**Guinea Pig Immunoglobulin E (IgE) ELISA Kit Instruction Manual**
**Kit Specifications:**
Available in 48-well or 96-well configurations.
- Standard Dilution: 1.5 mL × 1 bottle
- Enzyme Standard Reagent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
This reagent is intended for **research use only**. To determine the concentration of IgE, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis to create a standard curve. Use this curve to estimate the sample concentration based on its OD value. Alternatively, calculate the linear regression equation from the standard data and input the sample OD into the equation to obtain the actual concentration, then multiply by the dilution factor.
**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
- Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Developer A: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Chromogen B: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Stop Solution: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Concentrated Washing Solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well)
**Storage Conditions:**
- 2–8°C for all components.
- Shelf Life: 6 months from the date of receipt.
**Principle of Operation:**
The kit uses a double-antibody sandwich ELISA method to quantify Guinea Pig IgE in the sample. The microplate is pre-coated with purified anti-IgE antibodies. After adding the sample, IgE binds to the immobilized antibody. HRP-conjugated secondary antibody is then added, forming a complex. TMB substrate is used for color development, which changes from blue to yellow under acidic conditions. The intensity of the color is directly proportional to the IgE concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the concentration is determined using a standard curve.
**Purpose:**
This ELISA kit is designed to detect the level of anti-endometrial antibodies (EMAb) in Guinea Pig serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Sample Preparation and Handling:**
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 well, and centrifuge similarly.
3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
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. **Storage:** Samples should be processed immediately after collection. If not tested right away, store at -20°C, avoiding repeated freezing and thawing. Avoid samples containing NaN3, as it may inhibit HRP activity.
**Procedure Steps:**
1. **Standard Dilution:** Prepare a serial dilution of the standard solution and add to designated wells.
2. **Sample Loading:** Add 40 μL of sample diluent followed by 10 μL of the sample to each test well (final 5-fold dilution).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted washing buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well (except blank).
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.
**Notes:**
- Equilibrate the kit at room temperature before use.
- Store unopened enzyme strips in a sealed bag.
- Ensure accurate pipetting and avoid cross-contamination.
- Always prepare a standard curve and run duplicates for better accuracy.
- Avoid exposure of substrates to light.
- Follow instructions strictly and validate results with a microplate reader.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Linear regression correlation coefficient (R) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch 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 delivery.
This guide ensures accurate and reliable detection of Guinea Pig IgE levels. Proper handling and following the protocol will maximize experimental success.
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