SA-ELISA II (EIA Test Kit)

Manufacturer: Centaur

Equine Infectious Anemia Antibody Test

U.s. Vet. Lic. No.

320

Contents

Reagents: Except for the PBS Wash Concentrate, all materials are provided ready-to use.

Materials Provided

1. Microplate wells, coated with synthetic EIAV antigen and recombinant core antigen.

2. Conjugate, of both synthetic EIAV antigen and recombinant core antigen and horseradish peroxidase in phosphate buffered saline with 10% fetal bovine serum (contains 0.02% thimerosal as a preservative).

3. Substrate A, TMB (Tetramethylbenzidine) Peroxidase Substrate.

4. Substrate B, Hydrogen Peroxide, 0.02% in citric acid buffer.

5. PBS (Phosphate Buffered Saline) Wash Concentrate (contains 0.4% thimerosal as a preservative). Must be diluted prior to use (see Preliminary Steps, number 4).

6. Positive Control Serum (contains 0.1% thimerosal as a preservative).

7. Negative Control Serum (contains 0.1% thimerosal as a preservative).

8. Stop Solution (contains 1.0 M phosphoric acid).

Materials Required But Not Provided

1. Graduated cylinder for dilution of PBS Wash Solution.

2. Wash bottle or microplate wash system.

3. Micropipettor (50 microliter) and disposable tips.

4. Optional microplate reader.

SA-ELISA II (EIA Test Kit) Indications

Synthetic antigen enzyme-linked immunosorbent assay.

Test Principles

In the SA-ELISA II test, the wells of the microplate are coated with a synthetic peptide antigen which is chemically identical to a section of an EIAV envelope protein and a recombinant core antigen. The antigens contain multiple epitopes of EIAV which bind antibody present in the serum specimen.

A sample of serum is added to a well and after an incubation period, the well is washed and a conjugate of both the synthetic antigen and the recombinant core antigen and horseradish peroxidase is added. Upon completion of a second incubation period, the well is again washed and TMB (tetramethylbenzidine) as a substrate is added to the well.

If antibodies are present in the test specimen, color develops in the well. If antibodies are not present, no color or only minimal color develops. Positive and negative control sera are included with each group of serum specimens to provide representative color development.

Sample Collection

Specimen Collection and Preparation: Horse serum is recommended for use with the SA-ELISA II test kit. Whole blood may be collected by venipuncture and the serum fraction separated by centrifugation for 10 minutes at approximately 2500 rpm. Serum separators may be used. Anticoagulants are not recommended.

Specimens may be stored at refrigerator temperature (2 to 7°C) for five days. If longer storage is needed, specimens should be frozen at -20°C. Repeated freezing and thawing should be avoided. Frozen samples should thaw at room temperature and should be mixed by gentle inversion before testing begins.

Test Procedure

Assay Methods: Preliminary Steps

1. Remove the SA-ELISA II test kit from the refrigerator about one hour before use to allow reagents and microplate wells to come to room temperature.

2. Use a single microplate well for each serum specimen, Positive Control Serum and Negative Control Serum. Return the remainder of microplate wells in the plate to the storage bag with the desiccant and return the wells to the refrigerator.

3. Identify and record a well location for each control and serum specimen. Alternatively, identification may be written directly on the side of the well.

4. Dilute the 30 mL PBS Wash Concentrate with 570 mL distilled water. Mix thoroughly to ensure that the Wash Concentrate is completely solubilized. Other volumes may be used for convenience. The dilution of one part PBS concentrate with 19 parts distilled water must be maintained. Label the diluted PBS Wash Solution with an expiration of 30 days from the date of dilution. Do not use the Wash Solution after that date. Store diluted PBS Wash Solution at 2 to 7°C.

5. Washing the wells is a critical step in the assay procedure and should be done with care and diligence. Where the Testing Procedure requires the wells to be washed, the contents of the wells should be discarded and the wells completely filled with PBS Wash Solution. After all wells have been filled, empty the wells over a suitable container and immediately tap the inverted wells vigorously on absorbent material to remove any residual wash solution. It is important to maintain the plate in an inverted position during the interval between emptying and tapping the wells. Each well should be washed five times as indicated below (see Testing Procedure).

Testing Procedure

1. Add 50 microliters of control or serum specimen to the appropriate well. Use a new pipette tip for each sample.

2. Gently tap the side of the microplate to dislodge any trapped air bubbles. Incubate the uncovered wells at room temperature (23 to 29°C) for fifteen (15) minutes.

3. At the end of the incubation period, invert the well strip and discard the serum. Wash wells five (5) times with diluted PBS Wash Solution as described above. The washing operation must be done with care to minimize the possibility of nonspecific reactions or cross contamination of wells.

4. Add one drop (0.05 mL) Conjugate to each microplate well. Incubate at room temperature for fifteen (15) minutes.

5. Wash wells five (5) times with diluted PBS Wash Solution. Add one drop (0.05 mL) of Substrate A to each test microwell and then add one drop (0.05 mL) of substrate B to each well.

6. Incubate for fifteen (15) minutes to allow for blue color development.

7. Add one drop (0.05 mL) Stop Solution to each well. The blue color will now become yellow. The use of a white background facilitates the observation of color. Alternatively, the microplate may be read spectrophotometrically at 450 nm.

Quality Control: The Positive Control Serum produces an intense color in the assay. When read spectrophotometrically with the spectrophotometer blanked on air, the Positive Control Serum should yield an O.D. of 0.5 to 2.0. If color development in the Positive Control Serum is weak or appears to be less than usual to the experienced worker, the assay should be repeated. A lack of color development in the Positive Control Serum could be due to the use of expired reagents or wells. The Negative Control Serum produces either no color or only a faint tint. When read spectrophotometrically, the Negative Control Serum should yield an O.D. of 0.0 to 0.15. The presence of color could be due to cross contamination during the test procedure.

Interpretation Of Results

Results: For visual determination, any test well yielding color development greater than the Negative Control Serum should be considered positive for antibodies to EIAV. Wells which visually show color development equal to or less than the Negative Control Serum should be considered to contain serum specimens which are free of detectable antibody to EIAV.

For spectrophotometric determination, blank the spectrophotometer on air. Add 0.05 OD to the negative control value to establish the test cutoff value. Specimen wells with values above the cutoff should be considered positive for antibodies to EIAV. Specimen wells with values equal to or below the cutoff should be considered negative for antibodies to EIAV. It Is recommended that any positive ELISA evaluation be confirmed using the agar gel immunodiffusion (AGID) test. Discrepant samples should be retested and sent to the National Veterinary Services Laboratory for confirmation before being reported as positive.

Storage

Storage and Stability: All materials provided in the test kit should be stored at 2 to 7°C. Wells of the microplate should be stored with the desiccant in the bag provided. After dilution, PBS Wash Solution should again be stored at 2 to 7°C.

Important! Warm reagents to room temperature (23 to 29°C) prior to each use. Materials should not be used after the expiration date shown on the package label.

SA-ELISA II (EIA Test Kit) Caution(s)

1. Do not mix reagents or microplate wells of one kit with those of another kit.

2. For veterinary use only and for sale only to USDA/APHIS approved laboratories.

Warning(s)

Extreme care must be taken in washing microplate wells to avoid cross contamination of adjacent wells which could cause false results.

Discussion

Summary and Explanation: The SA-ELISA II kit utilizes synthetic envelope antigen and a recombinant core antigen in an enzyme-linked immunosorbent assay (ELISA) for the qualitative determination of antibodies to Equine Infectious Anemia Virus (EIAV) in horse serum. The synthetic viral peptide antigen and recombinant core antigen are coated onto the microwells and conjugated to horseradish peroxidase. In field trails, the results of testing by the EIAV antibody test show a high correlation with the agar gel immunodiffusion test of Coggins (1). Because there is no cure and no effective vaccine for EIAV, disease control measures are limited to the identification and isolation of infected animals (2,3). The SA-ELISA II antibody test provides a rapid and reliable method for the detection of EIAV antibodies.

Technical Services: If you have any questions regarding the use of this test, please call the manufacturer, Viral Antigens, Inc. technical services in the USA at (901) 382-8716.

References

Available upon request.

Manufactured By

Viral Antigens, Memphis, TN 38134 USA

09/99 V2111

Presentation

96 wells.

Nac No.

14880120