Human β-CGRP ELISA Kit

**Human β-CGRP ELISA Kit – For the quantitative in vitro determination of Human β-calcitonin gene-related peptide (β-CGRP) concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. This kit is intended for laboratory research use only and not for diagnostic procedures.** The ELISA kit operates on a colorimetric principle, where the reaction produces a yellow color, and the intensity is measured at 450 nm using a spectrophotometer. To quantify β-CGRP levels in samples, this kit includes a set of calibration standards. These standards are used alongside the samples to generate a standard curve, plotting optical density (OD) against β-CGRP concentration. The sample concentration is then determined by comparing its OD value to the standard curve. **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. Remove serum and 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 at 2000×g, 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, then assay immediately or store at -20°C. Ensure no hemolysis or granules are present. **Materials Required (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 **Reagents Provided (Stored at 2–8°C):** - Microtiter Strip Plate (12×8 strips / 12×4 strips) - Standard (6 vials, 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 units) - User Manual (1 copy) - Sealed Bags (1 each) **Note:** Standard concentrations are 200, 100, 50, 25, 12.5, and 6.25 pg/mL. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay. **Precautions:** - Do not mix reagents from different kit lots. Standards, conjugate, and plates are matched for optimal performance. - Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not thaw samples or reagents in water baths. - Do not use reagents past their expiration date. - Only use reagents provided by the manufacturer. - Keep unused strip wells in the sealed bag with desiccant. - Wear gloves during handling, especially when working with blood derivatives. - All samples should be treated as potentially infectious. - Waste must be inactivated for at least 30 minutes before disposal. - Chromogen solutions contain acetone; keep away from heat and flame. - Always allow reagents to warm to room temperature before use. **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 one month. **Assay Procedure:** 1. Prepare all reagents before starting the assay. Run standards and samples in duplicate. 2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition. 3. Add 100 µL of HRP-conjugate reagent to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C. 4. Wash microtiter plate 4 times manually or automatically. After washing, invert and blot dry. 5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protecting from light. 6. Add 50 µL of Stop Solution to each well. Mix gently if color change is uneven. 7. Measure OD at 450 nm. Generate a standard curve by plotting average OD values vs. concentration. **Interpretation:** - Calculate mean OD for each standard and sample. Subtract blank OD before interpretation. - Construct the standard curve and determine sample concentration by locating the OD on the Y-axis and drawing a line to the curve. - Intra-assay and inter-assay CVs are <15%. - Assay range: 6.25–200 pg/mL. - Sensitivity: <1.0 pg/mL. - Cross-reactivity: No significant cross-reactivity with other peptides. **Storage:** - Store at 2–8°C for frequent use. For long-term storage, keep at -20°C. Shelf life: 6 months at -20°C. **Important Note:** This kit is for research purposes only. Follow all safety protocols and handle all samples with care.

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