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Human calprotectin (CAL) ELISA detection kit instruction manual
Human Calprotectin (CAL) ELISA Detection Kit The following information is provided by Kamaishu (Shanghai Hi) Biotechnology. The detection principle kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with human calprotectin (CAL) capture antigen, add specimens, standards, and HRP-labeled detection antibodies in sequence, incubate and wash thoroughly. The color is developed with the substrate TMB, which is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The color depth is positively correlated with human calprotectin (CAL) in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration. Sample collection, processing and storage methods 1. Serum: Use a test tube that does not contain pyrogens and endotoxins. Avoid any cell stimulation during the operation. After collecting blood, centrifuge at 3000 rpm for 10 minutes to quickly separate serum and red blood cells carefully. 2. Plasma: EDTA, citrate or heparin anticoagulation. Take the supernatant by centrifugation at 3000 rpm for 30 minutes. 3. Cell supernatant: centrifuge at 3000 rpm for 10 minutes to remove particles and polymers. 4. Tissue homogenate: Add tissue to the right amount of saline and mash. Take the supernatant by centrifugation at 3000 rpm for 10 minutes. 5. Preservation: If the sample is not tested in time after collection, please aliquot it in one dose and freeze it at -20 ℃ to avoid repeated freezing and thawing. Thaw at room temperature and ensure that the sample is thawed evenly and fully. Self-supplied items 1. Microplate reader (450nm) 2. High-precision sampler and pipette tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL Equilibrate at room temperature for 20 minutes at 2-8 ° C before use. The concentrated washing liquid taken out of the refrigerator will have crystals, which is a normal phenomenon. The water bath is heated to completely dissolve the crystals before use. 2. The slats not used in the experiment should be immediately returned to the ziplock bag, sealed (dry at low temperature) and stored. 3. The sample after pretreatment does not need to be diluted, just take 10μL to add sample. 4. Perform the incubation operation strictly in accordance with the time, amount of liquid and sequence indicated in the manual. 5. Shake all liquid components thoroughly before use. Kit composition name 96-well configuration 48-well configuration Remarks Microwell microplate 12-well × 8 strips 12-well × 4 strips No standard (200 ng / mL) 0.5mL 0.5mL Dilute according to the instructions Standard dilution 6mL 3mL Dilute the sample according to the instructions. Dilute the sample 6mL 3mL. Dilute according to the instructions. Detection antibody-HRP 10mL 5mL No 20 × Wash buffer 20mL 20mL Dilute according to the instructions Substrate A 6mL 3mL No substrate B 6mL 3mL No stop solution 6mL 3mL No sealing membrane 2 sheets, 2 sheets without instructions, 1 copy, 1 copy, 1 ziplock bag, 1 set, 1 set Dilution of washing buffer: Distilled water is diluted 1:20, ie 1 part of 20 × washing buffer plus 19 parts of distilled water. Plate washing method 1. Manual plate washing: throw away the liquid in the hole, fill each hole with the washing liquid, let the liquid in the hole drain after standing for 1 min, pat dry on absorbent paper, and wash the plate 5 times in this way. 2. Automatic plate washing machine: Inject 350μL of washing liquid into each well, soak for 1min, and wash the plate 5 times. Operation steps 1. Take out the required slats from the aluminum foil bag after equilibrating at room temperature for 20min, and the remaining slats shall be sealed in a ziplock bag and put back at 4 ℃. 2. Set up standard wells, sample wells, and blank wells, and add nothing to the blank wells; add 50 μL of standard products with different concentrations to the standard wells; 3. Add 10 μL of the sample to be tested to the sample well, and then add the sample diluent 40μL; 4. Then add 50μL of detection antibody labeled with horseradish peroxidase (HRP) to the standard wells and sample wells (the blank wells are not added). Seal the reaction wells with a sealing plate, 37 ° C water bath or constant temperature The box was incubated for 60 minutes. 5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1min, shake off the washing solution, pat dry on the absorbent paper, and repeat washing the plate 5 times (you can also wash the plate with a washing machine). 6. Add 50 μL of substrate A and B to all wells, and incubate at 37 ° C in the dark for 15 minutes. 7. Add 50μL of stop solution to all wells, and measure the OD value of each well at 450nm wavelength within 15min. The result is judged to draw the standard curve: In the Excel worksheet, the standard product concentration is used as the abscissa, and the corresponding OD value is used as the ordinate, the standard product linear regression curve is drawn, and the concentration value of each sample is calculated according to the curve equation. Kit performance 1. Accuracy: The linear regression between the standard product and the expected concentration R value is greater than or equal to 0.9900. 2. Sensitivity: The minimum detection concentration is less than 1.0 ng / mL. 3. Specificity: Does not cross-react with other soluble structural analogs. 4. Repeatability: The coefficients of variation within and between panels are less than 15%. 5. Storage: 2-8 ℃, protected from light and moisture. 6. Validity period: 6 months 7. Detection range: 6.25 ng / ml -200ng / ml Disclaimer 1. The kit is for research use only and should not be used in clinical or human experiments, otherwise all consequences arising from the experimenter The company is not responsible for it. 2. Strictly follow the instructions, different batch numbers are not interchangeable, the experimenter violates the instructions, and the consequences will be borne by the experimenter.