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.
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