**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 PROCEDURES.**
This ELISA kit is designed for the accurate measurement of Human HLA-DPA1 levels in various biological samples. The principle of this assay is based on a sandwich immunoassay format, where specific antibodies are used to capture HLA-DPA1 from the sample. A horseradish peroxidase (HRP)-conjugated antibody then binds to the captured antigen, forming a complex. The reaction is terminated with a stop solution, which changes the color from blue to yellow. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration of HLA-DPA1 in the sample is determined by comparing it to a standard curve.
**Intended Use:**
This HLA-DPA1 ELISA Kit is strictly for research purposes and not intended for diagnostic or therapeutic use. It is suitable for quantifying HLA-DPA1 in a variety of biological matrices, including but not limited to serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids.
**Test Principle:**
The kit employs a competitive or sandwich ELISA method depending on the specific antibody pair used. In this case, a sandwich approach is utilized, where the microtiter plate is pre-coated with anti-HLA-DPA1 antibodies. After incubation with the sample, HRP-conjugated secondary antibodies bind to the captured HLA-DPA1. A chromogenic substrate is added, producing a color change proportional to the amount of HLA-DPA1 present. The reaction is stopped, and the OD is measured to determine the concentration.
**Storage and Handling:**
All reagents should be stored at 2–8°C and used before the expiration date indicated on the label. Samples should be stored at -20°C, and repeated freeze-thaw cycles should be avoided. For optimal results, centrifuge all liquid samples to remove particulates before testing. Hemolysis, lipemia, or turbidity may interfere with the assay and should be avoided.
**Materials Required (Not Included):**
- 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 (Stored at 2–8°C):**
- MicroELISA Strip Plate (12×8 or 12×4 strips)
- Standard (6 vials, 0.5 ml/vial) – Concentrations: 800, 400, 200, 100, 50, 25 pg/ml
- Sample Diluent (6.0 ml or 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml or 5.0 ml)
- 20X Wash Solution (25 ml or 15 ml)
- Chromogen Solution A (6.0 ml or 3.0 ml)
- Chromogen Solution B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 bag)
**Preparation and Storage:**
- **Wash Solution (1X):** Dilute 1 volume of 20X Wash Solution with 19 volumes of distilled or deionized water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents and allow them to reach room temperature (20–25°C).
2. Add 50 µl of standards or samples to the appropriate wells. Include a blank well without any addition.
3. Add 100 µl of HRP-conjugate reagent to each well except the blank. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times using either manual or automated washing methods. Ensure complete removal of unbound conjugate.
5. Add 50 µl of Chromogen Solution A and 50 µl of Chromogen Solution B to each well. Incubate for 15 minutes at 37°C, protecting from light.
6. Add 50 µl of Stop Solution to each well. The color will shift from blue to yellow. If uneven color development occurs, gently tap the plate to ensure mixing.
7. Measure the OD at 450 nm using a microplate reader. Generate a standard curve by plotting average OD values against corresponding HLA-DPA1 concentrations.
**Interpretation of Results:**
- Calculate the mean OD for each standard and sample. Subtract the blank OD value from all readings.
- Plot the standard curve and interpolate the unknown sample concentrations.
- Intra-assay and inter-assay CV% are less than 15%.
- Sensitivity: <1.0 pg/ml. No cross-reactivity or interference observed.
**Storage Conditions:**
- Store unused strips at 2–8°C in sealed bags with desiccant.
- Long-term storage: -20°C for up to six months.
**Safety and Disposal:**
- All biological materials should be treated as potentially infectious. Follow good laboratory practices.
- Liquid waste must be inactivated with 1.0% sodium hypochlorite for at least 30 minutes before disposal.
- Substrate B contains 20% acetone; keep away from heat or flame.
**Note:** Always read the full user manual before starting the assay. This kit is not intended for diagnostic use.
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