Pract. No.

4-Latex Agglutination Test

Aim: To learn the technique of agglutination

Introduction:

Agglutination is a reaction of clumping together of antigen-bearing cells,

microorganisms or particles in the presence of specific antibodies (agglutinins) in a
suspension. Reaction time for agglutination to occur is shorter compared to other antigen-
antibody interactions. Latex agglutination makes use of latex particles which are built from
different organic materials to a desired diameter, and may be functionalized with chemical
groups to facilitate attachment of molecules. Latex agglutination tests have been in use since
1956 to detect a wide range of analytes in the clinical laboratory. The first description of a
test based on latex agglutination was the ‘Rheumatoid Factor Test’ proposed by Singer and
Plotz in 1956. It can be used for detection of both antigen and antibody.

Principle:

All methods of detecting or quantitating antigen or antibody take advantage of the fact
that they react to form a complex. At the optimum antigen-antibody concentration, this
complex precipitates out. However, if the antigen is particulate in nature, agglutination of
antigen-antibody complex is observed.

In the latex agglutination method, antigen is coated onto an inert matrix i.e., latex,
which behaves as particulate antigen. Hence, when antigen coated latex is mixed with an
antibody, agglutination occurs which can be visualized by the naked eye. However, if the
antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited; this
is because free antibodies are not available for agglutination.
Materials Required:
Equipment:Microcentrifuge
Reagent:Distilled water.
Other Requirements:Micropipette, Tips, 1.5 ml vials, Tooth picks.

 Reconstitute the antigen for coating with 1.25 ml of glycine-saline buffer. Mix well.
Store at 4°C and use within 3 months.
 KT53A: Reconstitute the test antiserum with 0.1 ml of glycine-saline buffer. Store at
4°C and use within 3 months.

Procedure:

Day1: Coating of Latex

1) Add 40 µl of glycine-saline buffer to 20 µl of latex beads taken in a 1.5 ml vial.

2) Add 60 µl of antigen to the latex.
3) Incubate at 37°C for 2 hours.
4) After 2 hours, spin down at 5000 rpm for 10 minutes and carefully aspirate the
supernatant.
5) Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10
minutes. Repeat the washing once more.
6) Add 90 µl of blocking buffer to the pellet, mix well and incubate at 4°C, overnight

Day 2: Agglutination Test

7) Dilute the test antiserum 50 times with glycine-saline bufferi.e., to 200 µl of glycine-
saline buffer add 4 µl of test antisera.

Note: Use this diluted antiserum on the same day.

8) Add 50 µl of antigen to 50 µl of diluted antiserum in a1.5 ml vial, mix well and

incubate at room temperature for 10 minutes. This will be used for testing the
agglutination inhibition reaction.
9) Pipette 10 µl of coated latex onto a glass plates, as shown below:
 10) Add 10 µl of diluted test antiserum to ‘a’.
11) Add 10 µl of antiserum mixed with antigen (from step 8) to ‘b’.
12) Add 10 µl of glycine-saline buffer to ‘c’.
13) Mix each of them with a separate tooth pick and gently swirl the plate by hand,
observing for any signs of agglutination. Agglutination is observed within 2 minutes
as clumping of latex particles leaving a clear liquid phase(curdling). Absence of
agglutination is seen as a homogenous suspension (milky).

Observation:

Note down your observations as follows:

Case Agglutination
a
b
c

Denote + ve: Presence of agglutination

- ve: Absence of agglutination.

Interpretation:

 Agglutination is observed in the first case (a) in which coated latex is mixed with
antiserum, indicating that the antiserum has antibodies against the antigen used for
coating.
 In the second case (b) no agglutination is observed because having pre-incubated
antibody with the antigen, antigen-binding sites on the antibodies are already
occupied by the antigen. Since free antibodies are not available for agglutination,
inhibition is observed. This also confirms that the agglutination seen in case (a) is
specific.
 In the third case (c) no agglutination is observed as antibody was not added and only
buffer was used.