the respective antigens. After washing with wash buffer, protein A conjugated gold sol reagent is added to reveal

 

Accessories Pack

Glass slide with six reaction circles, mixing sticks, rubber

teats, sample dispensing pipettes.

Additional Material Required

Test tubes (10 × 75 mm), pipettes, isotonic saline, stop

watch, direct light source, 5% 2-mercaptoethanol solution.

Test Procedure

Bring reagent and samples to room temperature before

use. Dilute sample to be tested 1: 16 with 0.9% saline

(0.1 mL of serum+1.5 mL of 0.9% saline).

Qualitative Method

1. Place one drop of diluted serum on the reaction circle

of the glass slide using a disposable pipette provided

with the kit.

Serology/Immunology 669

2. Add one drop of well mixed latex reagent to the drop

of diluted serum sample.

3. Using a mixing stick, mix the sample and the latex

reagent uniformly over the entire circle.

4. Immediately start a stopwatch. Rock the slide

gently back and forth. Observe for agglutination

macroscopically at 5 minutes.

Semiquantitative Method

1. Using isotonic saline, prepare serial dilutions of the

serum samples positive in the qualitative method

starting frorn 1:32, 1:64, 1:128, 1:256 and so on.

2. Pipette each dilution of the serum sample onto

separate reaction circles of the slide.

3. Add one drop of well mixed latex reagent to each

dilution of the serum sample.

4. Using a mixing stick, mix the sample and the latex

reagent uniformly over the entire circle.

5. Immediately, start a stopwatch. Rock the slide

gently back and forth. Observe for agglutination

macroscopically at 5 minutes.

Interpretations of Results

Qualitative Method

Agglutination is a positive test result and indicates

presence of diagnostically significant level of antibodies

to Toxoplasma gondii. No agglutination is a negative test

result and indicates absence of diagnostically significant

level of antibodies to Toxoplasma gondii.

Semi Quantitative Method

The highest dilution of serum showing agglutination

corresponds to the titer of antibodies to Toxoplasma gondii.

Differentiation IgG - IgM

By previous treatment of the sera with reducing agents,

such as 2-mercaptoethanol, it is possible to observe the

type of immunoglobulins responsible for the reaction.

Add 50 µL of the 2-mercaptoethanol solution to 1 mL of

1:16 diluted serum under test. Incubate for 60 minutes at

37°C.

At the end of the incubation period, proceed using the

semiquantitative test procedure as outlined above.

When antibody titer drops 2 or more dilutions after

mercaptoethanol treatment, it can be considered IgM

positive.

Significance of Test Results

a. Serum samples that test negative in 1:16 dilution

indicate absence of diagnostically significant antitoxoplasma titer.

b. Serum samples positive at 1:16 dilution indicate

residual titer due to past exposure.

c. Positive titers from 1:32 -1:128 dilution should be

suspect of incipient toxoplasmosis. Evolution of titer

3 weeks later should be determined. Increase of at

least two dilutions should be considered indicative of

acute toxoplasmosis.

d. Titer of 1:256 or more suggest possible active

infection.

e. Determination of IgM antibodies is also advisable in

(c) and (d) cases.

Remarks

1. Markedly lipemic, hemolyzed and contaminated

serum samples could give rise to non-specific

result.

2. Use of plasma rather than serum can lead to false

positive results.

3. Positive and negative controls should be run with each

series of tests to validate the results.

4. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

RAPID IMMUNOCONCENTRATION TEST

FOR HIV-1 AND HIV-2 ANTIBODIES FLOW

THROUGH METHOD RETROQUICK-HIV

(Courtesy: Tulip Group of Companies)

Introduction

Retroquick-HIV is a membrane based flow through

immunoassay for the detection of antibodies to HIV-1

and HIV-2 in human serum and plasma. Highly purified

synthetic peptides of gp 120 and gp 41 (HIV-1) and gp 36

(HIV-2) corresponding to the immunodominant regions

of the HIV 1 and HIV 2 utilized in the test system assist in

visual, qualitative, simultaneous detection and differentiation of antibodies to HIV-1 and 2.

Summary

Acquired Immunodeficiency syndrome (AIDS) is caused

by at least two retroviruses, the HIV-1 and the HIV-2,

collectively referred to as HIV-1/2. Antibodies to HIV-1

envelope protein (gp 120), transmembrane protein (gp 41)

and HIV-2 transmembrane protein (gp 36) are prevalent

in sera of individuals with AIDS or ARC or who are at high

risk of contracting AIDS. Detection of these antibodies

indicates exposure to the HIV 1/2 virus.

670 Concise Book of Medical Laboratory Technology: Methods and Interpretations Principle

Retroquick-HIV test comprises of a test device striped

with distinct bands of purified gp 120 and gp 41 synthetic

peptide specific to HIV-1 at test region ‘1’ and gp 36

synthetic peptide specific to HIV-2 at test region ‘2’. The

third band striped at region ‘C’ corresponds to the assay

performance control. First the membrane assembly is

hydrated with wash buffer and then the specimen is added.

Antibodies to HIV-1 and/or 2 if present, are captured by

the respective antigens. After washing with wash buffer,

protein A conjugated gold sol reagent is added to reveal

the presence/absence of bound antibodies. Post final

wash a positive reaction is visualized by the appearance of

purple colored bands at the test region ‘1’ and/or ‘2’. The

absence of bands at test region ‘1’ and ‘2’ is a negative test

result. The appearance of control band serves to validate

sample addition, reagent and assay performance.

Reagents and Materials Supplied

Kit Components

Retroquick-HIV immunoconcentration test kit for HIV1 and HIV-2 antibodies comprises of the following