Usage

The pseudorabies ELISA antibody test kit is used for the detection of the pseudorabies antibodies in swine serum; assessment the immunity conditions against pseudorabies in the pig farm, investigation the epidemiology of the pseudorabies.

Brief Introduction

The test kit is made from the antigen coated microplate, HRP goat-anti-pig IgG and other reagents. It applies the indirect ELISA principle to test the antibodies against pseudorabies virus in pig serum.

Principle

The test kit is the antigen coated microplate made through the inactivation, purification and concentration of the pseudorabies virus. In the test, the diluted control serum and sample are added, then incubate. If pseudorabies specific antibodies exist in the sample, it will be bound with the antigens on the microplate, but the unbound antibodies and other components will be  removed by washing. Next adding enzyme conjugations(anti-pig IgG-HRP) to specifically bind with the compound on the microplate. Then the unbound conjugations are removed by washing, add the TMB substrate to the wells, react with HRP to a blue product. At last, end the reaction by adding stop solution.

Reagents of the Test Kit

1  Antigen coated microplate......................................... 2 pieces  (96 wells)

2  Negative (green lid)/positive (red lid) serum.......... 1 tube each (1ml/tube)

3  Anti-pig IgG-HRP combination(yellow lid)................. 1 bottle (22ml/bottle)

4  20× concentrated washing solution (white lid)........ 1 bottle (30ml/bottle)

5  Substrate A (red lid)/B (black lid)....................... 1bottle each (12ml/bottle)

6  Stop solution(blue lid)............................................... 1 bottle (12ml/bottle)

7  Sample diluent solution(transparent lid).................... 1 bottle (50ml/bottle)

Material Required But Not Provided

50μl-, 100μl-, 1000-micropipettes, adjustable.

Distilled water.

Constant temperature box or incubator.

Microtiter plate spectrophotometer(630nm)(or ELIASA).

Absorbent paper.

Warning and Procautions for the Users

-All reagents should be adjusted to the room temperature before using the test kit.

-Never mix up reagents from different test kits in batch number.

-Prevent the microplate to get damp.

Preparation of Wash Solution

Detergent:  dilute 20×concentrated washing solution with distilled water. The compounded detergent can be kept for 7 days at 4℃.

Test Procedure

1   Take out the pre-coated microplate. Dilute the target sample at 1:40 with the sample diluent, then add to the wells, 100μl each. Dilute the negative control serum at 1:10, add to 2 wells, 100μl each. Dilute the positive control serum at 1:10, add to 2 wells, 100μl each. Set another blank control well, and add in 100μl diluent. Shake softly, then incubate at 37℃ for 30 min.

2       Pour the liquid out of the wells, add detergent to each well, 300μl/well, static for 3min. Repeat 3 times, then pat to dry on absorbent paper.

3        Add 100μl enzyme conjugations(anti-pig IgG-HRP combination) to each well, incubate at 37℃ for 30 min.

4        Repeat step 2.

5        Add substrate A and B one drop(50μl) respectively to each well, mix properly, react for 10 min at room temperature at dark.

6        Add stop solution one drop (50μl) in each well, and measure the result within 10 min.

Results

Set zero for the blank well and test the OD630nm value on the ELIASA. The conditions for the test to be tenable are that the positive control wells’ average OD630nm value is higher than or equal to 0.6, and the negative control wells’ average OD630nm value is less than 0.2.

If the sample’s OD630nm value is greater than 0.42, it is judged to be positive; between 0.38 and 0.42, skeptical; and if lower than 0.38, negative.

Specifications : 96 wells × 2.

Expiry date: 6 months.

Storage: Storing at 2~8℃, don’t expose in strong light.

Production Date: See outer-packing of the test kit.