Human supersensitive C-reactive protein (hs-CRP) ELISA kit

Instructions for use of the human high-sensitivity C-reactive protein (hs-CRP) ELISA kit One-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with hypersensitive C-reactive protein (hs-CRP) antibody, 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 the hypersensitive C-reactive protein (hs-CRP) 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-supplied items: 1.37 ° C incubator 2. Microplate reader (450nm) 3. High-precision sampler and pipette tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL Operating notes: 1. 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. The 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 High Sensitive C-Reactive Protein (hs-CRP) ELISA Kit: Name 96-well configuration 48-well configuration Remarks Microwell enzyme plate 12 well × 8 strips 12 well × 4 strips without standard 0.3mL * 6 tubes 0.3mL * 6 tubes without sample diluent 6mL 3mL without detection antibody-HRP 10mL 5mL without 20 × washing buffer 25mL 15mL Dilute according to the instructions Substrate A 6mL 3mL without substrate B 6mL 3mL without stop solution 6mL 3mL without sealing plate 2 sheets 2 Zhang Wu Instructions 1 copy 1 copy without ziplock bag 1 set 1 no Note: The concentration of the standard (S0-S5) is: 0, 1, 2, 4, 8, 16 mg / L reagent preparation: 20 × washing buffer Dilution of liquid: Distilled water is diluted 1:20, that is, 1 part of 20 × washing buffer plus 19 parts of distilled water. Plate washing method: 1. Automatic plate washing machine: Inject 350μL of washing solution into each well, soak for 1min, and wash the plate 5 times. 2. Manually wash the plate: throw away all 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 the absorbent paper, and wash the plate 5 times in this way. 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. Kit performance 1. Accuracy: The linear regression between the standard product and the expected concentration of the correlation coefficient R value is greater than or equal to 0.9900. 2. Sensitivity: the minimum detection concentration is less than 0.1 mg / L. 3. Specificity: does not cross-react with other soluble structural analogs. 4. Repeatability: The coefficients of variation within and between plates are less than 15%. 5. Storage: Store at 2-8 ℃, protected from light and moisture. 6. Validity: 6 months

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