Bovine insulin-like growth factor binding protein-5 (IGFBP-5) enzyme-linked immunosorbent kit
**Bovine Insulin-Like Growth Factor Binding Protein-5 (IGFBP-5) ELISA Kit Instruction Manual**
This reagent is for research use only. The kit is designed to quantitatively measure the levels of Bovine Insulin-Like Growth Factor Binding Protein-5 (IGFBP-5) in bovine serum, plasma, and related biological fluids.
**Principle of the Assay**
The ELISA kit employs a sandwich immunoassay technique. A microtiter plate is pre-coated with a specific antibody against IGFBP-5. After adding the sample, the target protein binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex. Following washing steps, a TMB substrate is introduced, which changes color in the presence of HRP. The reaction is stopped, and the absorbance at 450 nm is measured. The intensity of the color is directly proportional to the concentration of IGFBP-5 in the sample. A standard curve is constructed using known concentrations of IGFBP-5 to calculate the unknown sample concentrations.
**Kit Components**
- 48-well or 96-well configuration
- Coated microplate (1 × 48 or 1 × 96)
- Standard (27 μg/L) – 0.5 mL × 1 bottle
- Standard Diluent – 1.5 mL × 1 bottle
- Enzyme Label Reagent – 3 mL × 1 bottle
- Sample Diluent – 3 mL × 1 bottle
- TMB Substrate A & B – 3 mL × 1 bottle each
- Wash Buffer (20×) – 20 mL × 20 times or 20 mL × 30 times
- Sealing Film – 2 pieces (48 wells), 2 pieces (96 wells)
- Storage: All components should be stored at 2–8°C.
**Sample Preparation**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge similarly.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant**: Centrifuge after collection. For intracellular components, lyse cells by repeated freezing and thawing before centrifugation.
- **Tissue Homogenate**: Weigh the tissue, homogenize in PBS, centrifuge, and collect the supernatant.
**Notes for Sample Handling**
- Samples should be processed as soon as possible after collection.
- Avoid repeated freeze-thaw cycles. Store at -20°C if not used immediately.
- Do not use samples containing NaN₃, as it may inhibit HRP activity.
**Procedure Summary**
1. Prepare standards and samples according to instructions.
2. Add samples and standards to the microplate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add HRP-labeled antibody and incubate again.
6. Add TMB substrate and incubate for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure OD at 450 nm using a microplate reader.
**Data Analysis**
Plot the standard curve using concentration vs. OD values. Calculate sample concentrations based on the standard curve and apply the dilution factor. Ensure proper calibration and use duplicate wells for accuracy.
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
**Safety and Disposal**
All reagents and waste should be handled as biohazardous materials. Follow local regulations for disposal.
**Important Notes**
- Equilibrate the kit to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Always prepare a standard curve for each run.
- Ensure accurate pipetting and avoid contamination during all steps.
This manual provides detailed guidance for the reliable detection of bovine IGFBP-5 using a sensitive and specific ELISA method. Proper execution of the protocol ensures accurate and reproducible results.
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