**Mouse Tyrosine Hydroxylase (TH) ELISA Kit – Instructions for Use** This kit is intended for research purposes only and is designed to quantitatively measure tyrosine hydroxylase (TH) levels in mouse serum, plasma, and other biological fluids. The assay is based on the sandwich ELISA method, using two specific antibodies to capture and detect TH in the sample. **Principle of Operation:** The microtiter plate is pre-coated with a purified monoclonal antibody against mouse TH. After adding the sample, TH binds to the immobilized antibody. A second HRP-conjugated antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, TMB substrate is introduced, and the reaction is stopped with an acidic solution. The color change from blue to yellow is directly proportional to the TH concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the TH concentration is determined by comparing the sample OD value to a standard curve. **Kit Components (48-well and 96-well configurations):** - Coated Microplate: 1×48 or 1×96 wells - Standard: 0.5 mL × 1 bottle (900 pg/mL) - Standard Diluent: 1.5 mL × 1 bottle - Enzyme Label Reagent: 3 mL × 1 bottle - Sample Diluent: 3 mL × 1 bottle - TMB Substrate A & B: 3 mL × 1 bottle each - Wash Buffer (20×): 20 mL × 20 times or 20 mL × 30 times - Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well) - Sealed Bag: 1 per kit **Storage Conditions:** All components should be stored at 2–8°C. The kit is valid for 6 months when stored properly. **Sample Preparation Guidelines:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. - **Plasma:** Use EDTA, citrate, or heparin as anticoagulant. Mix and centrifuge similarly. - **Urine:** Centrifuge after collection to remove debris. - **Cell Culture Supernatant:** Centrifuge to remove cells; for intracellular components, lyse cells via freeze-thaw cycles. - **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. **Procedure Summary:** 1. Prepare standards by serial dilution. 2. Add samples and blanks to the microplate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate 5 times with diluted wash buffer. 5. Add enzyme-labeled antibody and incubate again. 6. Add TMB substrate and incubate for 15 minutes. 7. Stop the reaction with stop solution. 8. Measure OD at 450 nm. 9. Calculate concentrations using a standard curve. **Important Notes:** - Allow all reagents to reach room temperature before use. - Avoid cross-contamination by using separate tips for each step. - Always include blank and duplicate wells for accuracy. - If sample OD exceeds the highest standard, dilute the sample before testing. - Handle all waste as biohazardous material. - Do not mix reagents from different batches. - Follow the manual strictly for accurate results. **Performance Specifications:** - Sensitivity: 30 pg/mL - Dynamic Range: 30–700 pg/mL - Correlation Coefficient (R²): ≥ 0.95 This ELISA kit provides a reliable and sensitive method for detecting TH levels in various biological matrices, making it ideal for research applications in neuroscience, pharmacology, and metabolic studies.

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