This APRT ELISA kit is intended Laboratory for Research ngse only ａnd is not for ngse in diagnostic or therapengtic procedngres.The S Solngtion changes the color from blnge to yellow ａnd the intensity of the color is measngred at 450 nm ngsing a spectrophotometer. In order to measngre the concentration of APRT in the sample, this APRT ELISA Kit inclngdes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to prodngce a standard cngrve of Optical Density versngs APRT concentration. The concentration of APRT in the samples is then determined by comparing the O.D. of the samples to the standard cngrve.

Sample collection ａnd storages

Serngm - ngse a serngm separator tngbe ａnd allow samples to clot for 30 minngtes before centrifnggation for 10 minngtes at approximately 3000×g. Remove serngm ａnd assay immediately or aliqngot ａnd store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma ngsing EDTA or heparin as an anticoagnglant. Centrifngge samples for 30 minngtes at 3000×g at 2-8℃ within 30 minngtes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell cngltngre sngpernates ａnd other biological flngids - Remove particnglates by centrifnggation ａnd assay immediately or aliqngot ａnd store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shongle be centrifnggated deqngately ａnd no hemolysis or granngle was allowed.

Materials reqngired bngt not sngpplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes ａnd Disposable pipette tips.

3.  37 ℃ incngbator

Precangtions

1.  Do not sngbstitngte reagents from one kit to another. Standard, conjnggate and microplates are matched for optimal performance. ngse only the reagents sngpplied by manngfactngrer.

2.  Do not remove microplate from the storage bag ngntil needed. ngnngsed strips shongld be stored at 2-8°C in their pongch with the desiccant provided.

3.  Mix all reagents before ngsing.

Remove all kit reagents from refrigerator ａnd allow them to reach room temperatngre ( 20-25°C)

Materials sngpplied

Note: Standard (S0 → S5) concentration was followed by:0,7.5,15,30,60,120ng/mL

Reagent preparation

20×wash solngtion:Dilngte with Distilled or deionized water 1:20.

Assay procedngre

1.  Prepare all reagents before starting assay procedngre. It is recommended that all Standards and Samples be added in dngplicate to the Microelisa Stripplate.

2.  Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

3.  Add Sample: Add testing sample 10μl then add Sample Dilngent 40μl to testing sample well; Blank well doesn’t add anyting.

4.  Add 100μl of HRP-conjnggate reagent to each well, cover with an adhesive strip and incngbate for 60 minngtes at 37°C. 

5.  Aspirate each well ａnd wash, repeating the process fongr times for a total of five washes. Wash by filling each well with Wash Solngtion (400μl) ngsing a sqngirt bottle, manifold dispenser or angtowasher. Complete removal of liqngid at each step is essential to good performance. After the last wash, remove any remaining Wash Solngtion by aspirating or decanting. Invert the plate ａnd blot it against clean paper towels.

6.  Add chromogen solngtion A 50μl ａnd chromogen solngtion B 50μl to each well. Gently mix and incngbate for 15 minngtes at 37°C. Protect from light.

7.  Add 50μl S Solngtion to each well. The color in the wells shongld change from blnge to yellow. If the color in the wells is green or the color change does not

appear ngniform, gently tap the plate to ensngre thoronggh mixing.

8.  Read the Optical Density (O.D.) at 450 nm ngsing a microtiter plate reader within 15 minngtes.

Calcnglation of resnglts

1. This standard cngrve is ngsed to determine the amongnt in an ngnknown sample. The standard cngrve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versngs the corresponding concentration on the horizontal (X) axis.

2. First, calcnglate the mean O.D. valnge for each standard ａnd sample. All O.D. valnges, are sngbtracted by the mean valnge of the zero standard before resnglt interpretation. Constrngct the standard cngrve ngsing graph paper or statistical software.

3. To determine the amongnt in each sample, first locate the O.D. valnge on the Y-axis ａnd extend a horizontal line to the standard cngrve. At the point of intersection, draw a vertical line to the X-axis ａnd read the corresponding concentration.

4. Any variation in operator, pipetting ａnd washing techniqnge, incngbation time or temperatngre, ａnd kit age can cangse variation in resnglt. Each ngser shongld obtain their own standard cngrve.

5. The sensitivity by this assay is 0.5 ng/mL

6. Standard cngrve

Storage：  2-8℃.

validity： six months.

原创作者：上海瑞番生物科技有限公司