Drug Names

Generic Name：MouseTB IgG ELISA Kit.

Purpose

This kitallows for the determinationof TB IgG in Mouse serum, and other biological fluids.

Principle of the assay

The kit assayMouse TB IgGlevel in the sample，use Purified TB IgG antibodyto coat microtiter plate wells, make solid-phase antibody, then add TB IgG to wells, Combined With TB IgG, afterwashing and removing non-combinative antibody and other components ,then Combined TB IgG which with HRP labeled become antibody - antigen - enzyme- antibody complex,after washing Completely, Add TMB substrate solution,, TMB substratebecomes blue color At HRP enzyme-catalyzed, reaction is terminated by theaddition of a sulphuric acid solution and the color change is measured spectrophotometricallyat a wavelength of 450 nm.Compared with theCUTOFF value, according to this to judge TB IgG exist in the sample or not.

Materials provided with the kit

Specimen requirements

1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain.

2. plasma-use suitedEDTA or citrate plasma as ananticoagulant,mix 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugalagain.

3. Urine-collectsue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain.The Operation of Hydrothorax and cerebrospinal fluidReference to it.

4. cell culture supernatant-detect secretory components,collect sue a sterile container, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant,detect the composition of cells, Dilut cellsuspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeatedfreeze-thaw cycles, damage cells and release of intracellularcomponents, centrifugation 20-min at the speedof 2000-3000 r.p.m.remove supernatant, If precipitation appeared, Centrifugalagain.

5. Tissuesamples- After cutting samples, check theweight,add PBS（PH7.2-7.4）, Rapidly frozen with liquidnitrogen, maintainsamples at 2-8℃aftermelting,add PBS（PH7.4）,Homogenized by hand or Grinders, centrifugation 20-min atthe speed of 2000-3000r.p.m. remove supernatant.

6. extract as soon as possible after Specimen collection,and according to the relevant literature,and should be experimentas soon as possible after the extraction. If it can’t, specimen can be kept in-20 ℃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 microtitrationwell and NumberSequence, each plate should be set feminine comparison 2 wells, masculine comparison 2wells, blank comparison 1 well(don’t add sample and HRP-Conjugatereagent to blankcomparison well, other each step the operation are same).

2.add sample：separately add Positive control and Negative control 50μl to the Positive and Negative well . add Sample dilution 40μl to testingsample well, then add testing sample 10μl. add sample to the bottom of ELISA platescoated well , don’ttouch the well wall as far as possible, and Gently mix.

3.Incubate: After closing plate with Closure platemembrane ,incubate for 30 min at37℃. 

4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted30-fold (or 20-fold) with distilled water until 600ml,and reserve.

5.washing：Uncover Closure platemembrane, discard Liquid, dry by swing, add washing buffer toevery 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 A50ul and ChromogenSolution B to each well, evade the light preservationfor 15 min at37℃

10.Stop thereaction：Add Stop Solution50μl to each well, Stop the reaction(theblue 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

Testvalidity: the average of Positive control well≥1.00; the average of Negativecontrol well≤0.15.

Calculate Critical(CUT OFF) : Critical= the average of Negativecontrol well +0.20.

Negative control: sample OD< Calculate Critical(CUTOFF) is TB IgG Negative control.

Positive control: ample OD≥ Calculate Critical(CUT OFF) is TB IgG Positive control.

Important notes

1.Please according to use instruction strictly, Donot mix reagents with those from other lots.

2.The kit takes out from the refrigerationenvironment should be balanced 15-30 minutes in the room temperature  then use, ELISA platescoated if has not use up after opened, the plate should be stored in Sealedbag.

3.washing buffer will Crystallization separation,it can be heated the water helps dissolve when dilute . Washing does not affectthe result.

4.Closure plate membrane only limits the disposable use, in order toavoid the overlapping pollution

5.The substrate please evade the lightpreservation.

6.The test result determination must take the microtiterplate reader as astandard, when use dual-wavelength to assay, Reference wavelength is 630nm.

7.All samples, washing buffer and each kind ofreject should according to infective material process. Stopp Solution is2Msulphuric acid. You must pay attention tosafe when use .

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.