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0032 (0)16 41 44 07
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0032 (0)16 41 44 07
The Human Immunodeficiency Virus Type 1 (HIV-1) is recognized as the etiologic
agent of acquired immunodeficiency syndrome (AIDS). The virus is transmitted by
sexual contact, exposure to infected body fluids or tissues, and from mother to
fetus or child during perinatal period. After exposure to the virus, HIV-1
infection is characterized by an early period of antigenemia in which HIV-1
antigens (Ag) are detectable in blood. In most individuals, the antigen level
becomes undetectable for a period of time; late in disease, increasing failure
of the immune system and increasing levels of virus may again result in
detectable levels of antigen. One of the viral components in blood during
antigenemia is the core protein, p24, the major internal structural protein of
The HIV-1 p24 Antigen Assay is an enzyme immunoassay (EIA, or Enzyme-linked
Immunosorbant Assay, ELISA) developed for detection and quantitation of the
HIV-1 p24 core protein. The HIV-1 p24 Antigen Assay uses a murine monoclonal
antibody to HIV-1 p24 antigen coated onto microtiter strip wells. A specimen of
plasma, serum or tissue culture media and lysis buffer are added to a coated
well and incubated. If present, the virus antigen particles bind to the
monoclonal antibody on the microtiter well. Following a wash step, biotinylated
human anti-HIV-1 IgG is added to the well which during incubation, binds to any
HIV-1 p24 antigen bound to the well. Following another wash step,
streptavidin-horseradish peroxidase is added which complexes with biotinylated
antibodies. In a final step, a substrate reagent containing tetramethylbenzidine
(TMB) and hydrogen peroxide is added which reacts with complexed peroxidase to
form a blue color. The reaction is terminated by the addition of acid, and the
absorbance is measured spectrophotometrically. The intensity of the color
development is directly proportional to the amount of uncomplexed p24 antigen in
the plasma, serum or tissue culture media. The quantity of free HIV-1 p24
antigen in a specimen is determined by comparing its absorbance with that of
known HIV-1 p24 antigen standard curve.
2. SPECIMEN REQUIREMENTS
2.1 Serum, tissue culture supernatant, or plasma collected in
acid-citrate-dextrose (ACD), citrate-phosphate-dextrose with adenine (CPDA-1),
EDTA, sodium citrate or heparin may be used and should be tested as soon as
possible following collection. If the situation limits the ability to test the
sample quickly, the specimen can be held in refrigeration (2-4° C) for a maximum
of 7 days. If the period of time will be greater, the sample can be held at
-20°C or -85°C for long-term storage.
2.2 Remove the serum from the clot or plasma from the red cells as soon as
possible to avoid hemolysis.
2.3 Heat-inactivated specimens or specimens with obvious microbial contamination
2.4 Specimens containing particulate matter may give inconsistent results. Such
specimens should be clarified by centrifugation prior to assay.
2.5 Avoid subjecting specimens to repeated freeze thaw cycles.
2.6 Bring all specimens to room temperature (15-30°C) prior to assay.
3.1 Reagents included in HIV-1 p24 Antigen Assay Kit, 96 (PN 6604534) or 2400
(PN 6607051), include the following:
3.1.1 HIV p24 Antibody-coated Microtiter Strips. Store at 2-8°C. Note
• Bring pouch containing HIV-1 p24 antibody coated microtiter strips to room
temperature (15-30°C) before opening to avoid condensation on the strips.
• The plate consists of 12 removable strips of 8 wells each. Any partial use of
strips commits all 8 wells to the assay. Antibody coated strips may be used only
once. When using a 96 well plate washer and fewer than 12 strips are needed,
place uncoated strips in the remaining positions.
• Unused strips may be placed back into the pouch and sealed with the desiccant
provided and stored at 2-8°C for 60 days.
3.1.2 Anti-HIV (human) Biotin Reagent. Store at 2-8°C. Note manufacturer’s
outdate. The reconstituted reagent is stable for 2 months. Bring to room
temperature (15-30°C) prior to assay.
• Add 21 mL of distilled water to the Biotin Reagent vial and recap the vial.
• Gently invert vial to mix contents. Allow 5 minutes for the contents to
3.1.3 Normal Human Serum (NHS). Store at 2-8°C. Note manufacturer’s outdate.
3.1.4 SA-Buffer (Tris buffer for SA-HRPO). Store at 2-8°C. Note
3.1.5 SA-HRPO (Streptavidin conjugated to horseradish peroxidase). Store at
2-8°C. Note manufacturer’s outdate. Prepare SA-HRPO Working Dilution as follows.
• Within 15 minutes prior to use, prepare the SA-HRPO Working Dilution. For a
complete 96 well plate, add 21 μL of SA-HRPO reagent to 21 mL of SA-HRPO Buffer.
Mix well and use.
• If a partial plate is used, prepare enough SA-HRPO Working Dilution as shown
No of Tests SA Buffer (mL)
• Discard unused portion at the end of the day.
3.1.6 TMB Diluent. Store at 2-8°C. Note manufacturer’s outdate.
3.1.7 TMB Reagent in Dimethyl sulfoxide. Store at 2-8°C. Note manufacturer’s
outdate. Prepare TMB-substrate Solution as follows:
• Within 15 minutes prior to use, prepare the TMB-Substrate Solution. For a
complete 96 well plate add 21mL to the TMB Diluent into a clean disposable
plastic container and add 210 μL of TMB Reagent. Mix well and use.
• If a partial plate is used, prepare enough TMB-Substrate as follows:
No of Tests TMB Diluent (mL)
• Discard unused portion at the end of the day.
• Note: TMB-Substrate Solution should appear colorless and, when combined with
CSR-1 Solution, should have an absorbance value less than 0.050 at 450 nm
450/570 nm when compared wit a distilled water blank.
3.1.8 Lysis Buffer. Store at 2-8°C. Note manufacturer’s outdate.
3.1.9 Wash Buffer. Store at 2-8°C. Note manufacturer’s outdate. Prepare
working Wash Buffer as follows:
• To prepare 700 mL of Wash Buffer, dilute 35 mL of 20X Wash Buffer with 665 mL
of distilled water.
• Discard any unused portion at the end of the day.
3.1.10 Stop Reagents-1 (CSR-1) (4N H2SO4). Store at 2-30°C. Note manufacturer’s
3.2 Reagents required but not provided:
3.2.1 5% Hypochlorite solution (household bleach) diluted 1/100 or appropriate
3.2.2 Dionized or distilled water.
3.2.3 Standards and controls for the assay provided by the Virology Quality
Assurance Laboratory (VQA):
18.104.22.168 VQA SQC (Serum Quality Control). A set of five concentrations. Store at
• Just prior to set up, thaw 1 vial of each of the 5 concentrations.
• Mix well and use.
22.214.171.124 VQA 400pg/mL standard, diluent and Media Correction Control (MCC) for
• Prepare 2 fold serial dilutions from 400pg/mL Standards using the VQA diluent.
• Run 0 (diluent), 12.5, 25, 50 and 100pg/mL standard curve in duplicate, run
MCC (30pg/mL) in duplicate
4. SUPPLIES AND EQUIPMENT
4.1 Lab coat
4.3 Micropipette(s) capable of delivering 10 μL, 20 μL, 50 μL, 200 μL volumes
4.4 Multichannel pipette(s) capable of delivering 10 μL, 20 μL, 50 μL, 200 μL
4.5 Disposable pipette tips suitable for the above pipettes
4.6 Disposable reagent reservoirs
4.7 Uncoated 96 well microtiter plate
4.8 Incubator without CO2 capable of maintaining 37°C +/- 1°C
4.9 Timer capable of measuring times up to 60 minutes
4.11 Graduated cylinders and beakers
4.12 Serological pipettes
4.13 ELISA microtiter plate washer with waste trap and vacuum source
4.14 ELISA microtiter plate reader capable of measuring absorbance at 450 nm
with reference at 570 nm
5.1 Plate Set-up
5.1.1 Bring all reagents and samples to room temperature.
5.1.2 Create an EIA template in the virology data-management software (see
5.1.3 Position the required number of microtiter strips in the strip holder
reaction plate (8 wells per strip). If fewer than 12 strips are needed, use
uncoated strip(s) in the remaining positions when using a 96 well plate washer.
5.1.4 Add 20 μL of Lysis Buffer to each test well of the coated microtiter
5.1.5 Add 200 μL of each VQA SQC concentration and each specimen to the coated
microtiter plate according to the template. Cover the plate using an adhesive
5.1.6 Incubate at 37°C for 1 hour + 5 minutes.
5.1.7 Wash as follows: Aspirate the solution from the wells. Add 300 μL pf Wash
Buffer Working Dilution to each well. Allow wells to soak for 25-30 seconds.
Aspirate the solution from the wells. Wash five (5) more times for a total of 6
washes. After the final wash step, grasp the plate firmly along the edges,
invert plate over absorbent paper and tap the plate gently to remove any
IMPORTANT: The time between the wash step and the next reagent must be less than
five (5) minutes.
5.1.8 Add 200 μL of reconstituted Biotin Reagent to all testing wells, except
the substrate blank well. Cover the plate using a new adhesive paper cover.
Incubate at 37°C + 2 for 1 hour + 5 minutes.
5.1.9 Wash as described above.
5.1.10 Add 200 μL of SA-HRPO Working Dilution to all testing wells, except the
substrate blank well. Cover the plate using new adhesive plate cover. Incubate
at 37°C + 2 for 30 + 2 minutes.
5.1.11 Wash as described above.
5.1.12 Add 200 μL of TMB-Substrate Solution to all wells. Cover the plate using
a new adhesive plate cover. Incubate at room temperature (15-30°C) for 30 + 2
5.1.13 Add 50 μL of CSR-1 to all wells.
IMPORTANT: Add CSR-1 to the wells in the same sequence and at the same rate of
speed that the TMB-Substrate Solution was added.
5.1.14 Read absorbance at 450 nm (reference at 570 nm if dual wavelength
Instrument is available) within 30 minutes of adding CSR-1 to the wells.
The HIV-1 p24 antigen concentrations may be generated from the LDMS. A weighted
linear least squares method using the VQA SQC or MCC-corrected concentrations is
used to estimate HIV-1 p24 antigen concentration for serum or culture samples.
7. QUALITY CONTROL
The OD values obtained from the spectrophotometer may be transferred into the
LDMS directly or indirectly using the remote read software. A run report may be
generated that includes the raw OD values and calculated p24 results in pg/mL
for each sample in the run. Assay validity must be determined by comparing the
obtained OD values for each control and standard to an established range.
Acceptable OD values must be established within each laboratory for each lot of
VQA controls. Kit controls may also be included in each run and should satisfy
the criteria outlined in the manufacturer’s package insert. The run should be
reviewed by the laboratory manager or director prior to data release. The
laboratory director/manager must determine the significance of any out of range
QC and resolve the situation prior to releasing any results.
8. PROCEDURAL NOTES
The incubation at 37°C is critical. If the temperature goes above 38°C,
coagulation of the samples may occur.
If a sample gels completely and the well still contains visible coagulated serum
proteins after washing, the results should be considered invalid and the sample