**Rat Bone Morphogenetic Protein-7 (BMP-7) ELISA Kit – User Manual**
This kit is intended for research purposes only. It is designed to quantitatively measure the concentration of bone morphogenetic protein-7 (BMP-7) in rat serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Principle of the Assay**
The kit utilizes a double-antibody sandwich ELISA method. A purified anti-BMP-7 antibody is pre-coated onto a microplate to serve as the solid-phase capture antibody. The sample is added, allowing BMP-7 to bind to the immobilized antibody. An HRP-conjugated secondary anti-BMP-7 antibody is then introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. After washing, TMB substrate is added, and the reaction is stopped with an acidic solution. The color intensity, measured at 450 nm, correlates directly with the concentration of BMP-7 in the sample. A standard curve is generated using known concentrations of BMP-7, and the sample concentration is calculated accordingly.
**Kit Components**
- Microplate: 48 or 96 wells
- Standard: 1×48 or 1×96, 0.5 ml per vial
- Standard Diluent: 1.5 ml per vial
- Enzyme-Labeled Antibody: 3 ml per vial
- Sample Diluent: 3 ml per vial
- TMB Substrate A & B: 3 ml each
- Stop Solution: 3 ml
- Washing Buffer: 20× concentrated (20 ml × 20/30 times)
- Sealing Film: 2 pieces
- Storage: 2–8°C
**Sample Preparation**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA, citrate, or heparin as anticoagulant; centrifuge after mixing.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by freeze-thaw cycles and centrifuge again.
- **Tissue:** Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
**Procedure**
1. **Standard Dilution:** Prepare a serial dilution of the standard from 600 pg/ml down to 50 pg/ml.
2. **Sample Addition:** Add 40 μl of sample diluent followed by 10 μl of sample (final 5× dilution).
3. **Incubation:** Seal and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted washing buffer.
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 TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μl of stop solution to each well.
9. **Reading:** Measure OD at 450 nm within 15 minutes.
**Notes**
- Equilibrate the kit at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Always prepare a standard curve and run duplicates.
- If sample OD exceeds the highest standard, perform a preliminary dilution.
- Keep all reagents away from light and handle with care.
- Discard all waste as biohazardous material.
**Performance**
- Sensitivity: 24 pg/ml
- Range: 24–800 pg/ml
- Correlation coefficient (R²): ≥0.95
- Intra- and inter-assay variation: <9% and <11%, respectively
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Validity: 6 months from the date of manufacture.
This manual provides detailed guidance to ensure accurate and reproducible results. Always follow the instructions carefully and verify results using a microplate reader.
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