**Human Prolactin (PRL) ELISA Kit Instruction Manual**
**Kit Specifications:**
This Human Prolactin (PRL) ELISA Kit is available in 48-well or 96-well configurations. The standard dilution is 1.5 ml × 1 bottle, and the enzyme standard reagent is 3 ml × 1 bottle (for 48-well) or 6 ml × 1 bottle (for 96-well). This kit is for research use only. The concentration of the standard is plotted on the x-axis, and the OD value is on the y-axis. A standard curve should be drawn based on the sample data, and the corresponding concentration can be determined from the curve. Alternatively, a linear regression equation can be calculated using the standard concentrations and their corresponding OD values, and the sample’s OD value can be substituted into the equation to calculate the actual concentration, which should then be multiplied by the dilution factor.
**Kit Composition:**
- 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) / 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 (48) / 20 ml × 30 times (96)
**Principle of Operation:**
The kit uses a double-antibody sandwich method to detect human prolactin (PRL) E in the sample. The microplate is coated with purified anti-PRL E antibody, and after incubation with the sample, HRP-labeled anti-PRL E antibody binds to form an antibody-antigen-enzyme complex. After washing, TMB substrate is added, and the reaction is stopped with a stop solution. The color change is measured at 450 nm, and the concentration is determined from a standard curve.
**Purpose:**
This kit is designed to quantify anti-endometrial antibodies (EMAb) in human serum, plasma, urine, and other biological fluids.
**Storage Conditions & Expiration Date:**
- Storage: 2–8°C
- Shelf Life: 6 months
**Sample Preparation Requirements:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix for 10–20 minutes, then centrifuge.
- **Urine:** Collect in a sterile tube, centrifuge, and collect supernatant.
- **Cell culture supernatant:** Centrifuge after collection, or lyse cells by repeated freezing and thawing.
- **Tissue samples:** Homogenize in PBS, centrifuge, and collect supernatant.
**Important Notes:**
- Samples should be processed as soon as possible after collection.
- Avoid repeated freezing and thawing.
- NaN3-containing samples cannot be tested due to inhibition of HRP activity.
**Operating Steps:**
1. Prepare standard dilutions and load them into the plate.
2. Add sample diluent and test samples to respective wells.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing buffer.
5. Add enzyme-labeled reagent and incubate again.
6. Add TMB substrate, incubate for 15 minutes, then add stop solution.
7. Measure OD at 450 nm within 15 minutes.
**Precautions:**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination; use a new sealing film for each experiment.
- Handle all reagents carefully and follow instructions strictly.
- All waste should be treated as biohazardous material.
- Do not mix components from different batches.
**Performance:**
- Linear regression correlation coefficient R ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery within the agreed time frame.
This manual provides detailed guidance for accurate and reliable detection of human PRL E levels in various biological samples. Always refer to the latest version of the manual for updates.
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