1Rat Estrogen（E）FOR RESEARCH USE ONLYAssay range：2.5 ng/L -80 ng/L 96 determinationsPurposeThis kit allows for the determination of E concentrations in Rat serum,cell culture supernates and other biological fluidsPrinciple of the assayThe kit assay Rat E level in the sample, use Purified Rat E antibody tocoat microtiter plate wells, make solid-phase antibody, then add E to wells,Combined E antibody which With HRP labeled,become antibody - antigen -enzyme-antibody complex, after washing Completely, Add TMB substratesolution,TMB substrate becomes blue color At HRP enzyme-catalyzed,reaction is terminated by the addition of a sulphuric acid solution and the colorchange is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of Rat E in the samples is then determined by comparing theO.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle2HRP-Conjugatereagent6ml×1 bottle 8Standard（160ng/L）0.5ml×1 bottle3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle4 Sample diluent 6ml×1 bottle 10 Instruction 15Chromogen SolutionA6ml×1 bottle 11Closure platemembrane26Chromogen SolutionB6ml×1 bottle 12 Sealed bags 1TSZ2Specimen requirements1. extract as soon as possible after Specimen collection,and according to therelevant literature, and should be experiment as soon as possible after theextraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoidrepeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRPactive.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:80ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent40ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent20ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent10ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent5ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent2.add sample：Set blank wells separately (blank comparison wells don’t addsample and HRP-Conjugate reagent, other each step operation is same).testing sample well. add Sample dilution 40μl to testing sample well, then addtesting sample 10μl (sample final dilution is 5-fold), add sample to wells ,don’t touch the well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30min at 37℃.4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold)with distilled water and reserve.5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing,add washing buffer to every well, still for 30s then drain, repeat 5 times, dry bypat.6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blankwell.37.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B 50ul toeach well, evade the light preservation for 10 min at 37℃10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(theblue color change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after AddingStop Solution and within 15min.Steps descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min at 37℃.Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.Add Stop Solution4Read absorbance at 450nm within 15 mincalculateCalculateTake the standard density as the horizontal, the OD value for thevertical ,draw the standard curve on graph paper, Find out the correspondingdensity according to the sample OD value by the Sample curve, multiplied bythe dilution multiple, or calculate the straight line regression equation of thestandard curve with the standard density and the OD value ,with the sampleOD value in the equation, calculate the sample density, multiplied by thedilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced15-30 minutes in the room temperature, ELISA plates coated if has not useup after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the waterhelps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently,avoids the experimental error. add sample within 5 min, if the number ofsample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD isbigger than the first standard well ),please dilute Sample (n-fold), Pleasediluente and multiplied by the dilution factor.（×n×5）.5. Closure plate membrane only limits the disposable use, to avoidcross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination5must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according toinfective material process.9. Do not mix reagents with those from other lots.Storage and validity1．Storage： 2-8℃.2．validity： six months