*Study of agglutination:-

Two bacteriologists, Herbert Edward Durham (-1945) and Max von Gruber (1853–1927), discovered specific agglutination in 1896

*Principle:-
The reaction  between a particulate antigen and an antibody results in visible clumping called agglutination.  
Since then, tests to detect microbial and viral infections, autoimmune diseases, hormones, drugs and serum proteins have been developed and marketed by many companies worldwide. Letex agglutination tests have been applied in clinical laboratories for the detection and diagnosing many infections such as Hepatitis B, H.influenzae, N. meningitidis, etc..
In rheumatoid arthritis (RA), IgG antibodies produced by lymphocytes in the synovial joint react with the IgM antibodies (RF, rheumatoid factor) to generate immune complexes that activate the complement and cause the tissue destruction. The RA is of diagnostic significance.

*Types of agglutination

1)Slide agglutination: Serotyping

2)Tube agglutination: e.g. Widal test.

3) Indirect (passive agglutination): where soluble antigens are coated on vehicle particle e.g. latex particle, RBCs.

*Protocol  :-
Coating of Latex  (For detection of antibodies)

To 20 μl of latex beads taken in a 1.5 ml vial add 40 μl of glycine-saline buffer.
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Add 60 μl of antigen to the latex and incubate at 37oC for 2 hours.
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Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant.
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Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes.
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Repeat the washing once more.
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Add 90 μl of blocking buffer to the pellet, mix well.
Incubate at 4oC, overnight

*Agglutination Test-:

To 200 μl of glycine-saline buffer taken in a vial, add 4 μl of test antisera. (50 times diluted).
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Add 50 μl of antigen to 50 μl of diluted antiserum in a 1.5 ml vial, mix well and incubate at room temperature for 10 minutes.
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Pipette 10 μl of coated latex onto a glass slides.
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Add 10 μl of diluted test antiserum to slide A.
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Add 10 μl of antiserum mixed with antigen (from step 1) to B.
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Add 10 μl of glycine-saline buffer to C.
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Take a tooth pick and mix the content in each slide.  (take a new one for the next slide).   
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After mixing, wait for 2 minutes to observe the result.
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