Rat insulin autoantibody (IAA) elisa kit instruction manual
2025-07-30 09:30:52
**Rat Insulin Autoantibody (IAA) ELISA Kit – Instructions for Use**
**Kit Specifications:**
This ELISA kit is available in 48-well or 96-well configurations. The standard dilution is 1.5 mL × 1 bottle. The enzyme standard reagent is 3 mL × 1 bottle (for 48 wells) or 6 mL × 1 bottle (for 96 wells). This reagent is intended solely for research use.
**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 to create a standard curve. Alternatively, calculate the linear regression equation using the standard concentrations and their respective OD values. Then, input the sample’s OD value into this equation to determine its concentration, multiplying by the dilution factor to obtain the actual sample concentration.
**Kit Composition:**
- Sealing film: 2 pieces (48) / 2 pieces (96)
- Standard: 2700 ng/L, 0.5 mL × 1 bottle
- Enzyme standard: 1×48 / 1×96
- Sample diluent: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Developer A: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Chromogen B: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Wash solution: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Concentrated washing solution: 20 mL × 20 times (48) / 20 mL × 30 times (96)
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of receipt.
**Principle of the Assay:**
This kit uses the sandwich ELISA method to detect rat insulin autoantibodies (IAA). The microplate is pre-coated with purified anti-IAA antibodies. After adding the sample, IAA binds to the coated antibody. HRP-labeled secondary antibodies are then added, forming an immune complex. After washing, TMB substrate is added, leading to a color change that correlates with the IAA concentration. The reaction is stopped, and absorbance is measured at 450 nm using a microplate reader. The IAA concentration is determined by comparing the sample’s OD value to the standard curve.
**Purpose:**
The kit is designed to quantify IAA levels in rat serum, plasma, urine, and other biological fluids.
**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.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge as above.
3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
4. **Cell culture supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again.
5. **Tissue samples:** Homogenize in PBS, centrifuge, and collect the supernatant.
6. **General:** Samples should be processed promptly. If not tested immediately, store at -20°C, avoiding repeated freezing and thawing. Avoid samples containing NaN3, as it may inhibit HRP activity.
**Operation Steps:**
1. **Standard Dilution:** Prepare serial dilutions of the standard, starting from 1800 ng/L down to 150 ng/L.
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 buffer (30× for 48-well; 20× for 96-well).
5. **Enzyme Addition:** Add 50 µL of enzyme conjugate to each well except blank.
6. **Second Incubation:** Repeat step 3.
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.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Avoid cross-contamination by using a new sealing film per test.
- Ensure accurate pipetting and avoid light exposure during TMB development.
- Always prepare a standard curve and run duplicates for better accuracy.
- Do not mix reagents from different batches.
- Follow the manual strictly for reliable results.
**Performance:**
- Linear correlation coefficient (R) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
We offer free technical support during working hours and sample testing services to ensure optimal experimental outcomes. Please contact us for assistance.
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