**Monkey B Virus IgG (BV IgG) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Instructions**
This kit is intended for research use only and is designed to detect the presence of Monkey B Virus IgG (BV IgG) in serum, plasma, and related liquid samples from monkeys.
**Principle of the Assay**
The kit utilizes a double-antibody sandwich ELISA method. A microplate is pre-coated with purified anti-BV IgG antibodies. The sample containing BV IgG is added, allowing it to bind to the immobilized antibody. After washing, horseradish peroxidase (HRP)-labeled anti-BV IgG antibodies are introduced, forming an antibody-antigen-enzyme complex. The reaction is developed using TMB substrate, which changes color under HRP catalysis, turning blue and then yellow upon acid addition. The optical density (OD) at 450 nm is measured, and results are compared against a CUTOFF value to determine positivity.
**Kit Components**
- 48-well configuration: 1 plate, 2 sealing films, 1 negative control (0.5 ml), 1 positive control (0.5 ml), 1 enzyme-labeled plate, 1 sample diluent (3 ml), 1 developer A (3 ml), 1 developer B (3 ml), 1 stop solution (3 ml), 1 concentrated wash solution (20×, 20 ml).
- 96-well configuration: Similar components, but with double quantities.
**Storage Conditions**
All reagents should be stored at 2–8°C. Do not freeze unless specified.
**Sample Preparation and Handling**
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully. If precipitate forms, centrifuge again.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well, then centrifuge similarly.
3. **Urine**: Collect in sterile tubes and centrifuge.
4. **Cell Culture Supernatant**: Centrifuge after collection. For intracellular components, lyse cells by freezing and thawing, then centrifuge.
5. **Tissue Samples**: Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C post-thawing.
6. **General Notes**: Process samples immediately after collection. Store at -20°C if testing is delayed. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3, as it may inhibit HRP activity.
**Procedure**
1. Label all wells. Include 2 negative controls, 2 positive controls, and 1 blank control per plate.
2. Add 50 µL of negative/positive control, 40 µL of sample diluent, and 10 µL of sample to each well. Mix gently.
3. Incubate at 37°C for 30 minutes.
4. Prepare wash buffer by diluting concentrated washing solution with distilled water.
5. Wash 5 times with wash buffer.
6. Add 50 µL of enzyme-labeled reagent to each well (except blank).
7. Incubate again at 37°C for 30 minutes.
8. Wash as before.
9. Add 50 µL of Developer A, then 50 µL of Developer B. Incubate at 37°C for 15 minutes.
10. Stop the reaction by adding 50 µL of stop solution.
11. Measure OD at 450 nm within 15 minutes.
**Result Interpretation**
- **Valid Test**: Positive control OD ≥ 1.00; Negative control OD ≤ 0.10.
- **CUTOFF Value**: CUTOFF = Mean of negative control + 0.15.
- **Positive Result**: Sample OD > CUTOFF.
- **Negative Result**: Sample OD ≤ CUTOFF.
**Note**: Always follow safety protocols when handling biological samples. Ensure proper disposal of waste materials.
For detailed instructions and further information, download the full manual [here](#).
*For procurement and support, visit the Shanghai Kamaishu Laboratory Research Reagents Network.*
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