Monkey Tuberculosis Antibody ELISA KitUse instruction
This kit is only used for researching.
Package size: :96
Purpose：
The kit is sued to assay the content of Monkey Tuberculosis Antibody in porcine serum, blood plasma, and other related Liquid samples.
Experimental principle：
The kit use ELISA to assay Monkey Tuberculosis Antibody level in the sample，use Purified Monkey Tuberculosis an antigen to coat microtitration wells, make solid-phase an antigen, add Monkey Tuberculosis Antibody in coated microtitration, Combined With HRP which labeled goat anti- Monkey antibody, become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution and color develops, TMB becomes blue color At HRP enzyme-catalyzed, And at the effect of acid the color finally become yellow, the depth of color and theMonkey Tuberculosis Antibody of sample were positively correlated, measure the optical densit （OD） at 450 nm with microtiter plate reader, calculate Monkey Tuberculosis Antibody concentration by standard curve.
Materials provided with the kit
Specimen requirements
1． extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediay, specimen can be kept in -20 ℃ to preserve, but repeated freezing and thawing should be avoided.
2． Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.
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 solutiondiluted 30-fold （or 20-fold） with distilled water until 600ml,and reserve.
5.washing：Uncover Closure plate membrane, discardLiquid, 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） isMonkey Tuberculosis Antibody Negative control.
Positive control: ample OD≥ Calculate Critical（CUT OFF） is Monkey Tuberculosis AntibodyPositive control.
Important notes
1. 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.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when
dilute . Washing does not affect the result.
3. 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 .
4. Please make specification curve when you assay, had better make duplicate well, if in the sample the testing material content is excessively high （The sample OD is bigger than the standard well OD 1.5 times ）,please use Sample dilution to dilute certain multiple （n times）,then assay. Please multiply total Dilution Times when calculate（×n×5）.
5. Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution
6. The substrate please evade the light preservation
7. The test result determination must take the enzyme sign meter reading as a standard
8. All samples, washing buffer and each kind of reject should according to infective material process.
9. This reagent which different batch number component do not mix .
10. If it’s different form English instruction, take English instruction as the standard.
Storage and validity:
1．Storage： 2-8℃.
2．validity： six months.
