**Human Anti-Alveolar Basement Membrane Antibody (ABM-Ab) ELISA Kit Instruction Manual**
**Kit Specifications:**
This ELISA kit is available in 48-well or 96-well configurations. The standard dilution is 1.5 ml × 1 bottle. The enzyme standard reagent is 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). This reagent is for research use only.
**Standard Curve Preparation:**
To determine the concentration of ABM-Ab in the sample, plot the standard curve by using the standard concentrations as the x-axis and the OD values as the y-axis. Alternatively, calculate the linear regression equation from the standard curve and input the sample OD value to determine the actual concentration. Multiply by the dilution factor to obtain the final result.
**Kit Components:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 ml × 1 bottle, 2700 ng/L
- Enzyme standard: 1×48 / 1×96
- Sample diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Chromogen B: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Wash solution: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Concentrated wash solution: 20 ml × 20 times (48) / 20 ml × 30 times (96)
**Storage Conditions:**
All components should be stored at 2–8°C. The shelf life is 6 months from the date of manufacture.
**Principle of the Assay:**
This kit uses a double-antibody sandwich method to detect human anti-alveolar basement membrane antibody (ABM-Ab) in the sample. The microtiter plate is pre-coated with a specific antibody against ABM-Ab. After incubation with the sample and HRP-labeled secondary antibody, the complex is formed. TMB substrate is added, and the color changes from blue to yellow under the action of HRP. The intensity of the color is directly proportional to the ABM-Ab concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the concentration is calculated based on the standard curve.
**Purpose:**
This ELISA kit is intended for the quantitative determination of ABM-Ab in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature, then centrifuge at 2000–3000 rpm for 10–20 minutes. Collect the supernatant.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes.
3. **Urine:** Collect using a sterile tube and centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
5. **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant. Store at 2–8°C or freeze at -20°C if not used immediately.
6. **General Note:** Avoid repeated freezing and thawing. Do not use samples containing NaN3, as it may inhibit HRP activity.
**Operation Steps:**
1. Prepare standard dilutions and load them into designated wells.
2. Add sample diluent and sample to the test wells.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing solution.
5. Add enzyme-labeled reagent to all wells except blank controls.
6. Incubate again at 37°C for 30 minutes.
7. Add TMB substrate A and B, incubate at 37°C for 15 minutes.
8. Stop the reaction with stop solution.
9. Measure absorbance at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Keep substrates away from light.
- Always prepare a standard curve and run duplicates.
- Follow the manual strictly and validate results with a microplate reader.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of discrepancies, the English manual takes precedence.
**Performance:**
- Correlation coefficient (R) ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
- Free technical support during working hours
- Free sample testing service available upon request
- Fast delivery after payment
- Ensure maximum experimental accuracy and reliability
**Note:** This manual is designed to guide users through the entire process of the ABM-Ab ELISA assay. Proper handling and execution are essential for accurate and reproducible results.
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