• Concentrated HIV-1 p24 Antigen Standard (0.5ml): Contains detergent disrupted, heat inactivated viral antigen at a concentration of 80 ng/ml, added protein, detergent and 2-chloroacetamide.

• Substrate (4 tablets): Contains OPD (orthophenylenediamine-HCl)

Materials Required but Not Supplied

• RETRO-TEK HIV-1 p24 ELISA (ZMC catalog #: 0801111)

• Test tubes and racks for preparing specimen and standard dilutions

• Adjustable micropipets, single and multichannel
• Incubator capable of maintaining 37º± 1º C

• Timer

• Graduated cylinders and assorted beakers

• Automatic microplate washer or manual vacuum aspiration equipment

• 1% sodium hypochlorite as disinfectant. May be prepared from household bleach.

• Distilled or deionized water

Storage:

Store all kit reagents at 2°-8°C.

OPD is a possible carcinogen. Avoid contact. Use gloves when handling tablets.

Use Universal Precautions when handling test specimens and when performing this test.*

Disposal: When examining human source material or other potentially infectious specimens, adhere to all applicable local, state and federal regulations regarding disposal of hazardous materials.

To avoid cross-contamination, use separate pipet tips for each specimen

*from MMWR, June 24,1988, Vol. 37, No.24, pp. 377-382, 387-8

Table 1
Preparation of HIV-1 p24 Antigen Standard

Any diluted standards remaining after the completion of the assay should be discarded.

Do not save diluted reagent.

OPD Substrate Solution: Dissolve 1 OPD tablet in 11.0 ml of the Substrate Buffer supplied in the RETRO-TEK HIV-1 p24 ELISA. Do not make up more than 20 minutes prior to use. Protect from light until use. Use the solution within 15 minutes of its preparation. Do not save diluted reagent.

1. Replace the standard curve supplied with the kit with the Extended Range Standard Curve.
    
2. Replace the liquid Substrate supplied with the p24 kit with the OPD Substrate Tablets.
    
3. Immediately after addition of OPD Substrate Solution to the plate, read in a kinetic plate reader. DO NOT add stop solution to the plate.

The suggested instrument set-up for kinetic readings is:

Total run time: 15 minutes

Time interval to read: 10 sec

Wavelength: 405 nm

OD limit: 1.0 OD

Lag time: 0

If kinetic reading capabilities are not available, the plate should be read every 2 minutes for 15 minutes. Upon examining these curves, select the curve which is most linear in the range of the samples to be quantified to perform calculations by point-to-point algorithm or linear regression. Use the Antigen concentration as the X values and the OD as the Y values.

TYPICAL STANDARD CURVE: This is an example of a typical standard curve. Variation may occur in individual labs due to pipetting, laboratory and incubator temperatures, etc.

• Host antibodies may mask the epitope reactive with capture
  MAb. Consequently, the optical density may be reduced.

• Host antibodies may mask epitopes that bind to the detector
  antibody, again reducing the optical density.

After immune complex dissociation and neutralization, host antibodies may recombine with p24 antigen. The recombination of host antibody with antigen is competitive with the capture of p24 by the MAb on the solid phase and thus may remask some of the p24. The rate of recombination is determined by the concentrations of p24 antigen and anti-p24 antibodies, the affinity of the antibodies, as well as temperature and time and is therefore difficult to control. Correlation of results for specimens between repeat test runs and among kits from different manufacturers may be adversely affected by such competitive recombination.

PREPARE REAGENTS

WASH PLATE

PIPET SPECIMENS, STANDARDS
AND CONTROLS

INCUBATE 2 HOURS AT 37º + 1ºC

WASH PLATE

PIPET DETECTOR ANTIBODY

INCUBATE 1 HOUR AT AT 37º + 1ºC

WASH PLATE

PIPET STREPTAVIDIN PEROXIDASE SOLUTION

INCUBATE 30 MINUTES AT AT 37º + 1ºC

WASH PLATE

PIPET SUBSTRATE SOLUTION

READ PLATE AT 405 NM