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Monkey Ebola virus antibody (Ebola virus-Ab) ELISA kit instruction manual
**Ebola Virus Antibody ELISA Kit Instructions for Use**
This reagent is intended for research use only. The kit is designed to detect the presence of Ebola virus antibodies (Ebola virus-Ab) in monkey serum, plasma, and related biological samples.
**Principle of the Assay**
The kit utilizes a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) method to quantify the level of anti-Ebola virus antibodies in the sample. The microtiter plate is pre-coated with purified monkey Ebola virus antigen, which binds to specific antibodies present in the sample. Unbound components are washed away, followed by the addition of horseradish peroxidase (HRP)-labeled Ebola virus antigen. This forms an antigen-antibody-enzyme complex. After thorough washing, TMB substrate is added, which turns blue under HRP catalysis and then changes to yellow when acid is introduced. The optical density (OD) is measured at 450 nm using a microplate reader. The result is compared with a cutoff value to determine the presence or absence of the antibody in the specimen.
**Kit Components**
- 48-well configuration: 1 × 48 coated plate, 2 sealing films, 1 negative control (0.5 ml), 1 positive control (0.5 ml), 1 enzyme-labeled reagent (3 ml), 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: 1 × 96 coated plate, 2 sealing films, 1 negative control (0.5 ml), 1 positive control (0.5 ml), 1 enzyme-labeled reagent (6 ml), 1 sample diluent (6 ml), 1 developer A (6 ml), 1 developer B (6 ml), 1 stop solution (6 ml), 1 concentrated wash solution (20×, 20 ml).
**Storage Conditions**
All components should be stored at 2–8°C. Ensure the kit is kept sealed and protected from light.
**Sample Preparation and Handling**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. If precipitation occurs, re-centrifuge.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Urine**: Collect in a sterile container and centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant.
- **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freeze-thaw cycles and centrifuge again.
- **Tissue Specimens**: Weigh the tissue, add PBS (pH 7.4), homogenize, and centrifuge at 2000–3000 rpm for 20 minutes. Store the supernatant at 2–8°C or freeze for later use.
**Important Notes**
- Samples should be processed as soon as possible after collection.
- Avoid repeated freezing and thawing. Store at -20°C if not tested immediately.
- Do not test samples containing NaN3, as it may inhibit HRP activity.
**Procedure**
1. Label all wells. Include 2 negative control, 2 positive control, and 1 blank control per plate.
2. Add 50 μL of negative and positive controls, 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 washing solution by diluting the concentrated wash solution with distilled water (1:20).
5. Wash the plate 5 times with washing solution, ensuring complete removal of liquid.
6. Add 50 μL of enzyme-labeled reagent to each well (except blank).
7. Incubate at 37°C for 30 minutes.
8. Wash again as described in step 5.
9. Add 50 μL of developer A and 50 μL of developer B to each well. Incubate at 37°C for 15 minutes.
10. Add 50 μL of stop solution to each well to terminate the reaction.
11. Measure OD at 450 nm within 15 minutes.
**Result Interpretation**
- **Validity Check**: Positive control OD ≥ 1.00; Negative control OD ≤ 0.10.
- **Cutoff Value**: Cutoff = Average of negative control + 0.15.
- **Negative Result**: Sample OD < Cutoff.
- **Positive Result**: Sample OD ≥ Cutoff.
**Download**
[Monkey Ebola Virus Antibody (Ebola Virus-Ab) ELISA Kit Instruction Manual](#)
**Company Information**
For more details, visit the booth of Kamaishu Biotechnology (Shanghai) Co., Ltd.
**Disclaimer**
All content provided is for informational purposes only. Unauthorized reproduction is prohibited. Please respect intellectual property rights.