Latex Agglutination Test

Definition:

It is a laboratory method that is used to check for certain antigen or

antibodies in clinical laboratories for the detection of infectious disease
such as Rheumatoid Arthritis, Hepatitis B, Haemophilus Influenza,
Enisseria Meningitidism etc.

Discovery:

 In 1956 Sanger and Plotz first described the Rheumatoid Factor Test. It
is a test based on latex agglutination. In rheumatoid arthritis 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.
 Agglutination: reaction between a particulate antigen and antibody
results in visible clumping called agglutination.
 Agglutinins: Antibodies that produce such reaction are known as
agglutinins.

 Principle:

The principle of agglutination reaction is similar to precipitation

reactions: they depend upon on the cross linking of polyvalent antigens.

Hemagglutination: when the antigen is an erythrocyte, it is called as

Hemagglutination. Theoretically all antibodies can agglutinate particulate
antigen, but IgM due to its high specificity is a particularly good
agglutinin.
Agglutination tests employ latex particles, gelatin beads, colloidal
particles, or preserved Mammalian or Avian blood cells to facilitate
visualization of agglutination.

In the agglutination test, sample that has to be tested is sent to the

laboratory where it is mixed with latex beads coated with a specific antigen
or antibody.
Materials:
1. 1.5 ml vials
2. Microcentrifuge
3. Pipette
4. Glycine buffer saline
5. Latex beads
6. Laboratory refrigerator
7. Blocking buffer
8. Test anti-serum
9. Beaker
10. Glass slide
11. Tooth picks
12. Antigen for coating
13. Micro tips
Procedure:
1. Coating of latex:
1. In 1.5 ml vial take 20 µl of latex beads
2. Add 40µl of Glycine saline buffer (GSB) in the vial containing latex
beads.
3. Add 60µl of antigen to the latex
4. Incubate the vial at 370C for 2 hours
 5. Centrifuge the vial down at 5000rpm for 10 min. carefully aspirate the
supernatant.
6. Resuspend the pellet in 1ml of blocking buffer& and spin down at
5000rpm for 10 min.
7. Repeat the washing once more.
8. Add 90 µl of blocking buffer to the pellet. Mix it well.
9. Incubate at 400 c overnight.

2. Agglutination test:
1. To 200µl of glycine saline buffer, add 4µl of test anti-serum (50
times diluted).
2. Add 50µl of antigen to 50µl of anti-serum in the 1.5ml vial.
3. Mix well& incubate it at room temperature for 10 min.
4. Pipette 10µl of coated latex bead on a clean glass slide.
5. Add 10µl of diluted antiserum to slide A.
6. Add 10 µl of anti-serum mixed with Ag to slide B
7. Add 10µl of glycine saline buffer to slide C
8. Take a tooth pick and mix contents on each slide,
9. Discard the toothpick after using for one slide and use new one
toothpick for next slide.
10. Wait for 2 minutes and observe result.
Result:
Positive result: Clumping of latex beads,(Agglutination) indicate the
presence of suspected particle. Observe in B slide.
Negative result: Absence of white clumps: slide A&C. (No agglutination)
Slide Material Clumping Result
A Diluted Antiserum No clumping , Absence of Ag Negative
B Antiserum+ Ag Visible clumping. Presence of Ag Positive
C Glycine saline buffer No clumping, Absence of Ag Negative
Advantages:

 It helps in detecting certain Ag or Ab in a variety of bodily fluid such as

blood, saliva, urine, cerebrospinal fluid.
 Ability to obtain semi-quantitative result
 A low individual test cost
 Relatively short time to obtain result