**Human β2-GP1 IgA ELISA Kit – For the Quantitative In Vitro Determination of Human β2-Glycoprotein 1 IgA in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This kit is designed for the quantitative measurement of Human β2-GP1 IgA in various biological samples using an enzyme-linked immunosorbent assay (ELISA) method. The principle of the test involves a competitive binding reaction between the sample β2-GP1 IgA and a pre-coated antibody on the microtiter plate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The intensity of the color at 450 nm is directly proportional to the concentration of β2-GP1 IgA in the sample.
To ensure accurate results, a standard curve is generated using a series of calibration standards with known concentrations of β2-GP1 IgA. By comparing the optical density (OD) values of the unknown samples to this standard curve, the exact concentration of β2-GP1 IgA can be determined.
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### **Sample Collection and Storage**
- **Serum:** Collect using a serum separator tube. Allow the blood to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Remove the serum and either assay immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid repeated freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs during processing.
*Note: Proper centrifugation is essential. Hemolyzed or particulate-laden samples may interfere with the accuracy of the assay.*
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### **Materials Required but Not Supplied**
- Incubator set at 37°C
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
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### **Reagents Provided (Stored at 2–8°C)**
| Reagent Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| MicroELISA Strip Plate | 12x8 strips | 12x4 strips |
| Standard (6 vials) | 0.5 ml/vial | 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 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
*Standard concentrations: 16, 8, 4, 2, 1, 0.5 ng/ml.*
*If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.*
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### **Precautions**
1. Do not mix reagents from different kit lots. All components are calibrated for optimal performance.
2. Allow all reagents and samples to reach room temperature (20–25°C) before use. Avoid using water baths for thawing.
3. Do not use reagents beyond their expiration date.
4. Keep microtiter plates in their sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant.
5. Always use fresh pipette tips for each step to avoid cross-contamination.
6. Handle plasma and other potentially infectious materials with care. Wear gloves and follow biosafety protocols.
7. All waste must be inactivated for at least 30 minutes before disposal.
8. Substrate solutions contain acetone and should be kept away from heat and flame.
9. Ensure all reagents are at room temperature before starting the assay.
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### **Reagent 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.
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### **Assay Procedure**
1. Prepare all reagents before beginning. Add 50 µL of standards or samples in duplicate to the microtiter plate. Include a blank well without any addition.
2. Add 100 µL of HRP-conjugate reagent to all wells except the blank. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times using either manual or automated washing methods.
4. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Incubate for 15 minutes at 37°C in the dark.
5. Add 50 µL of Stop Solution to each well. Gently mix if color change is uneven.
6. Measure OD at 450 nm using a microplate reader.
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### **Data Interpretation**
- Generate a standard curve by plotting average OD values (450 nm) against standard concentrations.
- Subtract blank OD values from all measurements before calculating sample concentrations.
- Locate the sample OD on the Y-axis and draw a line to intersect the standard curve. Read the corresponding concentration on the X-axis.
- Each user should generate their own standard curve due to possible variations in technique or environmental conditions.
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### **Performance Characteristics**
- **Sensitivity:** <0.1 ng/mL
- **Intra-assay CV(%):** <15%
- **Inter-assay CV(%):** <15%
- **Cross-reactivity:** No significant cross-reaction with other proteins was observed.
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### **Storage**
- Store unopened kits at 2–8°C.
- For frequent use, store at 2–8°C.
- For long-term storage, keep at -20°C.
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This kit is intended for research purposes only and should not be used for diagnostic or clinical applications. Always follow proper laboratory safety guidelines when handling biological samples and reagents.
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