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The concentration of Human Fk!a in the samples is then determined by comparing the O.D. of the samples to the standard curve. Materials provided with the kit 1wash solution20ml1bottle7Stop Solution6ml1 bottle2HRP-Conjugate reagent6ml1 bottle8Standard160U/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 cant, 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: 80U/L5 Standard150l Original density Standard+150l Standard diluent40U/L4 Standard150l 5 Standard+150l Standard diluent20U/L3 Standard150l 4 Standard+150l Standard diluent10U/L2 Standard150l 3 Standard +150l Standard diluent5U/L1 Standard150l 2 Standard +150l Standard diluent2. Add sample: Set 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. 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 by pat. 6. Add enzyme: Add HRP-Conjugate reagent 50l to each well, except blank well. 7. Incubate: Operation with 3. 8. Washing: Operation with 5. 9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evade the light preservation for 10 min at 37! 10. 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Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. 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 10 min at 37!. Add Stop 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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