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This Package Insert is provided for product evaluation purposes only and is
not intended to be used in place of the Package Insert shipped with the product.

RETRO-TEK
HIV-1 p24
Extended Range Kit
 

FOR RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC OR CLINICAL USE.

ZMC Catalog # : 0801137
   
INTENDED
USE
  The RETRO-TEK HIV-1 p24 Extended Range Kit is supplied for Research purposes only. It is not intended for use in the diagnosis or prognosis of disease or for screening and may not be used as a confirmatory test in diagnostic situations. It is to be used as an accessory to the RETRO-TEK HIV-1 p24 ELISA.
   
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 RETRO-TEK HIV-1 p24 ELISA (ZMC catalog #: 0801111) up to 4 ng/ml. Samples containing p24 antigen up to 4 ng/ml may be accurately quantified without extensive serial dilution by kinetic reading of the assay plate.
   
REAGENTS   Materials Supplied
  • 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 37 1 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 2-8C.

   
PRECAUTIONS  

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

   
PREPARATION
OF REAGENTS
  Extended Range Standard Curve: Prepare a series of seven standards from the Concentrated HIV-1 p24 Antigen Standard.The dilution scheme in Table 1 is recommended.

Table 1
Preparation of HIV-1 p24 Antigen Standard

Standard
Number

Concentration
of HIV-1 p24 (pg/ml)
Volume of conc.
AntigenStandard (l)
Volume of
Assay Diluent (l)

1

4000 50 l

950 l

2

2000 500 l of #1

500 l

3

1000 500 l of #2

500 l

4

500 500 l of #3

500 l

5

250 500 l of #4

500 l

6

125 500 l of # 5

500 l

7

0 0 l

500 l

 

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.

   
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 making the following changes:
  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

   
CALCULATIONS
AND
INTERPRETATIONS
OF RESULTS
  The HIV-1 p24 concentration of unknown samples may be determined by using a point-to-point algorithm, or linear regression. Only readings which fall within the range of the standard curve may be quantified. Use the Antigen concentrations as the X values and the mean slope/min as the Y values.

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.

HIV-1 Antigen
Standard Concentration

Mean
Slope

4.00 ng/ml

57.46

2.00 ng/ml

48.79

1.00 ng/ml

35.23

0.50 ng/ml

24.95

0.25 ng/ml

16.44

0.125 ng/ml

8.96

0.00 ng/ml 0.085

 

   
LIMITATIONS
OF THE
PROCEDURE
  Most HIV-1 infected individuals produce antibodies to p24 antigen. The concentration and microspecificity of these antibodies will vary from individual to individual and from bleed to bleed. The observed level of p24 antigen in any specimen containing p24 antibodies may be affected by the following:
  • 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.

   
PROCEDURAL
FLOW CHART
 

PREPARE REAGENTS

WASH PLATE

PIPET SPECIMENS, STANDARDS
AND CONTROLS

INCUBATE 2 HOURS AT 37 + 1C

WASH PLATE

PIPET DETECTOR ANTIBODY

INCUBATE 1 HOUR AT AT 37 + 1C

WASH PLATE

PIPET STREPTAVIDIN PEROXIDASE SOLUTION

INCUBATE 30 MINUTES AT AT 37 + 1C

WASH PLATE

PIPET SUBSTRATE SOLUTION

READ PLATE AT 405 NM

   

REV 04/00

 

For more information :: Bioxys and Gentaur BVBA :: Av. de l'Arme 68 B4 :: BE-1040 BELGIUM

Email: info@gentaur.com
 

International 

+32 (0) 16 58 90 45

+32 (0) 16 50 90 45

France

01 43 25 01 50

01 43 25 01 60

Italy

02 36 00 65 93

02 36 00 65 94

Germany

0241 6085 13140

0241 6085 33033

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Last modified: feb-07