Porcine epidemic diarrhea virus(PEDV)ELISA Kit

Porcine epidemic diarrhea virus(PEDV)ELISA Kit

FOR RESEARCH USE ONLY. Not for clinical diagnosis use

CATALOG #:11256

INTRODUCTION

? This kit allows for the determination of PEDV concentrations in Porcine serum

? Detection of species: Porcine

? Detection medium: serum, cell culture supernates.

PRINCIPLE OF TEST

The kit assay PEDV level in the sample，use Purified PEDV antibody to coat microtiter plate

wells, make solid-phase antibody, then add PEDV to wells, Combined With PEDV, after washing

and removing non-combinative antibody and other components ,then Combined PEDV antibody

which with HRP labeled become antibody – antigen - enzyme- antibody complex, after washing

Completely, 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 PEDV exist in the sample or not.

IDEXX Laboratories, Inc.

One IDEXX DriveWestbrook, Maine 04092United States

COMPOSITION OF THE KIT

Reagent Quantity

wash solution 20ml×1bottle

HRP-Conjugate reagen 6ml×1 bottle

Microelisa stripplate 12well×8strips

Sample Diluent 1×6ml bottle

Chromogen Solution A 1×6ml bottle

Chromogen Solution B 1×6ml bottle

Stop Solution 1×6ml bottle

Positive control 0.5ml×1 bottle

Negative control 0.5ml×1 bottle

Closure plate membrane 2

Sealed bag 1

Instruction 1

STORAGE CONDITIONS

? The unopened kit shall be stored at [2-8 ℃] .

? For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used recently,

the standard should be kept in -20 ℃.

WASHING METHOD

? Manually washing method: shake away the remained liquid in the enzyme

plates; place some bibulous papers on the test-bed, and flap the plates on the

upside down strongly. Inject at least 0.35ml after-dilution washing solution into the

well, and marinate 1~2 minutes. Repeat this process according to your

requirements.

? Automatic washing method: if there is automatic washing machine, it should

only be used in the test when you are quite familiar with its function and

performance.

IDEXX Laboratories, Inc.

One IDEXX DriveWestbrook, Maine 04092United States

SAMPLE PREPARATION

1. 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 ℃ to preserve, Avoid repeated freeze-thaw cycles.

2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

ASSAY PROCEDURE

Step 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).

Step 2: add sample：separately 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.

Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃.

Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled

water.

Step 5: Washing: Uncover the adhesive strip, discard liquid, Pipette washing buffer to

every well, still for 30s then drain, repeat 5 times.

Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank

well.

Step 7: Incubate: Operation with 3.

Step 8: Washing: Operation with 5.

Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each

well, avoid the light preservation for 15 min at 37℃

Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, Stop the reaction

(the blue change to yellow).

IDEXX Laboratories, Inc.

One IDEXX DriveWestbrook, Maine 04092United States

Step 11: Calculate: take blank well as zero, Read absorbance at 450nm after Pipetteing

Stop Solution within 15min.

CALCULATION OF 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 PEDV Negative control.

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

EXPIRATION

Six months [see label on the outer box for the specific date].

ATTENTION

? The kit takes out from the refrigeration should be balanced 15-30 minutes in the

room temperature, if the coated ELISA plates have not been used up after opening,

the plate should be stored in sealed bag.

? washing buffer will Crystallization separation, it can be heated the water helps dissolve

when dilute . Washing does not affect the result.

? Pipette sample with pipettors each step, and proofread its accuracy frequently to

avoid the experimental error. Pipette sample within 5 min, if the number of sample

is big, recommend using multichannel pipettor.

? The test result determination must take the microtiter plate reader as a standard, when use

dual-wavelength to assay, Reference wavelength is 630nm.

? Adhesive Strip only limits the disposable use to avoid cross-contamination.

? The substrate should evade the light to be preserved.

IDEXX Laboratories, Inc.

One IDEXX DriveWestbrook, Maine 04092United States

? Please refer to the user instruction strictly, the test result determination must take

the microtiter plate reader as a standard.

? The preparation of samples and all the reagents should refer to infective material

process.

? Do not mix reagents with those from other lots.