Rat insulin autoantibody (IAA) elisa kit instruction manual

**Rat Insulin Autoantibody (IAA) ELISA Kit – Instructions for Use** **Kit Specifications:** - 48-well or 96-well configuration - Standard Dilution: 1.5 mL × 1 vial - Enzyme Standard Reagent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - This reagent is intended solely for research purposes. **Standard Curve Preparation:** To determine the concentration of IAA in the sample, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Draw a standard curve on graph paper or using software. Alternatively, calculate the linear regression equation from the standard data and use it to compute the sample concentration based on its OD value. Multiply by the dilution factor to obtain the actual sample concentration. **Kit Components:** - Sealing Film: 2 pieces (48-well) / 2 pieces (96-well) - Standard: 2700 ng/L, 0.5 mL × 1 vial - Enzyme Standard: 1×48 / 1×96 - Sample Diluent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Developer A: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Chromogen B: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Stop Solution: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well) - Concentrated Washing Solution: 20 mL × 20 times / 20 mL × 30 times (per bottle) **Storage Conditions:** - Store at 2–8°C - Shelf Life: 6 months **Principle of the Assay:** This ELISA kit uses a double-antibody sandwich method to detect rat insulin autoantibodies (IAA). The microplate is coated with purified anti-IAA antibodies. After adding the sample, IAA binds to the immobilized antibody. An HRP-labeled secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, which changes color under HRP catalysis. The intensity of the color is proportional to the IAA concentration in the sample. The absorbance is measured at 450 nm, and the concentration is determined using a standard curve. **Purpose:** The kit is designed for the quantitative determination of IAA in rat serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Sample Processing Requirements:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. - **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix, centrifuge, and collect supernatant. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Cell Culture Supernatant:** Centrifuge to remove debris; for intracellular components, lyse cells and centrifuge again. - **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. - **Storage:** Avoid repeated freeze-thaw cycles. Store samples at -20°C if not tested immediately. **Important Notes:** - Do not use samples containing NaN3, as it inhibits HRP activity. - Prepare a standard curve for each experiment, ideally in duplicate. - Seal the plate after each step to prevent contamination. - Handle all reagents with care and follow safety protocols. - Wash thoroughly to ensure accurate results. - Use a microplate reader for precise OD measurements. **Operation Steps:** 1. **Standard Dilution:** Prepare a serial dilution of the standard, following the provided protocol. 2. **Sample Loading:** Add 40 µL of sample diluent and 10 µL of sample to each well (final dilution 5×). 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Wash 5 times with diluted washing solution. 5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well (except blank wells). 6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes. 7. **Color Development:** Add 50 µL of TMB A and B, incubate at 37°C for 15 minutes. 8. **Stop Reaction:** Add 50 µL of stop solution to each well. 9. **Measurement:** Read OD at 450 nm within 15 minutes. **Performance:** - Correlation coefficient (R) ≥ 0.95 - Intra-assay CV < 9%, Inter-assay CV < 11% - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** - Free technical support during working hours - Sample testing services available upon request - Delivery time: From payment to shipment **Caution:** Always refer to the English manual in case of discrepancies. Follow all instructions carefully to ensure reliable results.

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