Kit de detecção de anticorpos por enzima para vírus da língua azul

Instructions for use of the Blue Tongue Virus ELISA antibody test kit

Detection principle

This kit consists of a pre-coated bluetongue virus antigen, enzyme labels, and other supporting reagents. During experiments, the control serum and the sample to be tested are added to the plate. After incubation, if the sample contains blue tongue virus antibodies, it binds to the antigen on the plate, and after washing, removes any unbound components; Enzyme markers are then added to specifically bind to antigen-antibody complexes on the microplate labeled plates; Then, wash to remove unbound enzyme markers, add TMB substrate solution to the well, and react with the enzyme-labeled conjugate to form blue products. The depth of color development is positively correlated with the specific antibody content in the sample; After adding the termination solution to terminate the reaction, the product turns yellow; By measuring the absorbance value of each reaction well at a wavelength of 450nm using a microplate reader, it can be determined whether the sample contains blue tongue virus antibodies.

Composition of the kit

Storage and expiration date

Store at 2~8°C, shelf life is 12 months.

After opening, please store the blanket board at 2~8°C away from light to avoid moisture. The usage period is 2 months.

Applicable instruments

Includes a 450nm wavelength microplate reader, 37°C constant temperature equipment, and adjustable micropipettes.

Sample preparation

1. Prepare serum from whole animal blood using conventional methods, ensuring the serum is clear, free of hemolysis, and free of contamination. Samples can be stored at 2~8°C for 1 week, and should be stored at -20°C for extended periods.

2. Before use, the concentrated detergent should be returned to room temperature to dissolve the precipitate, then dilute 20 times with distilled or deionized water to form a working detergent (e.g., 19 parts distilled or deionized water plus 1 part concentrated detergent).

Inspection methods

1. Before use, place the kit at room temperature for 30 minutes to restore to room temperature.

2. Take the required amount of microplate-labeled slats, set one well each for negative and positive controls, seal unused slats as soon as possible, and store at 2~8°C.

3. Add 5μl of sample to the sample well first, then add 95μl of sample diluent, and mix thoroughly with shaking.

4. Add 100μl of negative and positive controls to the negative and positive control wells.

5. Mix well, cover with the cover film, and incubate in a 37°C incubator for 30 minutes.

6. Shake off the liquid inside the holes, add 250 μl of working cleaning solution to each hole, wash evenly by shaking for 60 seconds, then discard. Repeat the wash 5 times, then pat dry the liquid once.

7. Add 100μl of enzyme labeler to each well and react at 37°C in a dark place for 30 minutes.

8. Shake off the liquid inside the holes, add 250 μl of working cleaning solution to each hole, wash evenly by shaking for 60 seconds, then discard. Repeat washing 5 times, then pat dry once after washing.

9. According to the sample quantity, mix color-developing solution A and color-developing solution B in equal volume proportions to prepare the substrate working solution, ready for immediate use. Add 100ul per well, place at 37°C for a light-protected reaction for 10 minutes. If the overall color development speed is too fast, finish the color development 5 minutes early, and the maximum color development time should not exceed 15 minutes.

10. Add 100μl of stop solution to each well, mix well, and measure the absorbance value (OD value) of each well at 450nm.

Reference values

Under normal experimental conditions, the positive control OD versus negative control OD > 0.2.

Interpretation of test results

1. S/P = (sample OD - negative control OD) / (positive control OD - negative control OD).

2. S/P ≥ 0.4 indicates a positive result.

3. At 0.2

4. When S/P < 0.2, the result is interpreted as negative.

Limitations of the testing methods

1. This kit is only for the preliminary assessment of blue tongue virus antibodies in qualitative test samples.

2. This kit is for research purposes only and must not be used in clinical or human experiments.

Notes

1. Gloves and work clothes must be worn during experiments, and disinfection and isolation protocols must be strictly enforced and implemented. All experimental waste must be treated as infectious agents.

2. Terrators are corrosive and should be kept away from contact with skin and clothing. If accidentally touched, rinse immediately with plenty of tap water.

3. When microplate plates are removed from refrigeration, they should be returned to room temperature before opening. Unused plates should be stored in sealed bags containing desiccant.

4. Concentrated detergents tend to crystallize at low temperatures and should be returned to room temperature for complete dissolution during use.

5. Samples used for testing should be kept fresh.

6. Test results must be determined based on microplate reader readings.

7. Reagent components of different batches must not be mixed.