Mouse immunoglobulin G1 (IgG1) ELISA test kit instruction manual

**Mouse Immunoglobulin G1 (IgG1) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Manual** This kit is intended for research purposes only and is designed to quantify Mouse Immunoglobulin G1 (IgG1) in serum, plasma, and other biological fluids. The assay employs a double-antibody sandwich ELISA method. A microplate pre-coated with a specific monoclonal anti-IgG1 antibody is used as the solid phase. After incubation with the sample, the captured IgG1 binds to an HRP-conjugated secondary antibody, forming a complex. Following washing steps, TMB substrate is added, and the color develops in proportion to the amount of IgG1 present. The reaction is stopped with an acidic solution, turning the color from blue to yellow. The absorbance at 450 nm is measured using a microplate reader, and the concentration of IgG1 in the sample is determined by comparing it to a standard curve. **Kit Components:** - 48-well or 96-well plate - 2 sealing films (for 48-well) / 2 sealing films (for 96-well) - 1 enzyme-labeled plate - 1 vial of standard (360 μg/mL, 0.5 mL) - 1 vial of standard diluent (1.5 mL) - 1 vial of sample diluent (3 mL) - 1 vial of developer A (3 mL) - 1 vial of developer B (3 mL) - 1 vial of wash buffer (20x concentrated, 20 mL × 20 times or 30 times) - 1 vial of enzyme reagent (3 mL or 6 mL) **Storage Instructions:** All components should be stored at 2–8°C. The kit has a shelf life of 6 months when properly stored. **Sample Preparation:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Plasma:** Use anticoagulants such as EDTA, citrate, or heparin. Centrifuge after mixing for 10–20 minutes. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Discard any precipitate. - **Cell culture supernatant:** Centrifuge after collection. For intracellular components, lyse cells by repeated freeze-thaw cycles before centrifuging. - **Tissue homogenate:** Weigh the tissue, add PBS, homogenize, and centrifuge. Store the supernatant at 2–8°C or freeze for later use. **Precautions:** - Avoid samples containing NaN3, as they inhibit HRP activity. - Perform the assay as soon as possible after sample preparation. If not tested immediately, store at -20°C and avoid repeated freezing and thawing. - Always prepare a standard curve and run samples in duplicate for accuracy. **Procedure Summary:** 1. Prepare standard dilutions (240, 160, 80, 40, 20 μg/mL). 2. Add sample diluent and sample to the plate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate 5 times with diluted wash buffer. 5. Add HRP-conjugated antibody and incubate again. 6. Wash and add TMB substrate. 7. Stop the reaction with stop solution. 8. Measure OD at 450 nm within 15 minutes. **Data Analysis:** Plot the standard curve using concentration vs. OD values. Calculate the sample concentration using linear regression. Multiply by the dilution factor if applicable. **Notes:** - Allow the kit to reach room temperature before use. - Ensure accurate pipetting and proper mixing. - Do not reuse sealing films. - Keep substrates away from light. - Follow all instructions carefully for reliable results. **Performance:** - Correlation coefficient (R²) ≥ 0.95 - Intra-assay CV < 9%, Inter-assay CV < 11% - Detection range: 15–300 μg/mL **Safety:** Dispose of all waste materials as biohazardous. Avoid contact with skin and eyes. Wear appropriate personal protective equipment during handling.

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