**Human HLA-DPA1 ELISA Kit – For the quantitative in vitro determination of Human HLA-DPA1 concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
**INTENDED USE AND TEST PRINCIPLE**
This HLA-DPA1 ELISA kit is designed for research purposes only and is not intended for diagnostic or clinical use. The assay is based on a sandwich immunoassay principle, where the HLA-DPA1 antigen in the sample binds to specific antibodies coated on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody then binds to the captured antigen, forming a complex. After adding the chromogen solution, a color change occurs, which is stopped by the addition of a stop solution. The optical density (OD) at 450 nm is measured using a spectrophotometer, and the concentration of HLA-DPA1 is determined by comparing the sample OD values to a standard curve.
**STORAGE AND PREPARATION**
- Store all samples at -20°C. Avoid repeated freeze-thaw cycles.
- Centrifuge cell culture supernatants, tissue homogenates, and other biological fluids to remove particulates before use.
- Ensure no hemolysis or granules are present in the samples.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label before use.
- MicroELISA Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations)
- Standard (6 vials): 0.5 ml/vial
- Sample Diluent: 6.0 ml / 3.0 ml
- HRP-Conjugate Reagent: 10.0 ml / 5.0 ml
- 20X Wash Solution: 25 ml / 15 ml
- Chromogen Solution A: 6.0 ml / 3.0 ml
- Chromogen Solution B: 6.0 ml / 3.0 ml
- Stop Solution: 6.0 ml / 3.0 ml
- Closure Plate Membrane: 2 units
- User Manual: 1 unit
- Sealed Bags: 1 unit
**STANDARD CONCENTRATIONS**
Standard concentrations: 800, 400, 200, 100, 50, 25 pg/ml. If sample values exceed the highest standard, dilute the samples with sample diluent. Always use the provided reagents and match samples, standards, and microtiter plates for optimal performance.
**PRECAUTIONS AND SAFETY**
- Do not thaw samples or reagents using water baths.
- Do not use any kit components past their expiration date.
- Use only deionized or distilled water.
- Keep microtiter plates in sealed bags until needed. Store unused strips at 2–8°C with desiccant.
- Use fresh disposable pipette tips to prevent cross-contamination.
- Wear disposable gloves during the procedure. All biological materials should be treated as potentially infectious.
- Dispose of all samples and waste properly. Liquid waste should be treated with 1.0% sodium hypochlorite for at least 30 minutes before disposal.
**REAGENT PREPARATION**
- Wash Solution (1X): Dilute 1 volume of 20X wash solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**ASSAY PROCEDURE**
1. Prepare all reagents before starting.
2. Add 50 µl of standard or sample to each well (duplicate). Blank well contains no sample.
3. Add 100 µl of HRP-conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or using an automated washer.
- Manual washing: Fill each well with 1X wash solution, aspirate, repeat 4 times.
- Automated washing: Aspirate and wash 4 times with 1X wash buffer. Adjust brush and fill volume to 350 µl/well.
5. Add 50 µl of Chromogen A and 50 µl of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µl of Stop Solution to each well. The color changes from blue to yellow. If green or uneven color appears, gently tap the plate.
7. Measure OD at 450 nm. Plot the average OD of each standard against its concentration to create a standard curve.
**RESULT INTERPRETATION**
- Calculate the mean OD for each standard and sample. Subtract the blank OD value from all readings.
- Use the standard curve to determine the HLA-DPA1 concentration in unknown samples.
- Intra-assay and inter-assay CV% are less than 15%.
- Assay range: 25 pg/ml to 800 pg/ml.
- Sensitivity: <1.0 pg/ml. No cross-reactivity or interference observed.
**STORAGE**
- Store at 2–8°C for frequent use; for long-term storage, keep at -20°C for up to 6 months.
**NOTES**
- Always read the entire protocol before starting the experiment.
- Follow good laboratory practices to ensure accurate and reproducible results.
- This kit is not approved for clinical diagnostics.
**Please read this instruction carefully before use.**
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