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The kit contains 3 plastic combs, 3 development plates, Positive control, Negative control and Perforator.

Each comb has 6 pairs of teeth, one pair for each test (figure 3.5). Both teeth of a pair carry identical numbers. Each pair of teeth is sensitized with six reactive areas as follows.

The upper left tooth contains Human Immunoglobin (Internal Control), while the upper right tooth contains Recombinant HIV-1 env. Derived glycoprotein gp 120.

The middle left tooth contains Recombinant gag-derived core protein p24 (recognized by antibodies to HIV-1 and HIV-2), while the middle right tooth contains HIV-1 transmembrane env. Glycoprotein gp41-derived synthetic peptide.

The lower left tooth contains Recombinant pol-derived protein p31 (recognized by antibodies to HIV-1 and HIV-2), while the lower right tooth contains HIV-2 transmembrane env glycoprotein gp36-derived synthetic peptide.

The combs are provided in aluminium pouches containing a desiccant bag.

The kit also contains 3 developing plates, covered by aluminium foil. Each developing plate (figure 3.6) contains all reagents needed for the test. The Developing plate consists of 6 rows (A-F).

FIGURE 3.5 THE COMB

Row A is divided into 6 wells, whereas the other rows are divided into 12 wells each.

The contents of each row are as follows.

Row A specimen diluents Row B washing solution

RowC alkaline phosphatase-labelled goat anti-human IgG antibodies Row D washing solution

Row E washing solution

Row F chromogenic substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate(BCIB) and nitro blue tetrazolium (NBT).

FIG. 3.6 THE DEVELOPMENT PLATES

Positive Control- 1 vial (red-coloured cap) of 0.4 ml, diluted human plasma positive for anti-HIV-1 and anti-HIV-2 antibodies, inactivated by addition of b-propiolactone and by heat treatment.

Negative Control- 1 vial (green-coloured cap) of 0.4mls. Diluted heat-inactivated human plasma, negative for antibodies to HIV.

Perforator- for perforation of the aluminium foil, covering the wells of the Developing Plate.

TEST PROCEDURE

All the components, developing plates, combs, reagents and specimens are brought to room temperature. The kits are left at room temperature for 3 hours. The reagents are mixed by shaking the Development plates without removing the cover. The aluminium pouch of the Comb is torn to bring out the Comb. Each Comb shall be used for 6 tests, though part of it can be used by breaking the Comb.

After collecting about 50 micromillilitres of the specimen, the foil cover of one well in row A of the Developing plate is then perforated and the specimen is dispensed at the bottom of the well. It is then mixed by repeated refilling and ejecting the solution.

This step is repeated for the other specimens, including the Positive and Negative Control supplied with the kit.

The Comb is then inserted into the wells of row A containing specimens and controls. It is mixed by withdrawing and inserting the Comb into the wells several times. The comb in row A is left for exactly 30 minutes. Towards the end of 30 minutes, the foil of row B is perforated using the perforator. At the end of 30 minutes, the Comb in row A is taken out and the adhering liquid from the pointed tips of the teeth absorbed on a clean absorbent paper.

First Wash (Row B)

The Comb removed from row A is then inserted into the wells of Row B. Proper washing is ensured by withdrawing and inserting the Comb into the wells for at least 10 seconds to ensure proper washing. The agitation is continued for 2 minutes;

meanwhile the foil of row C is perforated. After 2 minutes, the comb is withdrawn and the adhering liquid absorbed.

Binding of conjugate (Row C)

The comb is inserted into the wells of Row C and mixed as usual. It is left for 20 minute. Towards the end of 20 minutes the foil on Row D is perforated. After 20 minutes, the comb is withdrawn and the adhering fluid absorbed.

Second Wash (Row D)

The comb is inserted into the wells of row D and repeatedly agitated for 2 minutes.

Meanwhile the foil of row E is perforated. After 2 minutes, the comb is withdrawn and the adhering liquid removed.

Third Wash (Row E)

The comb is inserted into the wells of row E and repeatedly agitated for 2 minutes.

Meanwhile the foil of row F is perforated. After 2 minutes the comb is withdrawn and the adhering liquid absorbed.

Colour Reaction (Row F)

The comb is inserted into the wells of row F and mixed. After 10 minutes the comb is withdrawn

Stop Reaction (Row E)

The comb is inserted again into row E for 1 minute. It is withdrawn and allowed to dry in the air.

Finally all used Developing plates; pipette tips, absorbent paper, and gloves are disposed as biohardazous wastes.

TEST RESULTS

In order to confirm that the tests function properly and to demonstrate that the results are valid, the following three conditions must be fulfilled.

1. The Positive control must produce six gray-blue coloured spots on the relevant pair of Comb teeth.

2. The Negative Control must produce an upper spot (internal control) on the left tooth of the pair and no other spots.

3. Each specimen tested must produce an upper spot (internal control) on the left tooth of the pair.

If any of the three conditions are not fulfilled, the results are invalid and the specimens and controls are retested (see figure 3.7)

FIGURE 3.7 TEST VALIDATION

Interpretation of the results

The sole appearance of the Internal Control indicates that the corresponding specimen is negative for antibodies to HIV-1 or HIV-2.

A specimen yielding only one isolated circular, coloured HIV antigen spot or both the p24 and p31 spots without a spot on the right tooth is considered indeterminate.

A specimen yielding a minimum of two circular, coloured antigen HIV-antigen spots, including one representing gp36 and presence of gp41 or gp36 (see figure 3.5) is defined as HIV-positive. In these cases differentiation between HIV-1 and HIV-2 may be obtained as follows:

.Absence of a spot for gp36 and presence of gp47 and/or gp120 indicates that the specimen is positive for antibodies to HIV-1

. Presence of a spot for gp36 indicates that the specimen is

positive for antibodies to HIV-2.