb’s test


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.

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 Acceptor
Universal donor

Anti-A

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

Drop of blood

Anti-Rh (anti-D)
If agglutination occures Rh+ve If No agglutination Rh-ve

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).


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.

Immunofluorescence

Direct
Indirect


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)



The test is used to detect Ab in patients’ sera.Fluorescein labelled anti-human Ig is used .Known Ag is fixed on a slideAdd the patient's serum & allow to react for some timethe excess is washed, add Fluorescein labelled anti-human Ig examine under UV.

positive test for rabies

This technique is ; Very sensitive The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is added as a quantitative measure of enzyme activity.


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


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)

Antigens

+
Antibody labelled with enzyme
wash
+
substrate
Antibodies
+

Direct ELISA (Sandwich 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).WashAdd the enzyme-labelled antihuman Ig wash the excess add the substrate, then quantitatively measure for the degree of color change.

Antigens

+
Antibody
=
wash
+
Ig enzyme labelled
=
+
substrate
=

Indirect ELISA