人穿孔素/成孔蛋白（PF/PFP）ELISA試劑盒

Reagent Kit.

120 times concentrated washing solution 20ml × 1 3ml× 1 bottles of 7 terminated liquid bottle

The 2 enzyme reagent 3ml ×1 bottles of 8 standard （ 960 μ g/L ） 0.5ml ×1 bottles

The 3 enzyme encapsulated plate 12 holes x 4 9 standard dilution of 1.5ml ×1 bottles

4 sample dilution 3ml ×1 bottles of 10 Manual 1

5 chromogenic reagent A fluid 3ml×1 bottles of 11 sealing plate film 2

6 chromogenic reagent B 3ml×12 1/ bottles of liquid sealing bag 1

Specimen requirements

1 specimens were collected after early extraction, extraction according to the related literature, after extracting the experiment should be conducted as soon as possible. If not immediay put the sample to test, can be stored at -20 ° C, but should avoid repeated freezing and thawing

2 could not be detected in the samples with NaN3, because NaN3 inhibition of horseradish peroxidase （ HRP ） activity.

Operation steps

Standard dilution: the kit provides raw times standard a, the user can according to the following chart in the small test tube dilution.

480 μ g/L 5 standard 150μl of the original times standard added 150 μ l standard dilution buffer

240 μ g/L 4 standard 150μL 5 standard added 150 μ l standard dilution buffer

120 μ g/L 3 standard 150μL 4 standard added 150 μ l standard dilution buffer

60 μ g/L 2 standard 150μL 3 standard added 150 μ l standard dilution buffer

30 μ g/L 1 standard 150μL 2 standard added 150 μ l standard dilution buffer

Sample: blank hole are respectively arranged （ blank hole without sample and enzyme reagent, the remaining steps the same ）, standard orifice, sample hole. In the enzyme coated board standard accurate sample adding 50 μ L, sample hole Zhongxian sample dilution of 40 μ L, then add the sample of 10μ L （ final dilution of the sample for 5 times）. Sample sample on the enzyme labeled plate hole bottom, try not to touch the wall holes, gently shake mix.

Incubation: sealing plate membrane sealing plate is 37 ℃incubation for 30 minutes.

Liquid : 20 times concentrated washing solution with distilled water after 20 times dilution standby

Washing: carefully off the sealing plate membrane, abandoned to the liquid, drying, each hole is filled with a washing liquid, standing for 30 seconds after discarding, so repeated 5 times, pat dry.

Enzyme: each hole joining enzyme reagent 50 μ L, with the exception of blank hole.

Incubation: operation with 3.

Washing: operation with 5.

Color: each hole to join the chromogenic reagent A50μ L, adding chromogenic reagent B50μ L, gently shake mix, 37 ℃light color for 15 minutes.

Termination: each hole and terminated liquid 50μ L, termination reaction （ the blue vertical turn yellow ）.

Determination of air conditioning: with blank zero, 450nm wavelength in order to measure the absorbance （ OD） hole. Determination should be in plus termination solution within 15 minutes after the for.

Calculation

With a standard concentration as abscissa, OD as ordinate, on coordinate paper draw the standard curve, according to samples by the standard curve was found corresponding concentration; multiplied by the dilution factor; or standard concentration was calculated with standard curve equation of linear regression, sample od into the equation, calculation out of sample concentration, multiplied by the dilution factor, namely the actual sample concentration.

Matters needing attention

The 1 kit from refrigerated removed should be at room temperature balance 15-30 minutes before use, enzyme encapsulated plate in Kaifeng such as unused, slats into sealed bag should be stored in.

2 concentrated washing solution may crystallization, when diluted in water bath heating dissolution, washing does not affect the result.

3 step sampling shall be use the sample injector, and often to check that the accuracy, in order to avoid the test error. A sample time best control in 5 minutes, such as specimen number, recommended the use of volley sample.

人穿孔素/成孔蛋白（PF/PFP）ELISA試劑盒

Please each measurement and standard curve, the best do complex holes. If the specimen substance to be determined in the content is too high （ sample od than standard hole of the first hole OD）, please use the sample dilution dilution of certain multiple （ n times） after the measurement, calculation when you finally multiplied by the total dilution （× n ×5）.

Sealing plate membrane only one-time use, to avoid cross contamination.

The 6 substrate please keep in dark place.

7 strictly in accordance with the manual operation, test results must be with the enzyme mark instrument readings shall prevail.

8 of all samples, the washing liquid and a variety of waste should be according to the infection treatment.

The 9 different reagent batch components not mix.

10 such as with English instructions are different, with English instructions shall prevail.

Preservation conditions and period of validity

The 1 Kit: 2-8℃preservation.

The 2 period: 6 months