Human N-terminal pro-brain natriuretic peptide (NT-proBNP) ELISA kit instruction manual
September 26 00:15:36, 2025
**Human N-terminal pro-brain natriuretic peptide (NT-proBNP) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed for the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in human serum, plasma, urine, and other biological fluids. The method employed is a double-antibody sandwich ELISA, ensuring high specificity and sensitivity.
**Principle of Operation**
The ELISA kit utilizes a solid-phase antibody coated on a microtiter plate to capture NT-proBNP from the sample. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured antigen, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, and the reaction is stopped with a stop solution. The color intensity, measured at 450 nm, is directly proportional to the concentration of NT-proBNP in the sample.
**Kit Components**
The kit includes:
- Microtiter plates (48 or 96 wells)
- Purified anti-NT-proBNP antibody-coated wells
- Standard solutions (0.5 ml × 1 bottle)
- Enzyme-labeled antibody
- Sample diluent
- TMB substrate (A and B solutions)
- Washing buffer (concentrated, 20×)
- Sealing film and storage bags
All components should be stored at 2–8°C and used within 6 months from the date of manufacture.
**Sample Preparation**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix well, and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant:** Centrifuge to remove debris before testing.
- **Tissue samples:** Homogenize in PBS, centrifuge, and collect the supernatant.
Avoid repeated freezing and thawing of samples. Do not use samples containing NaN3, as it may inhibit HRP activity.
**Procedure**
1. Prepare standard dilutions (from 60 ng/L down to 5 ng/L).
2. Add 40 μL of sample diluent and 10 μL of sample to each test well.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing buffer.
5. Add 50 μL of enzyme-labeled reagent to each well (except blank wells).
6. Incubate again at 37°C for 30 minutes.
7. Add TMB substrate A and B, incubate for 15 minutes in the dark.
8. Stop the reaction by adding 50 μL of stop solution.
9. Measure OD values at 450 nm within 15 minutes.
**Data Analysis**
Plot a standard curve using the OD values of standards vs. their concentrations. Calculate the sample concentration based on the standard curve and adjust for any dilution factor. It is recommended to run duplicate wells and prepare a new standard curve for each experiment.
**Notes**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a fresh sealing film for each test.
- Handle all reagents carefully and dispose of waste as biohazardous material.
- Do not mix reagents from different batches.
- Always follow the manufacturer’s instructions strictly.
**Performance Characteristics**
- Sensitivity: 2 ng/L
- Range: 2–80 ng/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-batch and inter-batch variability: <9% and <11%, respectively.
**Storage and Shelf Life**
Store the kit at 2–8°C. The shelf life is 6 months from the date of receipt. Do not freeze the kit unless specified.
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