Mouse immunoglobulin G1 (IgG1) ELISA test kit instruction manual

**Mouse Immunoglobulin G1 (IgG1) ELISA Kit – Instructions for Use** This kit is intended for research use only and is designed to quantify Mouse Immunoglobulin G1 (IgG1) in serum, plasma, and related biological samples. The assay is based on the double-antibody sandwich ELISA method. A microplate pre-coated with purified anti-IgG1 antibodies serves as the solid phase. After incubation with the sample, IgG1 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured IgG1, forming an immune complex. Following washing steps, TMB substrate is added, producing a blue color that turns yellow in the presence of acid. The intensity of the color is directly proportional to the concentration of IgG1 in the sample. Absorbance is measured at 450 nm using a microplate reader, and the IgG1 concentration is determined from a standard curve. **Kit Components:** - Microtiter Plate (48 or 96 wells) - Standard (360 μg/mL) - Standard Diluent - Enzyme Conjugate - Sample Diluent - TMB Substrate (A and B) - Wash Buffer (20x concentrated) - Sealing Film - Stop Solution **Storage Conditions:** - Store all components at 2–8°C. - Shelf life: 6 months from the date of manufacture. **Sample Preparation:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. - **Plasma:** Use anticoagulants like EDTA, citrate, or heparin. Centrifuge after mixing. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. - **Cell Culture Supernatant:** Centrifuge to remove debris. For intracellular components, lyse cells by freeze-thawing before centrifugation. - **Tissue Homogenate:** Homogenize tissue in PBS, centrifuge, and collect supernatant. **Important Notes:** - Avoid repeated freezing and thawing of samples. - Do not use samples containing NaN3, as it inhibits HRP activity. - Always prepare a standard curve and perform duplicate measurements. - Ensure proper dilution if the sample OD exceeds the highest standard. - Keep all reagents away from light and avoid cross-contamination. **Procedure Summary:** 1. Prepare standards by serial dilution. 2. Add samples and standards to the plate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate 5 times with wash buffer. 5. Add enzyme-conjugated antibody and incubate again. 6. Add TMB substrate and incubate for 15 minutes. 7. Stop the reaction with stop solution. 8. Measure absorbance at 450 nm. **Data Analysis:** Plot the standard curve using IgG1 concentrations vs. OD values. Calculate sample concentrations using linear regression or direct comparison. Multiply by the dilution factor to obtain the actual concentration. **Performance:** - Intra-assay CV <9%, Inter-assay CV <11% - Linear range: 15–300 μg/mL - Correlation coefficient (R²) ≥ 0.95 **Safety & Disposal:** Treat all waste materials as biohazardous. Follow local regulations for disposal. **Notes:** - Allow the kit to equilibrate to room temperature before use. - Use separate pipettes for each step to prevent contamination. - Maintain accurate timing during all steps, especially during incubation and color development. This kit provides a reliable and sensitive method for quantifying mouse IgG1 in various biological matrices. Always follow the instructions carefully to ensure accurate results.

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