Human anti-proliferating cell nuclear antigen antibody (PCNA) ELISA detection kit This reagent is for research use only Specimens: serum or plasma Test principle: PCNA kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Known PCNA Concentration standards and samples with unknown concentrations are added to microwell microplates for detection. First, PCNA and biotin-labeled antibodies are incubated simultaneously. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of PCNA in the sample. Kit contents and preparation kit components (stored at 2-8 ° C) 96-well configuration 48-well configuration 96/48 servings of enzyme labeling plate 1 plate (96T) half plate (48T) ready-to-use plastic membrane plate cover 1 half-block ready-to-use standard product: 400nmol / L 1 bottle (0.6ml) 1 bottle (0.3ml) Follow the instructions to carry out the dilute empty anti-proliferating cell nuclear antigen antibody control 1 bottle (1.0ml) 1 bottle (0.5ml) ie 1 bottle (5ml) of the standard dilution buffer 1 bottle (2.5ml) 1 bottle of biotin-labeled anti-PCNA antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use streptavidin-HRP 1 Bottle (10ml) 1 bottle (5.0ml) ready-to-use wash buffer 1 bottle (20ml) 1 bottle (10ml) Dilute according to the instructions Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use termination solution 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use specimen dilution 1 bottle (12ml) 1 bottle (6.0ml) ready-to-use Bring your own material distilled water. Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul. Oscillator and magnetic stirrer etc. Safety Avoid direct contact with the stop solution and substrates A and B. Once exposed to these liquids, rinse with water as soon as possible. Do not eat, drink, smoke or use cosmetics during the experiment. Do not use your mouth to absorb any ingredients in the kit. Handling precautions Reagents should be stored according to label instructions, and returned to room temperature before use. The sparse standards should be discarded and cannot be stored. The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty. Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and substrates A and B. Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use. When washing the enzyme-labeled plate, it should be fully patted dry. Do not put the absorbent paper directly into the enzyme-labeled reaction well to absorb water. Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed. The order of adding reagents should be the same to ensure that all wells are incubated for the same time. Perform the incubation operation according to the time, the amount and order of the liquid indicated in the instructions. Sample collection, processing and storage methods Serum-avoid any cell stimulation during the operation. Use test tubes free of pyrogens and endotoxins. After collecting blood, centrifuge at 1000 × g for 10 minutes to separate the serum and red blood cells quickly and carefully. Plasma ----- EDTA, citrate, heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles. The cell supernatant was centrifuged at 1000 × g for 10 minutes to remove particles and polymers. Tissue homogenate ----- Mash the tissue with appropriate amount of saline. Centrifuge at 1000 × g for 10 minutes, and take the supernatant for storage. If the sample is not used immediately, it should be divided into small parts and stored at -70 ℃ to avoid repeated freezing. If possible, do not use hemolysis or hyperlipidemia. If there are a large number of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 ° C or higher. Thaw at room temperature and ensure that the sample is thawed evenly and adequately. Reagent preparation standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows: 400 nmol / L (standard No. 6) The original concentration is added directly to 50ul without dilution. 200 nmol / L (No. 5 standard) 100ul of original standard is added to 100ul of standard dilution 100 nmol / L (No. 4 standard) 100ul of standard 5 is added to 100ul of standard dilution 50 nmol / L (Standard No. 3) 100ul No. 4 standard added 100ul standard dilution 25 nmol / L (No. 2 standard) 100ul No. 3 standard added 100ul standard dilution 12.5 nmol / L (No. 1 standard) Product) 100ul of No. 2 standard is added to 100ul of standard dilution solution 0 nmol / L (empty anti-proliferating cell nuclear antigen antibody control). The original concentration is directly added to 50ul without dilution. Dilution of washing buffer (50 ×): 50-fold dilution with distilled water. Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition. The number of slats required is determined by the number of samples to be tested plus the number of standard products. Each standard and empty anti-proliferating cell nuclear antigen antibody well are recommended to be re-welled. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible. The specimen was diluted 1: 1 with the specimen dilution solution and then added 50ul to the reaction well. Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37 ° C for 1 hour. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once. Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once. Add 50ul of substrate A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid light. Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately. The OD value of each well was measured at a wavelength of 450 nm. Recommended experimental standard concentration (nmol / L) A 400 400 sample sample sample sample sample sample sample sample sample sample B 200 200 sample sample sample sample sample sample sample sample sample sample C 100 100 sample sample sample sample sample sample sample Sample sample D 50 50 sample sample sample sample sample sample sample sample sample sample E 25 25 sample sample sample sample sample sample sample sample sample sample F 12.5 12.5 sample sample sample sample sample sample sample sample sample sample sample G 0 0 sample sample sample sample sample sample Sample sample sample sample sample H sample sample sample sample sample sample sample sample sample sample sample sample sample No. 6 or more results are non-linear, according to this standard curve can not get accurate results. Kit performance 1. Sensitivity: The minimum detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient between the linear regression of the sample and the expected concentration is 0.990. 2. Specificity: Does not react with other cytokines. 3. Repeatability: The coefficients of variation within and between plates are less than 10%. Judgment and analysis of results 1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450 nm. 2. Take the absorbance OD value as the ordinate (Y), and the corresponding PCNA standard concentration as the abscissa (X). To make the corresponding curve, the PCNA content of the sample can be converted to the corresponding concentration from the standard curve according to its OD value. 3. Detection value range: 0-400nmol / L4, sensitivity: 1.0 nmol / L
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