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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 purposes only. The kit is designed to detect the presence of Ebola virus antibodies (Ebola virus-Ab) in monkey serum, plasma, and related biological samples. The test is based on a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) method.
**Principle of the Test**
The microplate is pre-coated with purified monkey Ebola virus antigen, serving as a solid-phase capture agent. When the sample is added, any specific Ebola virus antibodies present will bind to the immobilized antigen. After washing away unbound components, horseradish peroxidase (HRP)-labeled Ebola virus antigen is introduced to form a complex of antigen-antibody-enzyme conjugate. Following another wash, the substrate TMB is added. Under HRP catalysis, TMB turns blue and then changes to yellow upon acidification. The optical density (OD) at 450 nm is measured using a microplate reader. By comparing the OD value with the cutoff value, the presence or absence of the antibody in the sample can be determined.
**Kit Components**
- 48-well configuration: 1 × 48 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 ml × 20 times).
- 96-well configuration: 1 × 96 plate, 2 sealing films, 1 negative control (0.5 ml), 1 positive control (0.5 ml), 1 enzyme-labeled plate, 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 ml × 30 times).
**Storage Conditions**
All components should be stored at 2–8°C. Do not freeze unless specified.
**Sample Preparation and Requirements**
- **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 precipitate forms during storage, re-centrifuge.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge for 20 minutes. Collect the supernatant.
- **Urine**: Collect in a sterile tube, centrifuge at 2000–3000 rpm for 20 minutes. Discard precipitate if present.
- **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freezing and thawing, then centrifuge again.
- **Tissue Samples**: Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. Store at 2–8°C after thawing.
- **General Notes**: Process samples as soon as possible after collection. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3, as it may inhibit HRP activity.
**Procedure**
1. Label all wells on the microplate. 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 the washing solution by diluting the concentrated wash solution with distilled water.
5. Wash the plate 5 times with washing buffer, ensuring complete removal of unbound substances.
6. Add 50 μL of enzyme-labeled reagent to each well except the blank control.
7. Incubate again at 37°C for 30 minutes.
8. Wash the plate again.
9. Add 50 μL of developer A, followed by 50 μL of developer B. Incubate at 37°C for 15 minutes.
10. Add 50 μL of stop solution to each well to halt the reaction.
11. Measure the absorbance at 450 nm within 15 minutes of stopping the reaction.
**Result Interpretation**
- **Validity Check**: The mean OD of the positive control should be ≥ 1.00; the mean OD of the negative control should be ≤ 0.10.
- **Cutoff Value**: Cutoff = Mean OD of negative control + 0.15
- **Negative Result**: Sample OD < Cutoff
- **Positive Result**: Sample OD ≥ Cutoff
For more information, please download the full instruction manual: [Monkey Ebola Virus Antibody (Ebola Virus-Ab) ELISA Kit Instruction Manual.rar](#)
For further assistance, visit the booth of Kamaishu Biotechnology (Shanghai) Co., Ltd.
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