Principle Of The Test

Many specimens and tissue culture specimens contain HIV-1 viral loads greater than the standard curve range of available antigen ELISA. To quantify such samples, a series of costly and time-consuming dilutions must be performed to adjust the sample concentration. The RETRO-TEK HIV-1 p24 Extended Range Kit includes reagents that extend the standard curve of up to 4 ng / mL for the HIV-1 p24 ELISA (Catalog No .: 0801111 or 0801200) or the HIV-1 p24 Antigen ELISA 2.0 (Catalog No: 0801002 or 0801008). Therefore, samples containing p24 antigen up to 4 ng / mL can be accurately quantified without extensive serialization dilution.

Reagents

Supplied materials

• Concentrated HIV-1 antigen p24 standard (0.3 ml): contains a concentration of heat-inactivated viral antigen, interrupted by detergent 80 ng / mL, added protein, detergent, and 2-chloroacetamide.
• Substrate (5 sets): SIGMAFASTTM OPD tablet sets.
  Contains: 5 OPD (o-phenylenediamine dihydrochloride) tablets [silver foil]
  Contains: 5 urea-hydrogen peroxide tablets [gold foil]
  SIGMAFASTTM is a registered trademark of Sigma-Aldrich Company.

Storage: Store all kit reagents at 2 ° -8 ° C.

Materials required but not provided

• HIV-1 p24 antigen ELISA (catalog number: 0801111 or 0801200) or
• HIV-1 p24 antigen ELISA 2.0 (Catalog # 0801002 or 0801008)
• Distilled or deionized water
• Test tubes and racks for preparing samples and standard dilutions
• Adjustable micropipettes, mono 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 a disinfectant. It can be prepared with homemade bleach.

Precautions

1. OPD is a possible carcinogen. Avoid contact. Wear gloves when handling the tablets.
2. Use universal precautions when handling HIV-1 p24 antigen standard and test specimens. *
3. Disposal – When examining the material of human origin or other potentially infectious samples, follow all applicable state and local regulations and federal regulations regarding the disposal of hazardous materials.
4. To avoid cross-contamination, use separate pipette tips for each sample.

Limitations Of The Procedure

Serum or plasma samples from most people infected with HIV-1 contain antibodies to the p24 antigen. Concentration and the microspecificity of these antibodies will vary from one individual to another and from one bleed to another. The observed level of p24 antigen in any sample containing p24 antibodies can be affected by the following:

• Antibodies from the host can mask the reactive epitope with the capture monoclonal antibody. Consequently, the optical density can be reduced.
• Antibodies from the host can mask epitopes that bind to the detector antibody, again reducing the optical density.
• After dissociation and neutralization of the immune complex, host antibodies can recombine with the p24 antigen. Recombination of the host of the antibody with the antigen is competitive with the capture of p24 by the monoclonal antibody on the solid phase and can therefore revert to mask 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 the temperature and the time and, therefore, it is difficult to control. Results correlation for samples between repeated test runs and between kits from different manufacturers may be adversely affected by such recombination.