PRINCIPLE OF THE TEST Many specimens and tissue culture samples contain HIV-1 viral loads greater than the range of the standard curve of commercially available antigen ELISA’s. In order to quantify such samples, a series of expensive and time consuming dilutions must be made to adjust the sample concentration. The RETRO-TEK HIV-1 p24 Extended Range Kit includes reagents that extend the standard curve of the up to 4 ng/mL for either the HIV-1 p24 ELISA (catalog #: 0801111 or 0801200) or the HIV-1 p24 Antigen ELISA 2.0 (catalog #: 0801002 or 0801008). Therefore, samples containing p24 antigen up to 4 ng/mL may be accurately quantified without extensive serial dilution.
REAGENTS
Materials Supplied
• Concentrated HIV-1 p24 Antigen Standard (0.3mL): Contains detergent disrupted, heat inactivated viral antigen at a concentration of 80 ng/mL, added protein, detergent and 2-chloroacetamide. • Substrate (5 sets): SIGMAFASTTM OPD tablet sets. Contains: 5 OPD tablets (o-phenylenediamine dihydrochloride) [silver foil] Contains: 5 urea-hydrogen peroxide tablets [gold foil]
SIGMAFASTTM is a registered trademark of the Sigma-Aldrich Company.
Storage: Store all kit reagents at 2°-8°C. Materials Required but Not Supplied
• HIV-1 p24 Antigen ELISA (catalog #: 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 specimen and standard dilutions • Adjustable micropipettes, 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
zeptometrix p24 extended range kit
PRECAUTIONS
• OPD is a possible carcinogen. Avoid contact. Use gloves when handling tablets. • Use Universal Precautions when handling test specimens and HIV-1 p24 Antigen Standard.* • 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
TEST PROCEDURE Allow all reagents to reach room temperature before use. Proceed with the RETRO-TEK HIV-1 p24 ELISA according to the product insert with the following changes:
1. Replace the HIV-1 p24 Antigen Standard supplied with the p24 ELISA kit with the HIV-1 p24 Antigen Extended Range Standard. 2. Substitute the TMB Substrate supplied with the p24 ELISA kit with the OPD Substrate Solution and incubate microplate for 30 minutes at room temperature. Please Note: Due to plate reader differences, the high standard absorbance values may be out of range. Read plate at 435nm or 405nm if necessary.
CALCULATION AND INTERPRETATION OF RESULTS Calculate the mean absorbance for each p24 standard and test sample. Plot the concentration of HIV-1 p24 Antigen Standard (pg/mL) on the X-axis versus the mean optical density (OD) for each standard on the Y-axis. The HIV-1 p24 concentration of unknown samples is best determined by using a 4-parameter logistic regression. Alternatively, a point-to-point algorithm may be used. Only readings which fall within the range of the standard curve may be quantified. Be sure to correct for all dilutions, including the 1.1 dilution made during the addition of Lysing Buffer. Below is an example of a typical standard curve for the RETRO-TEK HIV-1 p24 Antigen ELISA (ZEPTOMETRIX catalog # 0801111 or 0801200).
HIV Type 1 p24 Extended Range Kit (5 x 96 Determinations)
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HIV Type 1 p24 Extended Range Kit (5 x 96 Determinations)
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