This ELISA kit is exclusively intended for scientific research purposes and should not be used for medical diagnosis. It is designed to quantitatively detect Rabbit Heat Shock Protein 70 (HSP-70) in various biological samples using a double-antibody one-step sandwich ELISA method. The process involves coating microtiter wells with rabbit HSP-70 capture antibodies, followed by the sequential addition of standards, samples, and HRP-labeled detection antibodies. After incubation and washing, the substrate TMB is added, which changes color under the catalytic action of peroxidase. The final yellow color intensity is directly proportional to the concentration of HSP-70 in the sample. Absorbance is measured at 450 nm using a microplate reader, allowing for accurate quantification.
**Sample Collection, Preparation, and Storage**
1. **Serum**: Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Use anticoagulants such as EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell Culture Supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris.
4. **Tissue Homogenate**: Dilute tissue in saline, homogenize, and centrifuge at 3000 rpm for 10 minutes to collect the supernatant.
5. **Storage**: Store samples at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
**Required Equipment**
- Microplate reader (450 nm)
- Precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL)
- Incubator at 37°C
**Precautions**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the washing solution crystallizes after refrigeration, warm it gently in a water bath before use.
- Unused microtiter strips should be returned to the ziplock bag and stored at low temperature.
- Standard diluent can act as a negative control. Do not dilute the sample; add 10 µL directly.
- Follow the protocol strictly in terms of time, volume, and sequence.
- Shake all reagents thoroughly before use.
**Kit Components**
- Microwell ELISA Plate (96 well or 48 well configuration)
- Standards (400 pg/mL)
- Standard Diluent
- Sample Diluent
- Detection Antibody-HRP
- 20X Wash Buffer
- Substrate A & B
- Stop Solution
- Closure Film
- User Manual
**Reagent Preparation**
- Dilute the 20X Wash Buffer with distilled water at a 1:20 ratio.
**Washing Procedure**
- Manual: Discard liquid, fill each well with wash buffer, let stand for 1 minute, then blot dry. Repeat 5 times.
- Automatic: Add 350 µL wash buffer per well, soak for 1 minute, and repeat 5 times.
**Procedure**
1. Remove the required number of wells from the aluminum foil bag. Return unused wells to the ziplock bag and store at 4°C.
2. Add 50 µL of standard solutions with different concentrations to the standard wells.
3. Add 10 µL of sample and 40 µL of sample diluent to the test wells.
4. Add 100 µL of HRP-labeled detection antibody to each well. Seal and incubate at 37°C for 60 minutes.
5. Discard liquid, wash 5 times, and blot dry.
6. Add 50 µL of TMB substrate A and B, incubate in the dark for 15 minutes.
7. Add 50 µL stop solution and measure OD at 450 nm within 15 minutes.
**Results Analysis**
Plot standard concentrations against their corresponding OD values in Excel to create a standard curve. Calculate sample concentrations based on the regression equation.
**Performance Characteristics**
- **Accuracy**: Correlation coefficient ≥ 0.9900
- **Sensitivity**: Minimum detection <10 pg/mL
- **Specificity**: No cross-reactivity with other proteins
- **Repeatability**: Intra- and inter-assay CV <15%
- **Storage**: 2–8°C, protected from light and moisture
- **Shelf Life**: 6 months from the date of manufacture.
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