**Human Anti-Epidemic Hemorrhagic Fever Virus Antibody IgM (EHF) ELISA Kit Instruction Manual**
**Kit Specifications:**
- 48-well or 96-well configuration
- Standard Dilution: 1.5 ml × 1 bottle
- Enzyme Standard Reagent: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well)
- This reagent is intended for research use only.
**Standard Curve Preparation:**
To determine the concentration of the sample, plot the standard concentrations on the x-axis and their corresponding OD values on the y-axis. A standard curve is drawn, and the sample concentration is determined by matching its OD value to the curve. Alternatively, calculate the linear regression equation using the standard concentrations and OD values, then substitute the sample's OD into the equation to obtain the actual concentration, multiplied by the dilution factor.
**Kit Components:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 ml × 1 bottle, 2700 ng/L
- Enzyme Standard: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well), stored at 2–8°C
- Sample Diluent: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well), stored at 2–8°C
- Developer A: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well), stored at 2–8°C
- Chromogen B: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well), stored at -8°C
- Wash Solution: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well), stored at 2–8°C
- Concentrated Wash Solution: (20 ml × 20 times) × 1 bottle / (20 ml × 30 times) × 1 bottle, stored at 2–8°C
**Principle of the Assay:**
This kit uses a double-antibody sandwich method to detect human anti-EHF IgM antibodies in serum, plasma, or other biological fluids. The microtiter plate is pre-coated with a specific antibody against EHF IgM. After adding the sample, the antigen-antibody complex forms. An HRP-conjugated secondary antibody is then added, followed by TMB substrate. The reaction produces a blue color that turns yellow in the presence of an acid. The intensity of the color is directly proportional to the concentration of EHF IgM in the sample. The absorbance is measured at 450 nm, and the concentration is calculated based on the standard curve.
**Purpose:**
This ELISA kit is designed to quantify human anti-EHF IgM antibodies in various biological samples such as serum, plasma, urine, cell culture supernatants, and tissue homogenates.
**Storage Conditions & Expiration:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of manufacture
**Sample Preparation & Handling:**
1. **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 minutes. Store the supernatant at 2–8°C or -20°C.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes.
3. **Urine:** Collect in sterile tubes and centrifuge.
4. **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles before centrifugation.
5. **Tissue Samples:** Homogenize in PBS, centrifuge, and store the supernatant.
6. **General Notes:** Avoid repeated freezing and thawing. Do not use samples containing NaN3, as it inhibits HRP activity.
**Operation Steps:**
1. **Standard Dilution:** Prepare serial dilutions of the standard in the designated wells.
2. **Sample Loading:** Add 40 μl of sample diluent and 10 μl of sample to each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Rinse the plate 5 times with diluted washing solution.
5. **Enzyme Addition:** Add 50 μl of enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 μl of chromogen A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μl of stop solution to terminate the reaction.
9. **Measurement:** Read OD values at 450 nm within 15 minutes of stopping the reaction.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Keep all reagents away from light.
- Always perform a standard curve and ensure proper dilution if sample OD exceeds the standard range.
- Follow the instructions strictly and validate results with a microplate reader.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Correlation coefficient (R) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
This ELISA kit offers a reliable and accurate method for detecting human anti-EHF IgM antibodies, ensuring high sensitivity and specificity in diagnostic applications.
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