1. Lecture 2
Agglutination tests
Prepared by
DHuda Muharram

2. Agglutination reaction
When a particulate antigen is mixed with its
antibody in presence of electrolytes at
suitable temperature and pH, then the
particles are clumped or agglutinated .

3. Agglutination test
 Qualitative agglutination test - to assay
for the presence of an antigen or an
antibody.
 Quantitative agglutination test - to
quantitative the level of antibodies to
particulate antigens.

4. Uses
1. Identification of bacteria, e.g. serotyping
of salmonella and shigella with known
antisera.
2. Serological diagnosis of infection e.g.
widal test for typhoid
3. Haemagglutination test.

5. Agglutination tests can be performed
1. On slides or tiles
2. In tubes
3. In microtitration plate

6. The type of agglutination
 Active agglutination:- These are tests in which
there is a direct agglutination of Ag on the surface
of large insoluble particles ( as bacteria ) with it's
corresponding Ab.
 Passive agglutination :- These are tests in which
the specific Ab or known Ag is attached to inert
particles or cells (carriers ).

7. The substances and cells used as
carriers
1. Latex particles
2. Carbon particles
3. Stabilized staphylococcal cells
Specific Ab
protein A

8. Examples of latex agglutination tests
ASO latex slide agglutination test
Rheumatoid factors (RF) latex slide agglutination test
C- reactive protein (CRP) latex slide agglutination test
Pregnancy latex slide agglutination tests
Hepatitis surface antigen (HBsAg)

9. ASO latex slide agglutination test

10. Intended use
It is a rapid latex agglutination test for the
qualitative and semi-quantitative
determination of anti-streptolysin-O
antibodies (ASO) in serum.

11. Causative agent
 β-haemolytic Streptococci group A (Strept.
pyogenes )
 Streptolysin-O is one of the exotoxins liberated
from the bacteria that stimulates production
of ASO antibodies.

12. Diseases
 Sore throat
 Skin infection
 Scarlet fever
 Otitis media
 Complications
 Rheumatic fever (after sore throat)
 Acute glomerulonephritis (after skin infection)

13. SO
Principle
Latex particles coated with streptolysin O are
agglutinated when mixed with samples containing
ASO.

14. Reagents and material
1. ASO latex reagent
2. ASO Positive control
3. ASO Negative control
4. Stirrers & slide

15. Sample
 Fresh serum. Stable 8 days at 2-8ºC or 3 months
at –20ºC.
 Do not use highly hemolized or lipemic samples.

16. Procedure
 Qualitative method
 Semi-quantitative method

17. Qualitative
method
50 μL of the sample
+ One drop of latex
One drop of positive
control +
One drop of latex
One drop of negative
control +
One drop of latex
At room temperature
Mix than Place the slide on a mechanical rotator at 80-100 r.p.m. for
2 minutes

18. Result
Positive result :- ASO  200 IU/mL
Negative result :- ASO  200 IU/mL

19. Semi-quantitative method
1/8
1/4
1/2
Dilutions
50 μL
Sample
50 μL
50 μL
50 μL
Saline
50 μL
50 μL
200 X 8
200 X 4
200 X 2
ASO conc.
50 μL
The titer = the highest dilution showing a positive result

20. The approximate ASO concentration in the
patient sample
200 x ASO Titer = IU/mL
Calculation

21. End of lecture