HIV-1/HIV-2 plus O EIA
Test Principle and Clinical Relevance- EIA
The Genetic Systems HIV-1/HIV-2 plus O EIA marketed by BioRad is an enzyme immunoassay utilizing
recombinant proteins and synthetic peptides for the detection of antibodies to HIV-1(Groups M and O) and /or HIV-
2 in human serum, plasma, or cadavered serum specimens. It is indicated as a screening test for serum, plasma, or
cadavered serum specimens and as an aid in the diagnosis of infection with HIV-1 and/or HIV-2.
Summary and Explanation of the Test
The Genetic Systems HIV-1/HIV-2 plus O EIA incorporates highly conserved recombinant and synthetic peptide
sequences representing HIV-1 (groups M and O) and HIV-2. Despite some degree of immunological cross-reactivity
between types and subtypes of HIV, reliable detection of the more divergent strains may only be achieved by
incorporating specific sequences into the assay design. Serologic studies have also shown that the core proteins of
HIV-1 and HIV-2 display frequent cross-reactivity whereas the envelope proteins are more type specific. Any
specimen that reacts in an initial test with the Genetic Systems HIV-1/HIV-2 plus O EIA must be retested in duplicate
with the Genetic Systems HIV-1/HIV-2 plus O EIA. Initially reactive specimens that are reactive in wither one or
both duplicates from the repeat testing are referred to as repeatedly reactive. Repeatedly reactive specimens may
contain antibodies to either HIV-1 and /or HIV-2.
Therefore, additional, more specific or supplemental tests for antibodies to both HIV-1 and HIV-2 such as Western
blot or immune flourescence must be performed to verify the presence or absence of antibodies to HIV-1 and HIV-2.
Recommendations for appropriate use of such additional tests may be issued periodically by the united States Public
Health Service.
Biological Principles of the Procedure
The Genetic Systems HIV-1/HIV-2 plus O EIA is an immunoassay based on the principle of direct the antibody
sandwich technique. Microwell strip plates (the solid phase) are coated with purified antigens: gp160 and p24
recombinant proteins derived from HIV-1; a peptide representing the immunodominant region of the HIV-2
transmembrane glycoprotein, gp36; and a synthetic polypeptide mimicking an artificial (i.e., encoded by no existing
virus) HIV-1 group O specific epitope.
During the assay, specimens are evaluated for the presence of HIV-1 and HIV-2 antibodies by interaction with the
absorbed peptides in the wells. Specimens are diluted in specimen diluent and added to each well. The wells are
incubated and then washed. The next step is the addition of a Colored Conjugate Solution (green), which contains
peroxidase-conjugated antigens (peptides mimicking various immunodominant epitopes of the HIV-1 and HIV-2
trasmembrane glycoprotein, and a p24 recombinant protein). If antibodies to either HIV-1 or HIV-2 are present, they
bind to the adsorbed antigen and are not removed by washing. The working conjugate solution, peroxidase- labeled
goat anti-human immunoglobulin, is then added to the wells and binds with the antibody- antigen complex, if present.
Unbound conjugate is removed by a wash step. Working chromogen solution, TMB, is then added to the plate and
allowed to incubate. A blue or blue-green color develops in proportion to the amount of antibody that has been bound
to the antigen-coated plate. The enzyme reaction is stopped by the addition of acid, which results in a color change
to yellow. The optical absorbance of controls and specimens is determined with a spectrophotometer with wavelength
set at 450nm.
All repeatedly positive specimens are confirmed by Western blot (method at the end of this section).
Safety Precautions
The positive and negative controls are heat treated to inactivate viruses. However, handle assay specimens and
controls as if capable of transmitting infectious agents. Use of good laboratory practices and CDC-NIH
guidelines is recommended.
Test operators should adhere to the Occupational Safety and Health Administration (OSHA) regulations (29 CFR
19.10).
Keep testing area separate from areas in which blood or blood products for transfusion are stored.
Do not use reagents beyond the expiration date printed on the reagent label.
With the exception of Substrate Solution, Substrate Diluent, Wash Solution, and Stop Solution, do not interchange
reagents from different lots or kits.
Do not substitute Chromagen and/or Substrate Buffer with color development solutions from other Genetic
Systems tests.
Mix all liquid reagents by gently inverting 3 to 5 times, just prior to use.
Prior to performing the test, bring to room temperature only as many strips of microwells as needed to perform the
test run. Any strip of microwell that is not to be used in the current test run should be sealed in the foil bag with
desciccant and stored at 2-8°C.
Remove reagents from the refrigerator storage approximately 60 minutes before beginning assay. Bring kit reagents
to room temperature (15-30°C) prior to use. Return all kit components to their recommended storage conditions
immediately after use.
For the manual pipetting of controls and specimens, use individual pipette tips to eliminate carryover of samples.
Handle negative and positive controls in the same manner as patient specimens.
If a specimen is inadvertently not added to well, the assay result will read nonreactive.
Inadequate adherence to package insert instructions may result in erroneous or invalid results.
The Genetic Systems HIV-1/HIV-2 plus O EIA performance is highly dependent upon incubation times and
temperatures. Temperatures outside of the validated ranges may result in invalid assays. Incubation temperatures
should be carefully monitored using calibrated thermometers, or equivalent.
Use only adequately calibrated equipment with this assay.
Use of dedicated equipment is recommended if equipment performance validations have not precluded the
possibility of cross-contamination.
Use clean, disposable polypropylene( not polystyrene) tubes and reagent reservoirs for reagent preparation and
dispensing.
Avoid exposing Chromagen or Working TMB solution to strong light during storage and incubation. Do not
allow chromagen solutions to come into contact with an oxidizing agent.
Avoid contact with Stopping Solution. Do not allow Stopping Solution to come into contact with strong bases,
oxidizing agents or metals.
Care must be exercised when dispensing samples to avoid cross contamination through aerosols or carryover.
Use a clean tip for dispensing each individual sample.
Inadequate adherence to package insert instructions may result in erroneous results.
Use only adequately calibrated equipment with this assay.
The HIV-1 and HIV-2 positive controls are heat-treated to inactivate viruses. However, handle all the reagents as
though capable of transmitting infection. All tests should be conducted using the precautions recommended for
blood borne pathogens, as defined by OSHA regulations.
Do not pipette by mouth.
Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled.
Wear protective clothing and disposable gloves while handling, the kit reagents. Wash hands thoroughly after
performing the test.
Handle Chromogen Reagent with care since DMSO is readily absorbed through the skin.
The Stopping Reagent is an acid. Wipe up spills immediately and flush the area with water. If the Stopping Reagent
contacts the skin or eyes, flush with copious amounts of water and seek medical attention.
BIOLOGICAL SPILLS:
Spills not containing acid should be wiped thoroughly with an effective disinfectant. Disinfectants that can be
used include (but are not limited to) a solution of 10% bleach (O.5% solution of sodium hypochlorite), 70%
ethanol, or 0.5% Wescodyne. Spills containing acid should be wiped dry. The area of the spill should be wiped
with one of the chemical disinfectants. Materials used to wipe up spills should be disposed of as biohazardous
waste.
Note: DO NOT PLACE SOLUTIONS CONTAINING BLEACH IN THE AUTOCLAVE.
Dispose of all specimens and materials used to perform the test as though they contain an infectious agent. Disposal
should comply with all applicable waste disposal requirements.
Sodium azide is included as a preservative in the positive and negative controls. Sodium azide has been reported to
form lead or copper azide in laboratory plumbing. These azides are explosive. To prevent azide build-up if solutions
containing azide are disposed of in the sink after biological inactivation, flush plumbing with a large volume of water.
Computerization; Data System Management
HIV antibody results are manually entered into a Microsoft Excel result file spreadsheet. After a run is complete and
any additional corrections by the analyst are made, the Excel result file is prepared. Data is transmitted electronically
to Westat’s ISIS computer system weekly and transferred from there to NCHS.
Rejection
Serum, plasma or cadavered serum specimens may be used in the test.
The following anticoagulants have all been evaluated and found to be acceptable: EDTA, sodium and lithium
heparin, sodium citrate, CPD, CPDA 1, and ACD. Specimens which are collected into anticoagulant tubes should
completely fill the tube as label indicates to avoid improper dilution. Specimens with observable particulate matter
should be cleared by centrifugation prior to testing.
No clinically significant effect on assay results has been detected with increased levels of protein, lipids, bilirubin,
hemolysis, or after heat inactivation of patient samples.
Specimens may be stored at 2–8°C for 7 days. For long, term storage, the specimens should be frozen (at 20°C or
colder). Samples should not be used if they have incurred more than 5 freeze-thaw cycles. Mix samples thoroughly
after thawing.
If specimens are to be shipped, they should be packed in compliance with Federal Regulations covering the
transportation of etiologic agents. Studies have demonstrated that specimens may be shipped refrigerated (2–-8°C) or
at ambient temperature for up to 7 days. For shipments that are in transit for more than 7 days, specimens should be
kept frozen (20°C or lower).
The kit is not licensed for use with specimens other that serum, plasma, or cadavered serum specimens. This kit
is not intended for use on saliva/oral fluid or urine samples.
1. Procedures for Microscopic Examinations; Criteria for Rejection of Inadequately
Prepared Slides
Not applicable for this procedure
2. Equipment And Instrumentation, Materials, Reagent Preparation, Calibrators
(Standards), And Controls
Reagent Preparation
Working Conjugate Solution
Prepare a 1:11 dilution: Bring Conjugate Diluent to room temperature. Invert both the Conjugate Concentrate and
Conjugate diluent to mix before using. Prepare a 1:11 dilution for each strip to be tested by adding 100µL of
Conjugate Concentrate to 1ml Conjugate Diluent in a clean polypropylene tube. Mix well. Working Conjugate should
be green. Note Concentrate lot number,
Date and time of preparation, and date of expiration of the working Conjugate solution. Working Conjugate Solution
is stable for 8 hours at room temperature. Working Conjugate solution should be mixed prior to use.
Working Chromogen Solution
Prepare a 1:11 dilution for each strip being tested.
Bring chromogen reagent and chromogen diluent to room temperature. Invert the chromogen reagent and chromogen
diluent to mix before using. Prepare a 1:11 dilution for each strip to be tested by adding 100µL of chromogen reagent
to 1 ml of chromogen diluent in a clean polypropylene tube. (DO NOT USE A POLYSTYRENE CONTAINER.)
Note chromogen reagent lot number, date and time of preparation and time of expiration of the working chromogen
solution (8 hours from preparation). Mix working solution gently when combined. Working chromogen solution
should be kept in the dark at room temperature prior to use. Chromogen reagent should be colorless to very pale
yellow. Any other color indicates that the reagent is contaminated and should not be used. The working chromogen
solution should be colorless. A distinct blue color indicates that the reagent is contaminated. Discard the working
chromogen solution and prepare fresh reagent in a clean container. Prepare only the amount of the reagent to be used
within 8 hours, ensuring that the volume of diluted reagent will be adequate for the entire plate(s). Extra chromogen
reagent is provided.
Working Wash Solution
Prepare working wash solution as needed by adding one part wash solution concentrate (30×) to 29 parts of water
(e.g. 120 mL of wash solution to 3480 mL of water). Use deionized or distilled water. Clinical laboratory reagent
water Type I or Type II is acceptable. The working wash solution can be stored at room temperature for four weeks
in a plastic container. Note lot number, date prepared and expiration date. Discard if no foaming is evident in the
working-wash solution. Prepare a sufficient quantity of working wash solution to complete a full plate run.
Calibration and Calibration Verification Procedures
Validation of Results
Mean Negative Control absorbance value (NCx)
Determine the mean absorbance for the negative and positive controls by dividing the sum of their absorbance values
by the number of acceptable controls. The individual negative control absorbance values must be greater than equal
to 0.000 AU and less than or equal to 0.150 AU. One negative control absorbance value may be discarded if it is
outside this range. The NCx may be calculated from the two remaining values.
Determine the mean of the negative controls as shown in the example below. HIV-1 Negative
Control (HIV-1 NCx)
Sample Number Absorbance Total absorbance/3 = 0.239/3 = 0...080(HIV-1 NCx)
1 0.075
2 0.083
3 0.081
0.239
Calculation of Results:
Cut-off Value
Determine the cutoff by adding 0.250 to NCx, as shown in the example below: NCx = .080
Cutoff Value = 0.080 + 0.250 = 0.330
A. Assay Validation
A run is valid if the following criteria are met:
The absorbance value of each negative control is greater than or equal to 0.000 AU and less than or equal to 0.150
AU. One negative control value may be discarded, and the mean of negative controls (NCx) may be calculated from
the two remaining values. If two or more negative controls are out of limit, the plate is invalid and must be repeated.
The absorbance value of the HIV-1 Positive Control must be greater than or equal to 0.700 AU.
The absorbance value of the HIV-2 positive Control must be greater than or equal to 0.700 AU.
The absorbance value of the HIV-1 Group O positive Control must be greater than or equal to 0.700 AU.
Other Materials
Materials required but not provided:
Precision pipettes to deliver 20–200 µL, 1 mL, 5 mL and 10 mL, 25 ml (accurate within
+10%)and corresponding pipette tips, multichannel pipettors capable of delivering 25 µL and
100 µL (optional).
Appropriately sized graduated cylinders.
Dry heat incubator capable of maintaining 37 + 2°C. Calibrated thermometer.
Room temperature incubator 25 + 10 C. Calibrated thermometer
Microwell plate or strip washer qualified for use with this assay. The washer must be capable of dispensing at
least 400 µL per well, cycling 5 times, and soaking for 30-60 seconds between each wash.
Microwell strip reader qualified for us with this assay. The spectrophotometer should have the following
specifications at wavelength 450 nm:
Bandwidth: 10 nm HBW (half band width) or equivalent
Absorbance range: 0–2.0 AU (absorbance units)
Repeatability: + (0.5%+ 0.005) AU
Linearity or accuracy: 1% from 0 to 2.0 AU
The instrument should contain a reference filter for reading at 615–630 nm. An instrument without a reference filter
can be used; however areas in the bottom of the wells that are opaque, scratched or irregular may cause absorbance
readings that are falsely elevated.
Household bleach (5% to 8% sodium hypochlorite). Alternative disinfectants include: 70% ethanol or 0.5%
Wescodyne (West Chemical Products).
Reasons for non-repeatedly reactive specimens include:
Improper washing of microwell plates
Cross-contamination of non-reactive specimens with HIV-1 and/or HIV-2 Ab from a high titered specimen.
Contamination of the Working TMB solution
Contamination of then Stopping Solution
If, after repeated testing, the absorbance value of either of the duplicates is greater than or equal to
the cut-off value, the specimen must be considered repeatedly reactive.
Procedure Operating Instructions; Calculations; Interpretation of Results
The presence or absorbance of antibodies to HIV-1 and or HIV-2 is determined by relating the absorbance value of the
specimens to the cutoff value. The cutoff value is determined by adding
0.250 to the mean absorbance value of the negative controls.
Specimens with absorbance values that are less than 0.000 must be repeated.
Those with values greater than the upper linearity limits of the reader should be reported as reactive.
Specimens with absorbance values less than the cutoff value are considered non-reactive by the Genetic Systems
HIV1/HIV-2 plus O EIA and may be considered negative for HIV-1 (M and O groups) and HIV-2 antibodies. Further
testing is not required.
Specimens with absorbance values greater than or equal to the cutoff value are considered initially reactive by the
Genetic Systems HIV1/HIV-2 plus O EIA. Initially reactive specimens should be retested in duplicate to validate the
initial test results. If, after repeat testing, the absorbance values of both duplicate specimens are less than the cutoff
value, the original specimen may be considered non-repeatedly reactive and negative for HIV-1 (Groups M and O)
and HIV-2 antibodies.
Reasons for non-repeatedly reactive specimens include:
Improper washing of microwell plates
Cross contamination of non-reactive specimens with HIV-1 and/or HIV-2 serum of a high titered specimen.
Contamination of the Working TMB solution by oxidizing agents( sodium hypochlorite, hydrogen peroxide, etc)
Contamination of the Stopping solution
If after repeat testing, the absorbance value of either of the duplicates is greater than or equal to the cutoff value,
the specimen must be considered repeatedly reactive.
Repeatedly reactive specimens according to the algorithm will undergo further testing: Western blot and
Multispot.
Result entry
Open the NHANES master file on the shared drive. Enter the data being sure to match the correct specimen with
the correct specimen number.
Have an objective review of the data entry.
Report results: Open correct shipment file. Enter the date received, the date analyzed, the run number, the tech
initials, the result code, and the result comment into the spreadsheet. Save as reports in the CSV file format. Save,
ok, and yes. Do not save changes. Close shipment file.
