Human pituitary adenylate cyclase activating peptide (PACAP) elisa kit instruction manual Elisa kit Specifications: 48-well configuration / 96-well configuration Standard dilution: 1.5ml × 1 bottle Enzyme reagent: 3 ml × 1 bottle (48 ) / 6 ml × 1 bottle (96) [Human pituitary adenylate cyclase activating peptide (PACAP) elisa kit] This reagent is for research use only: the concentration of the standard is the abscissa, and the OD value is the ordinate , Draw a standard curve on coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration and OD value of the standard, and then The OD value of is substituted into the equation to calculate the sample concentration, and then multiplied by the dilution factor, which is the actual concentration of the sample. Kit composition: sealing film: 2 pieces (48) / 2 pieces (96) Instructions: 1 sealed bag: 1 standard product: 2700ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃ to store enzyme Standard coated plate: 1 × 48 1 × 96 2-8 ° C Store the sample diluent: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Store the developer A solution: 3ml × 1 bottle 6 ml × 1 bottle 2 -8 ℃ storage of developer B solution: 3ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ storage stop solution: 3ml × 1 bottle 6ml × 1 bottle 2-8 ℃ storage concentrated washing solution: (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ° C. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human pituitary adenylate cyclase activating peptide (PACAP) in the specimen. Microporous plates were coated with purified human pituitary adenylate cyclase activating peptide (PACAP) antibody to prepare solid phase antibodies, and pituitary adenylate cyclase activating peptide (PACAP) was added to the microwells coated with mAb in turn ), And then combined with HRP-labeled pituitary adenylate cyclase activating peptide (PACAP) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the pituitary adenylate cyclase activating peptide (PACAP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human pituitary adenylate cyclase activating peptide (PACAP) in the sample was calculated by a standard curve. Purpose: This kit is used to determine the content of pituitary adenylate cyclase activating peptide (PACAP) in human serum, plasma and related liquid samples. Service commitment:? Delivery period: payment to delivery. Free technical consultation and guidance during working hours? Please call to provide customers with sample testing services to maximize the validity of the experimental results (free substitute testing). Storage conditions and validity period: Kit storage: 2-8 ℃ | Validity period: 6 months [Human Pituitary Adenylate Cyclase Activating Peptide (PACAP) elisa kit] Sample processing and requirements: 1. Serum: blood coagulation at room temperature 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits HRP activity. Operating steps Dilution and sample loading of standards: set up 10 wells of the standard on the enzyme-coated plate, add 100 μl of the standard in the first and second wells, and then add the standard in the first and second wells 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentrations are 1800ng / L, 1200ng / L, 600ng / L, 300ng / L, 150ng / L). Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix. Incubation: Seal the plate with a sealing film and incubate at 37 ° C for 30 minutes. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds, then discard, repeat 5 times and pat dry. Enzyme addition: Add 50μl of enzyme label reagent to each well, except for blank wells. Incubation: The operation is the same as 3. Washing: The operation is the same as 5. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes. Termination: Add 50μl of stopper solution to each well to stop the reaction Li to yellow). Measurement: The absorbance (OD value) of each well was measured in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. [Human Pituitary Adenylate Cyclase Activating Peptide (PACAP) elisa kit] Note: The kit should be equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening When finished, the slats should be stored in sealed bags. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5). The sealing film is limited to one-time use to avoid cross-contamination. Please keep the substrate away from light. Carry out the operation in strict accordance with the instructions. The test result must be determined by the reading of the microplate reader. All samples, washing liquid and various wastes should be treated as infectious agents. The components of different batches of this reagent shall not be mixed. 10. If there is any difference with the English manual, the English manual shall prevail. Kit performance: 1. The linear regression of the sample and the expected concentration correlation coefficient R value is above 0.95. 2. In-batch and approval should be less than 9% and 11% respectively. Detection range: 0.2IU / L-6IU / L ELISA kits are available for on-site testing. The company has become an authoritative domestic supplier of Elisa kits and a professional sales platform for Chinese ELISA kits. 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