Pepsin (Pepsin) ELISA kit instruction manual The human pepsin (Pepsin) ELISA kit is for research use only. Detection principle: the kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with pepsin (Pepsin) 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 pepsin in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated. Sample collection, processing and storage methods: 1. Serum: Use test tubes that do 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 the 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: The tissue is crushed by adding appropriate amount of normal saline. 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 3.37 ℃ constant temperature box operation notes 1. Reagent kit Store at 2-8 ° C and equilibrate at room temperature for 20 minutes 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 (dried at low temperature) and stored. 3. S0 standard with a concentration of 0 can be regarded as a negative control or blank; the sample has been diluted 5 times when operated according to the instructions, and the final result is multiplied by 5 to be the actual concentration of the sample. 4. Carry out 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. Composition of human pepsin ELISA kit: Name 96-well configuration 48-well configuration Remarks Microwell enzyme plate 12 wells × 8 strips 12 wells × 4 strips No standard 0.3mL * 6 tubes 0.3mL * 6 tubes No sample dilution Solution 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 1 without instructions 1 piece without ziplock bag 1 piece without 1 Note: The concentration of the standard (S0-S5) is: 0, 3, 6, 12, 24, 48 ng / mL reagent preparation: 20 × dilution of 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, leave the hole in the hole for 1min, shake off the liquid in the hole, pat dry on absorbent paper, and wash the plate 5 times in this way. 2. Automatic plate washing machine: Inject 350μL of washing solution into each well, soak for 1min, 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. The remaining slats are sealed with a ziplock bag and put back at 4 ℃. 2. Set up standard wells and sample wells, and add 50μL of standard products of different concentrations to the standard wells; 3. Add 10μL of the sample to be tested to the sample wells, and then add 40μL of the sample diluent; no blank wells. 4. In addition to the blank wells, add 100 μL of horseradish peroxidase (HRP) -labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing plate, and incubate in a 37 ° C water bath or incubator 60min. 5 Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1min, shake off the washing liquid, pat dry on 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 for 15 min in the dark. 7. Add 50μL of stop solution to each well, and measure the OD value of each well at 450nm within 15min. Judgment of results: Draw a 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, a standard product linear regression curve is drawn, and the concentration value of each sample is calculated according to the curve equation. Human Pepsin (Pepsin) ELISA kit performance: 1. Accuracy: the linear regression of the standard product and the expected concentration correlation coefficient R value, 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: 6 months
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