**Human Heat Shock Protein 40 (HSP-40) ELISA Kit Instruction Manual**
**Kit Specifications:**
This ELISA kit is available in a 48-well or 96-well configuration. The standard dilution is 1.5 ml × 1 bottle, and the enzyme standard reagent is 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). The kit is intended for research use only.
**Calculation Method:**
To determine the concentration of HSP-40 in the sample, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. A standard curve is generated, and the sample concentration is determined based on its OD value. Alternatively, use linear regression to calculate the equation of the standard curve and input the sample’s OD value into the equation. Multiply the result by the dilution factor to obtain the actual sample concentration.
**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 (stored at 2–8°C)
- Sample diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) (stored at 2–8°C)
- Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) (stored at 2–8°C)
- Chromogen B: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) (stored at -8°C)
- Wash solution: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96) (stored at 2–8°C)
- Concentrated wash solution: (20 ml × 20 times) × 1 bottle (48) / (20 ml × 30 times) × 1 bottle (96) (stored at 2–8°C)
**Principle of the Assay:**
The kit employs a double-antibody sandwich ELISA method. Purified anti-HSP-40 antibody is coated onto microtiter plate wells. After incubation with the sample, HSP-40 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. TMB substrate is used to develop the color, which changes from blue to yellow under acidic conditions. The intensity of the color is directly proportional to the HSP-40 concentration in the sample. The absorbance is measured at 450 nm, and the concentration is calculated using a standard curve.
**Purpose:**
This ELISA kit is designed for the quantitative determination of human heat shock protein 40 (HSP-40) in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Storage Conditions & Expiration Date:**
- Storage: 2–8°C
- Shelf Life: 6 months
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant; if precipitate forms, centrifuge again.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge similarly.
3. **Urine:** Collect in a sterile tube and centrifuge for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by freezing and thawing, then centrifuge.
5. **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
6. **General Notes:** Process samples as soon as possible. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3, as it inhibits HRP activity.
**Operating Steps:**
1. **Standard Dilution and Loading:** Prepare serial dilutions of the standard and load them into the microplate.
2. **Sample Loading:** Add sample diluent and sample to the test wells.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute the concentrated wash solution and perform 5 washes.
5. **Enzyme Addition:** Add enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat incubation step.
7. **Color Development:** Add TMB reagents and incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add stop solution to terminate the reaction.
9. **Measurement:** Read OD values at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Keep all reagents away from light.
- Always prepare a standard curve and run duplicates for accuracy.
- Follow the manual strictly. Results should be based on instrument readings.
- Treat all waste materials as biohazardous.
- Do not mix components from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Correlation coefficient (R) of the standard curve ≥ 0.95
- Intra-assay and inter-assay variability < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
This kit provides a reliable and sensitive method for detecting HSP-40 levels, supporting research in stress response, disease biomarkers, and cellular function studies.
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