**Guinea Pig Immunoglobulin E (IgE) ELISA Kit Instruction Manual**
**Kit Specifications:**
The Guinea Pig IgE ELISA Kit is available in a 48-well or 96-well configuration. The standard dilution provided is 1.5 mL × 1 vial, while the enzyme standard reagent comes in 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well). This reagent is for research use only.
**Standard Curve Preparation:**
To generate a standard curve, plot the concentration of the standard on the x-axis and the OD value on the y-axis. Using the OD value from the sample, determine the corresponding concentration based on the standard curve. Multiply by the dilution factor to obtain the actual sample concentration. Alternatively, calculate the linear regression equation using the standard concentrations and their corresponding OD values, then substitute the sample’s OD into the equation to compute the concentration, again multiplying by the dilution factor.
**Kit Composition:**
- Sealing film: 2 pieces (48) / 2 pieces (96)
- Instructions: 1 part
- 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)
- Stop Solution: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Concentrated Washing Solution: (20 mL × 20 times) × 1 bottle (48) / (20 mL × 30 times) × 1 bottle (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 ELISA kit uses a double-antibody sandwich method to quantify Guinea Pig IgE in biological samples. A microtiter plate is pre-coated with purified anti-IgE antibodies. After incubation with the sample, HRP-conjugated secondary antibodies bind to the captured IgE, forming an antibody-antigen-enzyme complex. Following washing, TMB substrate is added, which turns blue under HRP activity and changes to yellow upon acid addition. The intensity of the color is directly proportional to the IgE concentration in the sample. Absorbance is measured at 450 nm, and the concentration is determined using a standard curve.
**Purpose:**
This kit is designed to detect the level of anti-endometrial antibodies (EMAb) in Guinea Pig serum, plasma, and other body fluids.
**Sample Processing and Requirements:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge and collect the supernatant.
- **Urine:** Collect in a sterile tube, centrifuge, and collect the supernatant.
- **Cell Culture Supernatant:** Centrifuge after collection; for intracellular components, lyse cells via freezing and thawing before centrifugation.
- **Tissue Specimens:** Homogenize in PBS, centrifuge, and collect the supernatant.
- **Storage:** Samples should be processed as soon as possible. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles. Avoid samples containing NaN3, as it inhibits HRP activity.
**Operation Steps:**
1. **Standard Dilution and Loading:** Prepare a serial dilution of the standard and add 100 µL to each well.
2. **Sample Loading:** Add 40 µL of sample diluent and 10 µL of sample to each test well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute the concentrated washing solution and wash 5 times.
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat the incubation step.
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 absorbance at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate to room temperature before use.
- The washing solution may crystallize; warm it gently if needed.
- Use a pipette accurately, and avoid cross-contamination.
- Always run a standard curve in duplicate. If the sample OD is too high, dilute the sample before testing.
- Do not mix reagents from different batches.
- All waste should be treated as biohazardous material.
- Follow the manual strictly, and rely on the microplate reader results.
- In case of discrepancy, the English version of the manual takes precedence.
**Kit Performance:**
- Correlation coefficient (R) for sample linear regression ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
This manual provides a detailed guide for accurate and reliable detection of Guinea Pig IgE levels in various biological samples. Always follow the instructions carefully to ensure consistent and reproducible results.
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