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Instruction Manual of Chicken Corticosterone (CORT) ELISA Test Kit
Chicken Corticosterone (CORT) ELISA Detection Kit Operating Instructions Detection Principle The kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with chicken corticosterone (CORT) capture antibody, add the specimen, standard, and HRP-labeled detection antibody 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 the chicken corticosterone (CORT) 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-provided items 1. Microplate reader (450nm) 2. High-precision sampler and pipette tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL 3. Cautions for operation of 37 ° C incubator 1. Reagent box storage 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 standard dilution can be regarded as a negative control or blank; the sample after pretreatment does not need to be diluted, just take 10μL and add it. 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 wells × 8 strips 12 wells × 4 strips No standard (800pg / mL) 0.6mL 0.6mL Dilute according to the instructions Standard dilution 6mL 3mL No sample Diluent 6mL 3mL No detection antibody-HRP 10mL 5mL No 20 × Wash buffer 25mL 15mL Dilute according to the instructions Substrate A 6mL 3mL No substrate B 6mL 3mL No stop solution 6mL 3mL No sealing film 2 sheets 2 sheets without instructions 1 One part, one part without ziplock bag, one part, no one. Note: Standards are diluted with standard dilutions in order as follows: 800, 400, 200, 100, 50, 25pg / mL. Preparation of reagents Dilution of 20 × washing buffer: distilled water Dilute 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 and sample wells, add 50μL of standard products of different concentrations to the standard wells; 3. Add 10μL of the test samples to the test wells, and then add 40μL of the sample diluent; 4. Then the standard wells and samples 100 μL of detection antibody labeled with horseradish peroxidase (HRP) was added to each well, and the reaction well was sealed with a sealing plate membrane, and incubated for 60 min in a 37 ° C water bath or incubator. 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 each well, and incubate at 37 ° C in the dark for 15 minutes. 7. Add 50μL of stop solution to each well, 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 vertical coordinate. 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 1.0pg / 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%.