[PRACTICAL 8] 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 8] MTEB 2404

RAPID PLASMA REAGIN (RPR) TEST

PRINCIPLE

Treponema pallidum, the etiological agent of syphilis, produces at

least two types of antibodies inliuman infections: treponemal antibodies
that can be detected by Fluorescent Treponemal Antibody-Absorption
(FTA-ABS) test and non-treponemal antibody (reagin) that can be detected
by RPR antigen card test.

The RPR card test is a non-treponemal test for the serological

detection of syphilis. Either clear unhemolyzed serum or plasma from
EDTA anticoagulated blood may be tested. The antigen suspension
consists of carbon particles and a cardiolipin-lecithin extract of beef heart.
This non-specific antigen detects reagin, an IgM or IgG immunoglobulin
present in the plasma or serum of patients with syphilis and occasionally
present in the serum or plasma of individuals with other acute or chronic
conditions.

If reagin is present it binds to the cardiolipin antigen resulting in

flocculation. The carbon particles become trapped in the flocculation and
appear agglutinated or as black clumps against a white background on
the test card. The agglutination can be seen macroscopically. Specimens
that are nonreactive, that is, do not contain reagin, have a uniform light-
gray color and even particle distribution (no clumping).

REAGENTS

1.RPR Carbon Antigen Suspension: 0.003% cardiolipin, 0.020-0.022%

lecithin, 0.09% cholesterol, 0.0125M EDTA, 0.01M Na2HP04, 0.1%
thimerosal, 0.0188% charcoal, and 10% choline chloride.

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2.RPR Reactive Control: human serum containing 0.1% sodium azide as

preservative.
3.RPR Non-reactive Control: human serum containing 0.1% sodium azide
as preservative. Stabilized liquid control, non-reactive with RPR antigen.
4.Test cards and disposable pipette stirrers.

PROCEDURE

QUALITATIVE CARD TEST

1.Bring the RPR carbon antigen suspension, controls and samples to room
temperature.
2.Hold pipette stirrer in vertical position and dispense one drop of serum
or plasma sample onto a separate circle on the test card with the
disposable stirrer pipettes supplied. Use a fresh pipette stirrer for each
sample.
3.Use the flat end of the stirrer pipettes and spread the sample over the
entire area of the test circle. Dispose of pipette stirrer. Repeat procedure
for number of specimens tested.
4.Shake the carbon antigen suspension dispensing bottle prior to use.
Place one (1) drop (20ul) of "free falling" antigen suspension onto test
circle containing the sample. DO NOT MIX the sample and the antigen.
5.Place test card on an automatic rotator and rotate for 8 minutes at 100
r.p.m.
6.Read results macroscopically under a high intensity incandescent lamp
or strong daylight.

RESULTS

Qualitative Test

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

RPR negative control

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

Control Control Sample Sample Sample

(+) (-) A B C

Test:

• Sample A: Positive (Agglutination observed)

• Sample B: Negative (No Agglutination)

• Sample C: Negative (No Agglutination)

DISCUSSION:
Rapid plasma reagin, a blood test for syphilis that looks for an
antibody that is present in the bloodstream when a patient has syphilis. A
negative (nonreactive) RPR is compatible with a person not having
syphilis, but in the early stages of the disease, the RPR often gives false
negative results.

Conversely, a false positive RPR can be encountered in infectious

mononucleosis, lupus, antiphospholipid antibody syndrome, hepatitis A,
leprosy, malaria and, occasionally, RPR (rapid plasma reagin) is a
screening test for syphilis. It looks for antibodies that are present in the
blood of people who have the disease. The test is similar to the venereal
disease research laboratory (VDRL) test.

The following conditions may cause a false positive test:

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• HIV
• Lyme disease
• Certain types of pneumonia
• Malaria
• Systemic lupus erythematosus

CONCLUSION:

On this experiment, sample A showed a positive of RPR test by forming an

agglutinate formed.

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