**Chicken Whitefly Antibody (PD) ELISA Test Kit – User Manual**
This ELISA test kit is designed for the quantitative detection of Chicken Whitefly Antibody (PD) in biological samples. The method employed is a double-antibody one-step sandwich ELISA. Microtiter plates are pre-coated with specific capture antibodies against Chicken Whitefly Antibody (PD). After adding the sample, standard, and HRP-labeled detection antibody, the plate is incubated, washed thoroughly, and then developed using TMB substrate. The reaction is stopped with an acidic solution, resulting in a color change from blue to yellow. The intensity of the color is directly proportional to the concentration of the target antibody in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the concentration is calculated based on a standard curve.
**Sample Collection and Preparation**
1. **Serum**: Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Use EDTA, citrate, or heparin as anticoagulant. Centrifuge at 3000 rpm for 30 minutes to collect plasma.
3. **Cell Culture Supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris.
4. **Tissue Homogenate**: Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to obtain supernatant.
5. **Storage**: Store samples at -20°C after aliquoting. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
**Required Equipment**
- Microplate reader (450 nm)
- Precision pipettes: 0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL
- Incubator at 37°C
**Important Notes**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the washing buffer crystallizes after removal from the fridge, warm it gently in a water bath before use.
- Unused strips should be resealed in the ziplock bag and stored at low temperature.
- No need to dilute the sample; add 10 µL directly.
- Follow the incubation time, volume, and sequence strictly.
- Shake all reagents well before use.
**Kit Components**
| Component | 96-well | 48-well | Notes |
|----------|---------|---------|-------|
| Microwells | 12×8 | 12×4 | — |
| Standards (200 pg/mL) | 0.5 mL | 0.5 mL | — |
| Standard Dilutions | 6 mL | 3 mL | As per instructions |
| Sample Diluent | 6 mL | 3 mL | As per instructions |
| Detection Antibody-HRP | 6 mL | 3 mL | — |
| 20× Wash Buffer | 20 mL | 20 mL | Dilute with distilled water (1:20) |
| Substrate A | 6 mL | 3 mL | — |
| Substrate B | 6 mL | 3 mL | — |
| Stop Solution | 6 mL | 3 mL | — |
| Seal Film | 2 sheets | 2 sheets | — |
| Instructions | 1 part | 1 part | — |
| Ziplock Bag | 1 piece | — | — |
**Reagent Preparation**
Dilute the 20× wash buffer with distilled water at a ratio of 1:20.
**Washing Procedure**
- Manual: Wash 5 times by filling each well with buffer, letting stand for 1 minute, and discarding. Dry on absorbent paper.
- Automated: Use 350 µL per well, soak for 1 minute, and repeat 5 times.
**Procedure**
1. Remove the required wells from the foil pouch after 20-minute equilibration. Re-seal unused wells.
2. Set up standard, sample, and blank wells. Add 50 µL of standards to the standard wells.
3. Add 10 µL of sample plus 40 µL of diluent to the sample wells.
4. Add 50 µL of HRP-labeled detection antibody to all wells except the blank. Seal and incubate at 37°C for 60 minutes.
5. Wash 5 times with wash buffer.
6. Add 50 µL of TMB A and B, incubate in the dark for 15 minutes.
7. Add 50 µL of stop solution, and measure OD450 within 15 minutes.
**Data Analysis**
Plot standard concentrations vs. OD values in Excel. Generate a linear regression curve and calculate sample concentrations accordingly.
**Kit Performance**
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 pg/mL
- Specificity: No cross-reactivity with other analogs
- Reproducibility: CV <15% between plates
- Storage: 2–8°C, protected from light and moisture
- Shelf Life: 6 months
- Detection Range: 6.25–200 pg/mL
**Disclaimer**
This kit is for research use only. Not suitable for clinical trials or animal testing. The company assumes no responsibility for misuse. Always follow the instructions carefully. Do not mix reagents from different batches. Any deviation may lead to inaccurate results.
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