The RETRO-TEK SIV p27 Antigen ELISA is an enzyme linked immunoassay used to detect Simian Immunodeficiency Virus (SIV) p27 antigen in research specimens, including cell culture media. It can also be used to monitor the purification and biochemical behavior of SIV.

Furthermore, the assay may augment or supplant reverse transcriptase measurements traditionally employed to detect the presence of SIV. Such enzymatic measurements are not SIV specific.

• SIV p27 Antibody Coated Microplate for 96 determinations,1 plate: 12x8 well strips.

• SIV p27 Detector Antibody, 1 vial, lyophilized: Contains biotin-labelled polyclonal antibody to SIV, milk, Tween^®, normal human serum and PBS.

• Detector Antibody Diluent, 25 ml: Contains ProClin 300^®.

• SIV p27 Antigen Standard, 0.5 ml: Contains detergent-disrupted, heat-inactivated viral antigen, goat serum, TritonX-100^® and sodium azide

• Lysing Buffer, 5 ml: Triton X-100^® in PBS and 2-chloroacetamide.

• Streptavidin peroxidase, 0.3 ml:  Contains streptavidin conjugated to horseradish peroxidase, PBS, goat serum, and 2-chloroacetamide.

• Assay Diluent, 100 ml: Contains goat serum, PBS, Triton X-100^® and 2-chloroacetamide.

• 10X Plate Wash Buffer, 125 ml: Contains PBS, Tween 20^® and 2-chloroacetamide.

• Substrate, 0.6 ml: Contains tetramethylbenzidine (TMB) and dimethyl sulfoxide.

• Substrate Buffer, 50 ml: Contains citrate/acetate buffer, hydrogen peroxide and 2-chloroacetamide.

• Stop Solution, 12 ml: Proprietary formulation.

• Plate Sealers, 10

• Resealable Plastic Bag.

^® Triton X-100 and ProClin 300 are registered trademarks of Rohm and Haas. Tween 20 is a registered trademark of Imperial Chemical Industries.

Storage:

Store all kit reagents at 2°-8°C. When stored properly the kit is stable until the date indicated on the box label.

Materials Required but Not Supplied:

• Disposable gloves

• Validated adjustable micropipets, single and multichannel

• Validated test tubes and racks for preparing specimen and control dilutions

• Graduated cylinders and assorted beakers

• Validated automatic microplate washer or manual vacuum aspiration equipment

• Validated incubator for 37°C ±1°C

• Validated microplate reader

• Timer

• 1% sodium hypochlorite as disinfectant. May be prepared from household bleach

• Distilled or deionized water

FOR RESEARCH USE ONLY. Not For in vitro Diagnostic Use.

• Prior to performing the assay, carefully read all instructions.

• Use universal precautions when handling kit components and test
  specimens.*

• To avoid cross-contamination, use separate pipet tips for each
  specimen.

• Disposal: When testing potentially-infectious human specimens, adhere to all applicable local, state and federal regulations regarding the disposal of biohazardous materials.

• Human source material used in the manufacture of the SIV Detector Antibody has been tested and found negative for Hepatitis B surface antigen. The viral lysate used to prepare the SIV p27 Antigen Standard has been inactivated by chemical disruption and heating. Handle these reagents as if capable of transmitting infectious agents.

• The SIV Antigen Standard contains sodium azide as a preservative. Sodium azide may react with lead or copper pipes to form explosive metal azides. Flush pipes with large quantities of water upon disposal to prevent azide buildup in drains.

• Stop Solution contains hydrochloric acid which may cause severe burns. In case of contact with eyes or skin, rinse immediately with water and seek medical assistance. Wear protective clothing and eyewear.

* from MMWR, June 24, 1988, Vol. 37, No. 24, pp. 377-382, 387-388.

SIV p27 Antigen Standard: Prepare a series of six standards from the SIV p27 Antigen Standard. Use the dilution scheme in Table 1. Any diluted SIV p27 Antigen Standard remaining after the completion of the assay should be disinfected and discarded. Do not save diluted reagent.

Table 1
Preparation of SIV p27 Antigen Standard

Streptavidin peroxidase and Substrate: To prepare the Streptavidin peroxidase Working Solution and the Substrate Working Solution, use the dilution schemes in Table 2. Use Substrate Working Solution within 15 minutes of its preparation. Any diluted Substrate Working Solution and Streptavidin peroxidase Working Solution remaining after the completion of the assay must be discarded.

Table 2
Preparation of Streptavidin peroxidase Working Solution
and Substrate Working Solution

Plate Wash Buffer: Dilute 10X Plate Wash Buffer 1:10 in distilled or deionized water prior to use. 1X Plate Wash Buffer may be stored at 2° - 8°C for up to 1 week. Additional bottles of 10X Plate Wash Buffer (ZMC Catalog #: 0801060) may be ordered.

Step 1: Treat specimens in a test tube by pipetting 50 µl of Lysing Buffer into 450 µl specimen and mix well.

Step 2: Wash each well of the microplate or the selected number of strips 6 times with 300µl of 1X Plate Wash Buffer and aspirate. Thoroughly blot by striking inverted microplate or strips on a pad of absorbent towels. Continue striking until no droplets remain in the wells. Do not allow washed plates to completely dry prior to sample addition. Drying will adversely affect test results.

Step 3: Leave one well of the microtiter plate empty during the assay. This well is used for a substrate blank.

Step 4: Pipet 200 µl of standards #1-6 into duplicate wells.

Step 5: Pipet 200 µl of each specimen, as prepared in Step 1, into duplicate wells.

Step 6: Cover microplate with a plate sealer and incubate as follows, either:

            a. Overnight at 37°C ± 1°C
                          or
            b. 2 hours at 37°C ± 1°C

Step 7: Aspirate and wash plate as described in Step 2.

Step 8: Pipet 100 µl of reconstituted SIV p27 Detector Antibody into each well, except the substrate blank. Cover the microplate with a sealer and incubate for 1 hour at 37°C ± 1°C.

Step 9: Aspirate and wash plate as described in Step 2.

Step 10: Pipet 100 µl of the Streptavidin Peroxidase Working Solution into each well except the substrate blank. Cover the microplate with a sealer and incubate for 30 minutes at 37°C ± 1°C.

Step 11: Aspirate and wash plate as described in Step 2.

Step 12: Pipet 100 µl of Substrate Working Solution into all wells and incubate uncovered for 30 minutes at room temperature (18°- 25°C). A  blue color will develop in wells containing viral antigen.

Step 13: Stop the reaction by pipetting 100 µl of Stop Solution into each well. A color change from blue to yellow will result.

Step 14: Within 15 minutes, read the optical density of each well at 450 nm using a microplate reader.

Determine the mean optical density values for each standard and specimen. For the test to be valid, it must meet the following criteria:

• The mean optical density of the 0 pg/ml standard and the substrate blank must be less than 0.200.

• The mean optical density of the 1000 pg/ml standard must be greater than or equal to 0.500.

Cut-off Determination for a Qualitative Assay:

• Determine the mean optical density of the media or normal plasma control wells. Add a factor of 0.05.

• Alternatively, run a series of normal specimens and determine cut-off as the mean plus five standard deviations.

To Quantitate Levels of SIV p27:

Using linear graph paper, plot the concentration of SIV p27 Antigen Standard (pg/ml) on the X-axis versus the mean optical densities for each standard on the Y-axis. Then determine the concentration of SIV p27 antigen in specimens by interpolation from the standard curve. Alternatively, the level of SIV p27 may be calculated by computer using a point-to-point algorithm. Be sure to correct for all dilutions, including the 1 to 1.1 dilution made during the addition of Lysing Buffer.

Table 3
Typical Standard Curve

INCUBATE OVERNIGHT AT 37º + 1ºC or INCUBATE 2 HOURS AT 37º + 1ºC

ADD STOP SOLUTION AND READ AT 450 NM