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Human ALB ELISA Kit
**Human Albumin (ALB) ELISA Kit – For the quantitative in vitro determination of Human microalbuminuria concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
Before using this product, please read this entire package insert carefully.
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### **INTENDED USE AND TEST PRINCIPLE**
This Human Albumin ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic purposes. The assay is based on the principle of a competitive enzyme-linked immunosorbent assay (ELISA). The color change from blue to yellow occurs upon addition of the Stop Solution, and the intensity of the color correlates with the concentration of ALB in the sample. A standard curve is generated by measuring optical density (OD) at 450 nm, and the ALB concentration in unknown samples is determined by comparing their OD values to the standard curve.
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### **SAMPLE COLLECTION AND STORAGE**
- **Serum:** Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. 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. Assay immediately or aliquot and store at -20°C. Ensure proper centrifugation and avoid hemolysis or granulation.
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### **MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
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### **REAGENTS PROVIDED**
All reagents should be stored at 2–8°C. Check the expiration date on the label.
| Reagent Name | 96 Determinations | 48 Determinations |
|--------------|-------------------|-------------------|
| Microtiter Strip Plate | 12×8 strips | 12×4 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: 400, 200, 100, 50, 25, 12.5 μg/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. Only use manufacturer-supplied reagents.
2. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not thaw using water baths.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for dilution.
5. Keep unused microtiter plate strips in the sealed bag with desiccant until needed.
6. Use fresh pipette tips for each transfer to prevent cross-contamination.
7. Wear disposable gloves during the procedure. Handle all biological samples with caution, as they may contain infectious agents.
8. Dispose of all samples and waste following local biosafety regulations.
9. Liquid waste must be treated with sodium hypochlorite (final concentration 1.0%) for at least 30 minutes before disposal.
10. Substrate solution is sensitive to contamination. If it appears bluish, do not use.
11. Chromogen B contains 20% acetone; keep away from heat or open flame.
12. Remove all reagents from the refrigerator and allow them to reach room temperature (20–25°C) before use.
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### **REAGENT PREPARATION AND STORAGE**
**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 1 month.
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### **ASSAY PROCEDURE**
1. Prepare all reagents before starting the assay.
2. Add 100 μL of standards, samples, and blank to the microtiter plate. Cover with an adhesive strip and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times manually or automatically.
- **Manual Washing:** Aspirate contents, fill each well with 1X Wash Solution, then aspirate again. Repeat 4 times. Invert and blot dry.
- **Automated Washing:** Aspirate, wash 4 times with 350 μL/well, and blot dry.
4. 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.
5. Add 50 μL of Stop Solution to each well. The color should turn from blue to yellow. If green or uneven, gently tap the plate.
6. Read OD at 450 nm within 15 minutes using a microplate reader.
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### **CALCULATION OF RESULTS**
1. Plot the average OD (450 nm) of each standard against concentration on a graph.
2. Calculate the mean OD for each standard and sample. Subtract the blank value before interpretation.
3. Locate the OD value on the Y-axis and draw a horizontal line to intersect the standard curve. Draw a vertical line to the X-axis to determine the ALB concentration.
4. Each user should generate their own standard curve due to potential variability in technique or reagent age.
5. Intra-assay and inter-assay CVs are less than 15%.
6. Assay range: 12.5–400 μg/mL.
7. Sensitivity: <10 μg/mL.
8. No significant cross-reactivity or interference observed.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
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**Always follow safety protocols and handle all materials with care.**
**For research use only. Not for diagnostic procedures.**