[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 Positive Control: A stabilized human serum containing at least 200 Negative Control: A stabilized human serum containing less than

streptococcal exoenzymes. MIX WELL BEFORE USING.
2.ASO

lU/mL of ASO reactive with the test reagent. Ready for use; do not dilute.
3.ASO

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 3. ASO Positive Control 5. ASO Negative Control 7. Glycine - Saline Buffer

2. Pipette/Stir Sticks. 4. Timer 6. Test Tubes, Rack 8. Serological Pipettes
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[PRACTICAL 6] 9. Reaction Slide.

MTEB 2404

PROCEDURE Qualitative Test: 1. Bring reagents and specimens to room temperature before use. #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.

2. Place one drop (50 ul) of ASO Positive Control on field
o

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 A

Sample B

Sample 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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