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[PRACTICAL 6] MTEB 2404

INDIRECT AGGLUTINATION ASSAY

PRINCIPLE

Indirect agglutination test involves the use of inert agglutinable


particles (e.g. red cells or latex particles) as passive carriers for small
soluble antigens or antibodies. In this case, the soluble antigens /
antibodies are adsorbed to, or covalently linked to, the surface of the inert
particles. Once the bound antigen / antibody are exposed to its specific
antibody (usually IgM) or antigen, antigen-antibody complexes will be
formed.

In the case of the particles with absorbed / bound antigens, due to


the presence of multiple Fab arms or the IgM, a single IgM may complex
with bound antigens from 2 adjacent particles. Meanwhile, in the case of
the particles with absorbed / bound antibody, the soluble antigens (in
sample) that have multiple antigenic determinants will bind to antibodies
from 2 adjacent particles. In both cases, an antibody- antigen-particle
network is formed through multiple repeats of the aforementioned
reactions. As a result, agglutination and clumping of the particles
occurred.

Indirect agglutination assay that uses erythrocytes as carrier of


antigens is termed "Passive Haemagglutination" while assays using
antibody-coated erythrocytes are termed "Reverse Passive
Haemagglutination".

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[PRACTICAL 6] MTEB 2404

ANTISTREPTOLYSIN O (ASO) LATEX SLIDE TEST

PRINCIPLE

ASO test method is based on an immunological reaction between


streptococcal exoenzymes bound to biologically inert latex particles and
streptococcal antibodies in the test sample. The reagent has been
adjusted in the way that presence of an ASO titer of 200 lU/mL or higher
in the serum gives a visible agglutination of the latex particles without
previous sample dilution.

REAGENTS

1.ASO Latex Reagent: A suspension of polystyrene particles coated with


streptococcal exoenzymes. MIX WELL BEFORE USING.
2.ASO Positive Control: A stabilized human serum containing at least 200
lU/mL of ASO reactive with the test reagent. Ready for use; do not dilute.
3.ASO Negative Control: A stabilized human serum containing less than
200 lU/mL of ASO non-reactive with the test reagent. Ready for use; do
not dilute.
4.Glycine-Saline Buffer (20x) pH = 8.2 ± 0.1. A diluent containing 0.1 M
glycine and 0.15 M NaCI. Dilute buffer according to instructions on the
label.

* All reagents contain 0.1% (w/v) sodium azide as a preservative. Store all reagents at 2
- 8°C. DO NOT FREEZE.

1. ASO Latex Reagent 2. Pipette/Stir Sticks.

3. ASO Positive Control 4. Timer

5. ASO Negative Control 6. Test Tubes, Rack

7. Glycine - Saline Buffer 8. Serological Pipettes

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[PRACTICAL 6] MTEB 2404

9. Reaction Slide.

PROCEDURE

Qualitative Test:

1. Bring reagents and specimens to room temperature before use.


2. Place one drop (50 ul) of ASO Positive Control on field
o #1 of the reaction slide. Place one drop (50 ul) of the ASO
Negative Control on field
o #2 of the reaction slide.
3. Use pipette/stir stick to deliver 1 drop (50ul) of undiluted test serum
sample to field #3. Continue likewise with additional unknowns.
Retain pipette/stir sticks for mixing step.
4. Gently resuspend the ASO Latex Reagent and add one drop to each
test field.
5. Mix well with the flat end of the pipette. Gently rock the slide for two
(2) minutes and read immediately under direct light.

RESULTS

Qualitative Test

1. Negative Reactions : Formed of milky suspension by comparing the


ASO negative control

2. Positive Reaction: Formed of small white particle agglutinate. A


positive reaction indicates the concentration of ASO is equal or
greater than 200 IU/ml which then compared to ASO negative
control.

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[PRACTICAL 6] MTEB 2404

Control Control Sample Sample Sample


(+) (-) A B C

Test:

• Sample A: Negative (No Agglutination)

• Sample B: Negative (No Agglutination)

• Sample C: Positive (Agglutination observed)

DISCUSSION:

The ASO-Latex test is a rapid slide agglutination test for the direct
detection and semi-quantitation of anti-streptolysin (ASO). The antigen, a
particulate latex suspension coated with streptolysin O, agglutinates in the
presence of specific antibodies present in the sera of patients with
Streptococcal- haemolytic infection (group A and C).

The reagent is prepared by reacting streptolysin O with a


bifunctional low molecular weight compound and, in the preferred
embodiment, with a gamma globulin or a Fab fragment thereof to form a
product, which product is adsorbed to latex particles.

However there are some limitations of the procedure which are:


• Positive results may be obtained in conditions other than
rheumatic fever and glomerulonephritis such as scarlet fever,
amigdalitis, various streptococcal infections and even in
healthy carriers.

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[PRACTICAL 6] MTEB 2404

• False negative reactions may occur in early primary infections


and children aged between 6 months and 2 years of age.
• A single ASO determination does not yield much information
on the contemporary significance of the disease. Therefore it is
advisable that titrations on questionable cases are carried out
at bi-weekly intervals for 4 to 6 weeks in order to ascertain the
evolution of the disease.

Conclusion:

On this experiment, sample C showed a positive of ASO test by forming an


agglutinate formed.

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