1. Antigen-Antibody reactions

2. Let’s start The immune system
The immune system is a system of biological structures and processes within an organism that protects against disease.
To function properly, an immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism’s own healthy tissue.

3. Antigens
Antigens are any substances that are capable, under appropriate conditions, of inducing the formation of antibodies and reacting specifically with the antibodies so produced.
These antigenic molecules may have several antigenic determinants, called epitopes, and each epitope can bind with a specific antibody. Thus, a single antigen can bind to many different antibodies with different binding sites
Substances that can be recognized by the immunoglobulin receptor of B cells, or by the T- cell receptor when complexed with MHC, are called antigens.

4. Types of antigens:
RBCs
WBCs
Bacteria
Latex particles
And any other foreign bodies.

5. Antibodies
An antibody is a protein produced by the body’s immune cells “ activated B-lymphocytes” when it detects a foreign antigen.
Classes of antibodies:
IgG : The main antibody in blood(70%), made in 2ry immune response and has a long half life”up to 20 years”.
IgM : accounts for(10%) of Igs, has a key role in 1ry immune response.
IgA : (20%) of Igs present in serum and secretions.
IgE : (0.001%), involved in allergy and parasitic infections.
IgD : (1%) present on the surface of B cells, so that it plays a role in induction of Ab production

7. Nature of antigen antibody reactions
Lock and Key Concept - The combining site of an antibody is located in the Fab portion binds with antigen’s epitope by Non-covalent and Reversible Bonds.
Types Ag/Ab reactions:
Agglutination
Direct agglutination
Indirect agglutination
Precipitation
Non-covalent Bonds: hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds
Since Ag-Ab reactions occur via non-covalent bonds they are by their nature reversible.

8. Agglutination
Agglutination Reactions are interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reactions are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens.
When the antigen is erythrocyte it is called hemagglutination
All antibodies can theoretically agglutinate particulate antigens but IgM due to its high valence is particularly good agglutinin.

9. b) Quantitative agglutination test :
a) Qualitative agglutination test (Direct)
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen.
b) Quantitative agglutination test :
(Indirect)
Used to semi-quantitate the level of antibodies to particulate antigens. In this test one makes serial dilutions of a sample to be tested for antibody and then adds a fixed number of red blood cells or bacteria or other such particulate antigen and determines the maximum dilution that gives visible agglutination which called the titer, the results is shown as the reciprocal of the maximum dilution that forms visible agglutination.

10. Advantages of agglutination tests:
Low indvidual test cost.
Ability to obtain semi-quantitative results.
Short time to obtain result.
Don’t need expensive instrument.
Disadvantages of agglutination tests:
May give false positive or negative results
False +ve due to:
Technical errors as using plasma instead of serum, hemolysed or lipemic sample or dirty working slide.
Cross reaction
False –ve due to: - not giving enough time for agglutination, - antibodies not yet formed in the body or - prozone phenomena.

11. Differences B/w latex and rose-waller agglutination
Antigen is suspended on sensitized RBCs
Antigen is suspended on polystyrene reagent
More sensitive
Less sensitive
Sensitized RBCs is more sensitive than latex particle, since it can react with low concentration of antibody, so sometimes the latex maybe negative while the sensitized RBCs is positive.

12. Applications of agglutination tests
Determination of blood types or antibodies to blood group antigens.
To assess bacterial infections e.g: OX-19, widal test and brucella.., etc
For some autoimmune diseases e.g: RF.

13. precipitation
Antibody and soluble antigen interacting in aqueous solution form a lattice that eventually develops into a visible precipitate. Antibodies that aggregate soluble antigens are called precipitins
Formation of an Ag-Ab lattice depends on the valency of both the antibody and antigen:
The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments.
The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope,or have different epitopes that react with different antibodies present in polyclonal antisera.
The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble Ag/Ab complexes.

14. Applications of precipitation Rxns
1. Radial Immunodiffusion
(Mancini test)
2. Immunoelectrophoresis
3. Countercurrent electrophoresis
Radial Immunodiffusion (Mancini) - In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed

15. Factors affecting Ag/Ab rxns
Buffer
Concentration of antigen
Concentration of antibody
Temperature
Electolytes

16. Prozone phenomena
Prozone effect - On occasion one observes that when the concentration of antibody is high (i.e. lower dilutions) there is no agglutination and then as the sample is diluted agglutination occur.
The lack of agglutination at high concentrations of antibodies is called the prozone effect. That occur due to antibody excess resulting in very small complexes which do not clump to form visible agglutination.
An excess of antibody inhibits precipitation and agglutination reactions.

17. “Prozone effect”

18. Volumes used in dilutions mustn't be below 10ul or more than 1000ul .
Dilution is decreasing the concentration of a solution by a calculated factor using an approved diluent . As well, dilution is used to prepare samples, buffers, and controls.
In serology tests it is Used to detect the titer of a specific Ab.
When a strong positive reaction is encountered, dilution should be made to detect the titer.
Volumes used in dilutions mustn't be below 10ul or more than 1000ul .

19. Serial dilution : for example: (1:4) 1 express the volume of sample
4 express the total volume( sample + reagent)
This is done by mixing 100 ul of sample with 300 ul of reagent.
Serial dilution :
100ul
sample
100ul
From 1
100ul
From 2
sample 1 2 3
300ul
saline
300ul
saline
300ul
saline
Total Dillution
1:4
1:16
=1/4 * 1/4
1:64
=1/4 * 1/16
Dillution of tube 1
Dillution of tube 2

20. Another example on serial dillution :
100ul
sample
100ul
From 1
100ul
From 2 1 2 3
sample
900ul
saline
900ul
saline
900ul
saline
Total Dillution
1:10
1:100
= 1/10 * 1/10
1:1000
=1/10 * 1/100
Dillution of tube 1
Dillution of tube 2

21. It can be done only by serial dilution… 9:25 * 1:10 *10 9 : 250
9:250??
It can be done only by serial dilution…
9:25 * 1: *10
9 : 250
9:25 * 1:10
90ul
100ul
From 1 1 2
sample
160ul
900ul
Total Dillution
9:25
9:250
1:10