Bovine Brucellosis Indirect ELISA Antibody Test Kit used for the detection of smooth Brucella antibodies in bovine serum.

Bovine Brucellosis Indirect ELISA Antibody Test Kit

Instructions for Use

Catalog No.: EW03-27

【Intended Use】

This product is used for the detection of smooth Bru- cella antibodies in bovine serum.

【Specification】

192 Wells / Kit

【Kit Components】

【Test Procedure and Interpretation】

1 Method

1.1 Sample preparation: Collect whole blood from ani- mals and separate the serum. The serum should be clear and free of hemolysis.

1.2 Preparation of 1× washing buffer: Before use, bring the 20× washing buffer to room temperature (20–25°C) and shake gently to dissolve any crystals (can be heated in a 37℃ water bath for 5–10 minutes if nec- essary). Then dilute with purified water at 1:20 and mix thoroughly.

1.3 Dilution of test serum and control serum: Dilute the test serum, negative control serum, and positive control serum with sample diluent at a ratio of 1:50 in the serum dilution plate,

1.4 Procedure

1.4.1 Sample addition: Take the antigen-coated plate (the strips can be separated and used in batches according to the number of samples).

Wash the plate once with 300 μl of 1× washing buffer per well, then discard the washing solution. Add the diluted test serum, positive control serum, and negative control serum into the corresponding wells of the ELISA plate, 100 μl per well. Each test serum is added to one well, while the positive control serum and negative con- trol serum are each added to two wells (Well 1 and Well 2). After sample addition, incubate at 37℃ for 30 min- utes. Remove the plate and discard the reaction solu- tion.

Add 300 μl of 1× washing buffer per well and wash three times.

After the final wash, remove residual liquid by shaking or tapping dry.

1.4.2 Addition of enzyme conjugate: Dilute HRP-la- beled rabbit anti-bovine IgG with its diluent at 1:200. Add 100 μl per well, then incubate at 37℃ for 30 min- utes. Wash three times according to the method de- scribed in 1.4.1, and remove residual liquid.

1.4.3 Color development: Mix substrate solution A and substrate solution B at a ratio of 1:1 (V/V) and immedi- ately add to the ELISA plate, 100 μl per well. Incubate at room temperature in the dark for 15 minutes for color development.

1.4.4 Reaction termination: Add 50 μl of stop solution per well to terminate the reaction.

2 Result interpretation: After stopping the reaction, measure the OD450 value using a microplate reader within 15 minutes.

2.1 Calculation

Average OD450value of positive control:

OD450(Well 1) + OD450(Well 2)

2

Average OD450value of negative control:

OD450(Well 1)+ OD450(Well 2)

2

OD450(Sample)

SP = X 100% Mean OD450(Positive Control)

2.2 Validity criteria: When the average OD450 of the positive control ≥ 0.8, and the average OD450 of the negative control < 0.1, and Average OD450 value of negative control/ Average OD450 value of positive con- trol × 100% < 10%, the test is considered valid.

2.3 Result determination

When the S/P value ≥ 20%, the sample is positive for bovine brucellosis antibodies.

When the S/P value < 20%, the sample is negative for bovine brucellosis antibodies.

【Precautions】

1. Follow the instructions strictly. Do not use compo- nents from different batches.

2. All reagents should be stored at 2–8℃ . Return to room temperature for 15–30 minutes before use. If the antigen-coated plate is not fully used after opening, the strips should be sealed in a sealed bag for storage.

3. The serum dilution plate is intended for single use only. If the serum dilution plate is not completely used, the used wells should be thoroughly washed and dried, and clearly marked to prevent confusion in subsequent testing.

4. During sample addition, avoid bubble formation and liquid splashing as much as possible to prevent cross-contamination between samples.

5. The substrate solution should be stored protected from light and must not come into contact with oxidiz- ing agents. Containers used for the substrate solution should be clean and free from contamination.

6. The stop solution contains 1 M hydrochloric acid (HCl) and is corrosive. Handle with care. Avoid contact with skin and eyes.

【Storage Conditions and Shelf Life】

1. Store at 2–8°C.

2. Shelf life: 12 months.