Bovine M.pt AbFOR RESEARCH USE ONLY

96 determinations
Purpose
This kit allows for the determination of M.pt Ab expression in Bovine serum, and other biological fluids.
Principle of the assay
The kit assay M.pt Ab level in the sample, use Purified antigen to coat microtiter plate wells, make solid-phase antigen, then add M.pt Ab to wells, Combined With M.pt Ab, after washing and removing non-combinative antibody and other components ,then Combined antigen  which with HRP labeled ,become antigen – antibody - enzyme- antigen 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 M.pt Ab 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 50ul to each well, evade the light preservation for 10min 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 M.pt Ab Negative control.
Positive control: ample OD≥ Calculate Critical(CUT OFF) is M.pt 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
1．Storage：  2-8℃.
2．validity： six months.