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INDIRECT AGGLUTINATION ASSAY

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MTEB 2404

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".

[PRACTICAL 7]

MTEB 2404

RHEUMATOID FACTOR (RF) LATEX SLIDE TEST

PRINCIPLE

The RF reagent is a suspension of polystyrene latex particles sensitized with specially prepared human IgG. The reagent is based on an immunological reaction between human IgG bound to biologically inert latex particles and rheumatoid factors in the test specimen.

When serum containing rheumatoid factors is mixed with the latex reagent, visible agglutination occurs. The RF latex reagent sensitivity has been adjusted to detect a minimum of 8 lU/mL of rheumatoid factors according with the WHO International Standard without previous sample dilution.

REAGENTS

1.RF Latex Reagent: A suspension of uniform polystyrene particles coated with IgG (human) in glycine buffer, pH 8.2; reagent sensitivity is standardized with the World Health Organization RF Standard. MIX WELL BEFORE USING. 2.RF Positive Control Serum: A stabilized, prediluted human serum containing at least 8 lU/mL of RF. 3.RF Negative Control Serum: A stabilized, prediluted human serum containing less than 8 lU/mL of RF. 4.Glycine-Saline Buffer (20x): pH 8.2 ± O.IM glycine and 0.15M NaCI. (Dilute buffer following instructions on the label before using.)

***Store all reagents at 2 - 8°C. DO NOT FREEZE.

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MTEB 2404

RF Latex Reagent

Pipette/Stir Sticks

RF Positive ControL

Timer

RF Negative Control

Test Tubes

Glycine-Saline Buffer

Serological pipettes

Reaction Slide

PROCEDURE

Qualitative Test:

1.Bring reagents and specimens to room temperature before use. 2.Place one drop (50 ul) of the RF Positive Control on field #l of the reaction slide. Place one drop (50 ul) of the RF Negative Control on field #2. The remaining fields are used for test specimens. 3.Using pipettes provided. Place one drop of the undiluted specimens on successive fields. Retain Pipette/Stir Sticks for mixing step. 4.Gently resuspend the RF Latex Reagent and add one drop to each test field. Use pipette/ Stir Stick to spread reaction mixture over entire test field. 5.Rotate the slide manually or with a mechanical rotor at 80-100 rpm for 2 minutes and read immediately under direct light.

3 | P a g e

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MTEB 2404

6.Presence of agglutination of the latex particle is a positive result (see figure 1). Agglutination indicates a RF concentration of equal or more than 8 IU/ml. Sera with positive agglutination should be run again with the Quantitative Test.

[PRACTICAL 7]

MTEB 2404

RESULTS

Qualitative Test

1. Negative Reactions:

A negative reaction is indicated by a uniform milky suspension with no

agglutination observed with the RF Negative Control.

2. Positive Reaction:

A positive reaction is indicated by any observable agglutination in the

reaction mixture. The specimen reaction should be compared to the RF Negative and Positive Controls.
reaction mixture. The specimen reaction should be compared to the RF
Negative and Positive Controls.
Control
Control
Sample
Sample
Sample
(+ve)
(-ve)
A
B
C

TEST:

Sample A: Negative (No Agglutination)

Sample B: Positive (Agglutination observed)

Sample C: Negative (No Agglutination)

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MTEB 2404

DISCUSSION:

Rheumatoid arthritis is a systemic chronic disease of unknown etiology. It is often characterized by inflammation and pain in articulations and by an inflammatory and degenerative process which involves the muscle or synoval tissue membrane.

As the specific cure has not yet been found, the early therapy is of great value in patients with difficulties in movement as to minimize the irreversible damage of joints. Therefore, an early diagnosis is fundamental. Rheumatoid arthritis is characterized by the presence of proteins, rheumatoid factors, both in blood and in the synovial fluid.

Rheumatoid factor test is based upon the immunological reaction between the rheumatoid factor (RF) in the serum and the corresponding human antibody IgG coated onto polystyrene latex particles. When the serum containing RF is mixed with the RF latex, a resulting agglutination will be observed.

However,the some cautions should be be consider such as :

1. RF Latex Reagent should be shaken well prior to use so as to secure a uniform suspension of the latex.

2. Thoroughly rinse slide with distilled water and wipe with lint free tissue. Traces of detergent or previous samples may affect the results.

3. Test the reagents with positive and negative controls so as to secure proper performance. Reagents can be damaged by extreme temperature during storage or transport.

4. Human serum components of the kit have been tested and found to be negative for hepatitis B surface antigen (HbsAg), for antibodies to human immunodeficiency virus type 1 (HIV- 1) and hepatitis C. Because no test method can offer complete assurance that HIV, hepatitis B

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MTEB 2404

virus, or other infec-tious agents are absent, samples and kit reagents should be handled as potentially infectious.

CONCLUSION:

On this experiment, sample B showed a positive of RF test by forming an agglutinate formed.