Preparation of human monocyte chemoattractant protein-4 ELISA test kit

This reagent is for research use only: serum or plasma Shanghai Fusheng Industrial Co., Ltd. specializes in supplying dextran gel HL-20, interleukin elisa kit, tumor necrosis factor elisa kit, human interleukin antigen B27, cell growth factor reagent Box, providing free proxy testing services to ensure the objective authenticity of the experimental results. Our company guarantees 100% of the quality of the kit. If there is a quality problem, we unconditionally return the test principle: The MCP-4 kit is a solid-phase sandwich method enzyme-linked immunosorbent assay (ELISA). A standard product with known MCP-4 concentration 3. Samples of unknown concentration are added to microwell microplate for detection. First, MCP-4 and biotin-labeled antibody 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 MCP-4 in the sample. 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. 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. It is recommended to make multiple holes for each standard and blank hole. 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 60ul of affinity 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. The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve. 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 MCP-4 standard concentration as the abscissa (X ), The corresponding curve is made, and the MCP-4 content of the sample can be converted to the corresponding concentration from the standard curve according to its OD value. 3. Detection range: 0-1600 pg / ml 4. Sensitivity: 1.0 pg / ml IL6Rβ / CD130 / gp130 Interleukin 6 receptor β antibody 0.2 ml IL-8 / CXCL8 (Interleukin-8) Interleukin 8 antibody 0.1 ml Insulin Protein / APC Fluorescein APC labeled porcine insulin protein 0.1ml human Fibrinogen / APC Fluorescein APC labeled human fibrinogen 0.1ml IL-8 / CXCL8 (Interleukin-8) Interleukin 8 antibody 0.2ml 8 antibody (large and mouse) 0.1ml IL-8 / CXCL8 (Interleukin-8) to rat, mouse interleukin 8 antibody (large and mouse) 0.2ml IL-7Ra / CD127 (Interleukin-7 Receptor-alpha) interleukin- 7 Receptor a antibody 0.2ml Mitofilin / IMMT Mitofilin inner membrane protein antibody 0.2ml ILK-1 (Integrin-linked protein kinase 1) integrin-linked kinase-1 antibody 0.2ml OVA / APC fluorescein APC labeled chicken ovalbumin 0.1ml HSA (Human serum albumin ) / APC fluorescein APC labeled human serum albumin 0.1ml AACT / APC fluorescein APC labeled chymotrypsin 0.1ml Goat anti-human Fibrinogen / APC fluorescein APC labeled sheep anti-human fibrinogen 0.1ml ILTV (Infectious Laryngotracheitis Virus) anti-chicken infection Sex throat Stomatitis virus antibody 0.2mlING1 / p33 (inhibitor of growth gene 1) an antibody growth inhibitory factor gene 0.2ml

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