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Bovine FMD-O-AgDrug Names
Generic Name锛欱ovine FMD-O-Ag ELISA Kit.
Purpose
This kit allows for the determination of FMD-O-Ag in Bovine serum, blood plasma, tissue and other biological fluids.
Principle of the assay
The kit assay FMD-O- Ag level in the sample锛寀se Purified FMD-O antibody to coat microtiter plate wells, make solid-phase antibody, then add FMD-O- Ag to wells, Combined With FMD-O- Ag, after washing and removing non-combinative and other components ,then Combined FMD-O 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 FMD-O- Ag exist in the sample or not.鐗涘彛韫勭柅O鍨嬫姉鍘�(FMD-O-Ag)瑭﹀姂鐩掗綂涓�鐢熺墿渚�
Materials provided with the kit
Materials provided with the kit 48determinations 96 determinations Storage
User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8鈩�
Negative control 0.5ml×1 bottle 0.5ml×1 bottle 2-8鈩�
Positive control 0.5ml×1 bottle 0.5ml×1 bottle 2-8鈩�
HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8鈩�
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8鈩�
Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8鈩�
Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8鈩�
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8鈩�
wash solution 锛�20ml×20 fold锛�
×1bottle 锛�20ml×30 fold锛�
×1bottle 2-8鈩�
Specimen requirements
1.serum- coagulation at room temperature 10-20 mins锛宑entrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
2.plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
3.Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.
4.cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS锛圥H7.2-7.4锛�, Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
5.Tissue samples- After cutting samples, check the weight,add PBS锛圥H7.2-7.4锛�, Rapidly frozen with liquid nitrogen, maintain samples at 2-8鈩� after melting,add PBS锛圥H7.4锛�, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.
6.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 鈩� to preserve, Avoid repeated freeze-thaw cycles.
7.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锛歴eparay 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鈩�.
4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted until 600ml,and reserve.
5.washing锛歎ncover 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锛欰dd HRP-Conjugate reagent 50μlto each well, except the blank well.
7.incubate锛歄peration with 3.
8.washing锛歄peration with 5.
9.color锛欰dd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37鈩�
10.Stop the reaction锛欰dd Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).
11. assay锛歵ake 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 FMD-O-Ag Negative control.
Positive control: ample OD≥ Calculate Critical(CUT OFF) is FMD-O-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
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