ࡱ> '` RnbjbjLULU2.?.?%nnnnz(z(z(8(f)L.*.*"P*P*P*---KKKKKKK$NhQ:?K0,-00?KnnP*P*_K^?^?^?0n8P*P*K^?0K^?^?VF@MGP*"* %Xz(V8F G<K0LFxPQJ<PQMGPQMG-h-J^?7.<s.)---?K?K(>6---L0000nnnnnn   Rat CRP FOR RESEARCH USE ONLY Assay range80g/L -2400g/L 96 determinations Purpose This kit allows for the determination of CRP concentrations in Rat serum, cell culture supernates and other biological fluids Principle of the assay The kit assay Rat CRP level in the sample use Purified Rat CRP antibody to coat microtiter plate wells, make solid-phase antibody, then add CRP to wells, Combined CRP antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Rat CRP in the samples is then determined by comparing the O.D. of the samples to the standard curve. Materials provided with the kit 1wash solution20ml1bottle7Stopp Solution6ml1 bottle2HRP-Conjugate reagent6ml1 bottle8Standard4800g/L 0.5ml1 bottle3Microelisa stripplate12well8strips9Standard diluent1.5ml1bottle4Sample diluent6ml1 bottle10Instruction15Chromogen Solution A6ml1 bottle11Closure plate membrane26Chromogen Solution B6ml1 bottle12Sealed bags1Specimen requirements extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can t, specimen can be kept in -20 ! to preserve, Avoid repeated freeze-thaw cycles. Can t detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay procedure Dilute and add sample:Dilute Original density Standard as follow table: 2400g/L5 Standard150l Original density Standard+150l Standard diluent1200g/L4 Standard150l 5 Standard+150l Standard diluent600g/L3 Standard150l 4 Standard+150l Standard diluent300g/L2 Standard150l 3 Standard +150l Standard diluent150g/L1 Standard150l 2 Standard +150l Standard diluent2.add sampleSet blank wells separately (blank comparison wells don t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (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 30 min at 37!. 4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve. 5.washingUncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 6.add enzymeAdd HRP-Conjugate reagent 50l to each well, except blank well. 7.incubateOperation with 3. 8.washingOperation with 5. 9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37! 10.Stop the reaction "2DFH^`dflpxzĴqbTIB>B9B>B hgo(hg hgaJo(h5CJaJo(hrh5CJPJaJh5CJOJQJaJo(hL`h5CJOJQJaJ"hL`h5CJOJQJaJo("hKh5CJ$OJQJaJ$o(hg5CJ$OJQJaJ$o(hYGh5CJ$OJQJaJ$*h*jxh*jx5CJ$OJQJaJ$mHnHu h*jxh*jx0jh5CJ$OJQJUaJ$mHnHo(u ho(FH } $$Ifa$gd@{ kdhWD'`kgd WD'`gd dhWD`gd WDd`gd*jx H WD`H gd  WD` gdmgdmnn $ & , . 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Steps description Standard, Sample diluent Add 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 30 min at 37!. Add Stopp Solution Read absorbance at 450nm within 15 min calculateCalculate Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density. Important notes The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley . if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.n5 . Closure plate membrane only limits the disposable use, to avoid cross-contamination. The substrate evade the light preservation. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard. All samples, washing buffer and each kind of reject should according to infective material process. Do not mix reagents with those from other lots. 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