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豬藍耳病抗原（PRRS Ag）英文說明書定性.pdf
閱讀：832 發布時間：2013-1-16Porcine PRRS Ag
FOR RESEARCH USE ONLY
96 determinations
Purpose
This kit allows for the determination of PRRS Ag expression in Porcine serum, and other
biological fluids.
Principle of the assay
The kit assay PRRS Ag level in the sample，use Purified antibody to coat microtiter plate
wells, make solid-phase antibody, then add PRRS Ag to wells, Combined With PRRS Ag, after
washing and removing non-combinative antibody and other components ,then Combined
antibody which with HRP labeled become antibody – antigen - enzyme- antibody complex, after
washing Compley, 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. Compared with
the CUTOFF value, according to this to judge PRRS Ag exist in the sample or not.
Materials provided with the kit
1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle
2 HRP-Conjugate reagent 6ml×1 bottle 8 Positive control 0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Negative control 0.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml×1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
Specimen requirements
1. 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.
2. 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 sample：separay add Positive control and Negative control 50μl to the Positive and
Negative well . add Sample dilution 40μl to testing sample well, then add testing sample 10μl.
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.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 50μlto each well, except the blank well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the
light preservation for 15 min at 37℃
10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color
change to yellow color）.
11. assay：take 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 well≥1.00; the average of Negative control well
≤0.10.
Calculate Critical（CUT OFF） : Critical= the average of Negative control well + 0.15.
Negative control: sample OD< Calculate Critical（CUT OFF） is PRRS Ag Negative control.
Positive control: ample OD≥ Calculate Critical（CUT OFF） is PRRS Ag 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
1．Storage： 2-8℃.
2．validity： six months.
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