The human insulin (INS) ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. This method involves pre-coated microwells with human insulin capture antibodies. The process begins by adding the sample, standard solutions, and HRP-labeled detection antibodies sequentially. After incubation and thorough washing, the substrate TMB is introduced to develop color. Under peroxidase catalysis, TMB turns blue and then yellow when an acid is added. The intensity of the color correlates directly with the concentration of human insulin in the sample. The absorbance at 450 nm is measured using a microplate reader to determine the sample concentration.
**Sample Collection and Handling:**
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 EDTA, citrate, or heparin as anticoagulants. Centrifuge at 3000 rpm for 30 minutes to collect plasma.
3. **Cell Supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris.
4. **Tissue Homogenate**: Mix tissue with physiological saline, then centrifuge at 3000 rpm for 10 minutes to obtain supernatant.
5. **Storage**: Store samples at -20°C if not tested immediately. Avoid repeated freezing and thawing. 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
**Operation Notes:**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the washing buffer crystallizes after removal from the fridge, warm it gently in a water bath before use.
- Unused wells should be resealed in the ziplock bag and stored in a dry, cool place.
- Do not dilute pre-treated samples; add 10 μL directly.
- Follow the incubation time, volume, and sequence strictly.
- Shake all liquid reagents well before use.
**Kit Components (96-well format):**
- Microwells: 12 × 8
- Standards (140 uIU/ml): 0.5 mL
- Standard Dilutions: 6 mL
- Sample Diluent: 6 mL
- Detection Antibody-HRP: 6 mL
- 20× Washing Buffer: 20 mL
- Substrate A: 6 mL
- Substrate B: 6 mL
- Stop Solution: 6 mL
- Sealing Film: 2 sheets
**Reagent Preparation:**
- Dilute 20× Washing Buffer 1:20 with distilled water (1 part buffer + 19 parts water).
**Washing Procedure:**
- Manual: Wash 5 times by filling each well with wash solution, letting stand for 1 minute, and discarding.
- Automatic: Use 350 μL per well, soak for 1 minute, and repeat 5 times.
**Procedure Steps:**
1. Remove the required microwells from the foil pouch and allow them to reach room temperature.
2. Set up standard, sample, and blank wells. Add 50 μL of standards and 10 μL of sample plus 40 μL diluent.
3. Add 50 μL of HRP-labeled antibody to each well. Seal with a membrane and incubate at 37°C for 60 minutes.
4. Wash 5 times with washing solution.
5. Add 50 μL of Substrate A and B, incubate in the dark for 15 minutes.
6. Add 50 μL of stop solution and measure OD at 450 nm within 15 minutes.
**Data Analysis:**
Plot standard concentrations against OD values in Excel. Generate a standard curve and calculate sample concentrations using the regression equation.
**Kit Performance:**
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 uIU/ml
- Specificity: No cross-reactivity with other analogs
- Repeatability: CV <15% between plates
- Storage: 2–8°C, protected from light and moisture
- Shelf Life: 6 months
- Detection Range: 4.35–140 uIU/ml
**Disclaimer:**
This kit is for research use only. Not suitable for clinical testing. The user assumes full responsibility for any misuse. Follow instructions carefully. Do not mix different batch numbers. Any deviation from the protocol is the user’s responsibility.
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