??? Blank well: don’t add samples and (D2HGDH)-antibodylabeled

with biotin, Streptavidin-HRP, only Chromogen solution AandB, and stop solution are allowed; other operations are thesame.

??? Standard wells: add standard 50μl, Streptavidin-HRP50μl(since the standard already has combined biotin antibody, it isnot necessary to add the antibody);

??? To be test wells: add sample 40μl, and then add both (D2HGDH)-antibody 10μl andStreptavidin-HRP 50μl. Then seal the sealing memberance, and gentlyshaking, incubated 60 minutes at 37 ???.

4.Confection: dilute 30 times the 30×washing concentratewith distilled water as standby.

5. Washing: remove the memberance carefully, and drainthe liquid, shake away the remaining water.

6. Add chromogen solution A 50μl, then chromogensolution B 50μl to each well. Gently mixed, incubate for 10 min at37??? away from light.

7. Stop: Add Stop Solution 50μl into each well to stopthe reaction(the blue changes into yellowimmediately).

8. Final measurement: Take blank well as zero , measurethe optical densit (OD) under 450 nm wavelength which should becarried out within 15min after adding the stopsolution.

9. According to standards’ concentration and thecorresponding OD values, calculate out the standard curve linearregression equation, and then apply the OD values of the sample onthe regression equation to calculate the corresponding sample’sconcentration. It is acceptable to use kinds of software to makecalculations.

 Summary procedures                                               

Preparing reagents, samples and standards

Add prepared samples and standards, antibodies labeledwith enzyme, reacting 60 minutesat 37 ???

Plate washed five times, adding Chromogen solution A, B,reacting 10 minutes at 37???

Add stop solution

measure the OD value within 10min

Calculation

 Calculate                                                                

Take the standard density as the

horizontal???the OD value forthe

vertical???draw the standardcurve

on graph paper, Find out the

corresponding density according to

the sample OD value by the Sample

curve (the result is the sample density)

or calculate the straight line regression equation ofthe standard curve with the standard density and the OD value ,withthe sample OD value in the equation, calculate the sampledensity.

Sensitivity???Assayrange                                                         

Sensitivity:5.125Ng/L

Assay range: 7ng/L-2000ng/L

Package size                                                                         

96T perbox

validity&Storage                                                             

six months (2-8???)  

???Our electronic version of the manual is not related to thekit performance, specificity and other relevant data andinstructions. Because we need to avoid data copying. So theelectronic version of the manual only as reference, The deliveryinstruction is taken as final instruction.