Enzyme linked immunosorbent assay of E-cadherin in rats

Instructions for use of the Kit Book kits are for research purposes only Drug name: general name: rat E-cadherin (E-cad) ELISA diagnostic kit purpose: this kit is used to determine the content of E-cadherin (E-cad) in rat serum, plasma or other related tissue fluid Experimental principle: the E-cadherin (E-cad) level of rats was determined by double antibody sandwich method The purified E-cadherin (E-cad) antibody was coated on the microplate to make solid-phase antibody, then E-cad antigen was added into the micropore of the coated McAb in turn, and then combined with E-cad antibody labeled by HRP to form antibody antigen enzyme antibody complex After thorough washing, TMB substrate was added to develop color TMB was converted to blue under the catalysis of HRP enzyme, and to Zui final yellow under the action of acid There was a positive correlation between the color and E-cadherin (E-cad) The absorbance (OD value) was measured at 450 nm with an enzyme reader The concentration of E-cadherin (E-cad) antigen in the sample was calculated by standard curve The kit consists of 1 20 times concentrated washing solution 20ml × 1 bottle 7 termination solution 6ml × 1 bottle 2 enzyme standard reagent 6ml × 1 bottle 8 standard (180ng / L) 0.5ml × 1 bottle 3 enzyme standard coated plate 12 holes × 8 strips 9 standard diluent 2ml × 1 bottle 4 sample diluent 6ml × 1 bottle 10 instructions 1 copy 5 developer a solution 6ml × 1 bottle 11 sealing plate membrane 2 copies 6 developer B solution 6ml × 1 / bottle 12 sealing bag 1 sample requirements 1 Conduct the experiment as soon as possible after the specimen collection 2 The sample containing NaN3 can not be detected because it inhibits the activity of horseradish peroxidase (HRP) 3 Specimens can be stored at - 20 ℃, but repeated freezing and thawing should be avoided Operation step 1 Dilution and sample adding of standards: set 10 holes of standards on the enzyme label coated plate, add 100 μ l of standards in the second hole and the second hole respectively, and then add 50 μ l of standards diluent in the second hole and the second hole respectively, and mix well; then add 100 μ l of standards diluent in the third hole and the second hole respectively to the third hole and the fourth hole, and then add 50 μ l of standards diluent in the third hole and the fourth hole respectively, and mix well; then add 50 μ l of standards diluent in the third hole and the fourth hole respectively, and Take 50 μ L from the fourth hole, discard it, add 50 μ l to the fifth hole and the sixth hole respectively, add 50ul of standard diluent to the fifth hole and the sixth hole respectively, and mix well; take 50 μ L from the fifth hole and the sixth hole respectively, add 50 μ l to the seventh hole and the eighth hole respectively, and then add 50 μ l of standard diluent to the seventh hole and the eighth hole respectively, mix well and then add 50 μ L from the seventh hole and the eighth hole respectively Add 50 μ l Standard diluent to the ninth and tenth holes respectively, mix well and discard 50 μ l Standard diluent from the ninth and tenth holes respectively (after dilution, the sample amount of each hole is 50 μ L, and the concentration is (120ng / L, 80ng / L, 40ng / L, 20ng / L, 10NG / L) 2 Sample adding: set blank hole (blank control hole without sample and enzyme standard reagent, the operation of other steps is the same) and sample hole to be tested Add 40 μ l of sample diluent to the sample hole on the enzyme coated plate, and then add 10 μ l of sample to be tested (the final dilution of sample Zui is 5 times) Add the sample to the bottom of the enzyme plate hole, try not to touch the hole wall, shake gently and mix 3 Warm Incubation: seal the plate with sealing film and leave it at 37 ℃ for 30 minutes 4 Solution preparation: dilute 25 times of concentrated washing solution with 25 times of distilled water for standby use 5 Washing: carefully remove the sealing film, discard the liquid, dry it, fill each hole with washing solution, leave it for 30 seconds, then discard it, repeat for 5 times, pat it dry 6 Enzyme addition: add 50 μ l of enzyme standard reagent into each hole, except for the blank hole 7 Warm Education: the operation is the same as 3 8 Washing: the same as 5 9 Color development: first add color agent A50 μ l to each hole, then add color agent B50 μ L, shake and mix gently, and develop color in 37 ℃ dark for 15 minutes 10 Stop: add 50 μ l of stop solution to each hole, and stop the reaction (at this time, the blue color turns to yellow vertically) 11 Measurement: measure the absorbance (OD value) of each hole in sequence with the blank air conditioning zero and the wavelength of 450nm The determination shall be carried out within 15 minutes after the addition of termination solution Calculation Take the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw the standard curve on the coordinate paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply the dilution ratio; or use the concentration of the standard substance and the OD value to calculate the linear regression equation of the standard curve, and substitute the OD value of the sample into the equation to calculate the concentration of the sample, and then multiply the dilution ratio, which is the sample The actual concentration of the product Note 1 The reagent box can be used only after it is taken out from the cold storage environment and balanced at room temperature for 15-30 minutes If the enzyme label package is not used up after being unsealed by the plate, the strip shall be stored in the sealed bag 2 The concentrated detergent may have crystallization and precipitation When diluted, it can be heated and solubilized in the water bath, and washing will not affect the results 3 The sample feeder shall be used in each step, and its accuracy shall be checked frequently to avoid test error The sample adding time Zui should be well controlled within 5 minutes If the number of samples is large, it is recommended to use the gun to add samples 4 Please make the standard curve at the same time of each measurement, so that Zui can do the re drilling If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than 1.5 times of the OD value of the standard sample hole), please dilute it with the sample diluent for a certain number of times (n times) before the determination, and then multiply it by the total Dilution Times (× n × 5) when calculating 5 The sealing film is only used once to avoid cross contamination 6 Keep the substrate away from light 7 The determination of test results must be based on the reading of enzyme scale instrument 8 All samples, washing solution and various wastes shall be treated as infectious substances 9 Components of different batches of this reagent shall not be mixed 10 In case of any difference from the English manual, the English manual shall prevail Detection range: 5ng / L - 150ng / L specification: 96 person / box storage conditions and validity period 1 Reagent box storage: 2-8 ℃ 2 Validity: 6 months