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Tissue samples- After cutting samples, check the weight,add PBSPH7.2-7.4 , Rapidly frozen with liquid nitrogen, maintain samples at 2-8! after melting,add PBSPH7.4 , Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant. 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 1.Number: to sample correspond microtitration well and Number Sequence, each plate should be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1 well(don t add sample and HRP-Conjugate reagent to blank comparison well, other each step the operation are same). 2.add sampleseparately add Positive control and Negative control 50l to the Positive and Negative well . add Sample dilution 40l to testing sample well, then add testing sample 10l. add sample to the bottom of ELISA plates coated well , 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 until 600ml,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 50lto each well, except the 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 reactionAdd Stop Solution50l to each well, Stop the reaction(the blue color change to yellow color). 11. assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. Determine the result Test validity: the average of Positive control welle"1.00; the average of Negative control well d"0.10. Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15. Negative control: sample OD< Calculate Critical(CUT OFF) is BR Ab Negative control. Positive control: ample ODe" Calculate Critical(CUT OFF) is BR Ab Positive control. Important notes 1.Please according to use instruction strictly, Do not mix reagents with those from other lots. 2.The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature then use, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag. 3.washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result. 4.Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution 5.The substrate please evade the light preservation. 6.The test result determination must take the microtiter plate reader as a standard, when use dual-wavelength to assay, Reference wavelength is 630nm. 7.All samples, washing buffer and each kind of reject should according to infective material process. Stopp Solution is 2M sulphuric acid. You must pay attention to safe when use . Storage and validity 1Storage 2-8!. 2validity six months.     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