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孔雀石綠英文說明書.pdf
閱讀：1651 發布時間：2012-9-28malachite green（MG） ELISA
1 Use purpose
This kit for Feed, aquatic samples, water samples in the MG remaining quantitative
detection.
2 Experimental principle
This kit adopts methods in competition ELISA In microplates coated with antigen
conjugated MG, add MG standard or samples, Free MG and pre-coated on strips of MG
conjugated antigen compete against MG antibody conjugates, With TMB chromogenic
substrate,the color is changed from blue to yellow after adding stop solution, enzyme
standard instrument 450nm wavelengths in testing, absorb light value and in the sample
was MG content is inversely proportional to the standard curve, through calculation
samples was MG concentrations.
3 Materials provided with the kit
3.1 Microelisa stripplate: 1 block （12well×8strips）.
3.2 MG standard: six vials of （1ml/ vial）, content is respectively: 0 PPB, 0.1 PPB, 0.3 PPB
and 0.9 PPB, 2.7 PPB, 8.1 PPB.
3.3 Anti- MG antibody conjugate: 1 vial （6ml）.
3.4 Chromogen Solution A: 1 vial （6ml）.
3.5 Chromogen Solution B: 1 vial （6ml）.
3.6 Stop Solution: 1 vial （6ml）, 2M sulphuric acid.
3.7 sample dilution: 1 vial （10 x, 6ml）, used for sample diluted with.
3.8 wash solution: 1 vial （20 x, 20ml）, used for washing board.
3.9 Instruction.
4 Need not provide materials
4.1 equipment
4.4.1 wavelength 450nm microplate.
4.1.2 shredder.
4.1.3 LiangTong.
4.1.4 oscillators.
4.1.5 funnel.
4.1.6 Whatman or equivalent filter paper.
4.1.7 trace remove liquid device.
4.2 reagent
4.2.1 the deionized water or distilled water.
4.2.2 methanol.
5 Storage
5.1 kit stored in 2 ~ 8 ℃, Don’t frozen
5.2 Don’t use up the Microelisa stripplate should be sealed drying preserve
6. Precautions
6.1 please read the instructions carefully , before useing the kit.
6.2 don't use expired kit.
6.3 before using the kit, please let the reagent recover to room temperature （25 + 2 ℃）,
the proposal for at least 2 hours to temperature.
6.4 the standard contain MG, pay special attention to, the operation, we should bring
gloves.
6.5 stop solution is containing sulfate, when using, prevent burns skin and corrosion
clothing.
6.6 different standard and sample suction head used cannot be mixed use, otherwise, it
will affect test results.
6.7 different batches of reagent kit not mix, Different standard and sample suction head
shall not be used in combination, otherwise, it will affect the experimental result.
6.8 diluted sample must use this kit of sample diluent, otherwise, it will affect the
experimental result
6.9 mixed reagents should avoid blistering.
7 Working liquid preparation
7.1Carbendazim standards: 0ppb, 0.1 PPB, 0.3 PPB and 0.9 PPB, 2.7 PPB, 8.1 PPB
7.2 wash solution: 1:20 with distilled water （1 +19） diluted. preparation
7.3 sample dilutions: 1:10 with distilled water （1 +9） diluted. preparation
7.3 Chromogen Solution reagent: already standby, avoid light straight as
7.4 stop solution: already termination aside
8 Sample processing program （sample in extraction process, must strictly
according to the operation of the extraction process should be accurate dilution, can
appear otherwise results are inaccurate, samples should be kept in a cool place to avoid
light and frozen keep）
8.1 Smash the samples taken 10g, add 20ml 70% methanol solution
8.2 powerful oscillation 3 minutes
8.3 Whatman filter with
8.4 Take the 25μl treatment of the sample by adding 25μl sample dilution in the wells
（sample dilution factor of 2）
9 Enzyme 2linded analysis steps
9.1 experimental guidelines
9.1.1 experiment begins prior to all reagent in boxes outside the room temperature （25
fully recovered to + 2 ℃）, time about 2 hours. Return to room temperature （25 + 2 ℃）
again after remove Strips, excess pore bar to seal immediay to the 2 ~ 8 ℃ drying
preserve
Note: be sure to temperature, otherwise fully guarantee the accuracy and precision of the
affecting detection.
9.1.2 after use please immediay reagent put back 2 ~ 8 ℃ preservation
9.1.3 please don't change analysis program
9.1.4 please use accurate trace remove liquid device
9.1.5 operation once started, please do not interrupt any program
9.1.6 ELISA results of repeatability of severe depends on operating procedures, please
strictly according to requirements operation
9.1.7 to avoid cross-contamination, each standard and samples should use different
suction head add samples
9.1.8 plus sample do make suck a head contact microporous the solution or inside surface
9.2 analysis steps
9.2.1 beforehand numbered, mark B0, standard and the sample position recommended
double orifice detection
9.2.2 take the required amount of the Strips （Strips detachable）, will spare attrib wattle
and sealed immediay put back again 2 ~ 8 ℃ preservation
9.2.3 sample dilutions （10）, wash solution x （20 x） dilution into working liquid （distilled
water or deionized water dilute）
In 9.2.4 B0 well joining 50μl 0.0 ng/ml standard
9.2.5 in each standard well joining 50μl standard
9.2.6 in each sample well joining 50μl sample solution
9.2.7 In all well joining 50 μl Anti- MG antibody conjugate
9.2.8 gently sloshing response board for a few seconds.
9.3 37 ℃ warm bath 30min （warm bath process sometimes pat reaction plate, can reduce
double orifice error）
9.3.1 Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer
to every well, still for 30s then drain, repeat 5 times, dry by pat..
9.4 reaction
9.4.1 washing procedure completed, immediay with liquid apparatus in every trace
move microporous first join 50 μl Chromogen Solution A, add 50μl Chromogen Solution B,
Slight sloshing response board make thoroughly incorporated
37 9.4.2 ℃ warm bath 10min
9.4.3 each well joining 50 μl Stop Solution, blending
9.4.4 in 450nm testing absorbency, result in 5min inside read.
10 The results calculated
10.1 quantitative analysis
10.1.1 obtained by each concentration standard solution and the average value of a
sample spectrophotometry （B） divided by the first standard （0 standard absorbency value
（B0） multiplying by 100%, namely percentage absorbency values.
B - standard solution or sample solution of average absorbance of the values
B0-0 μg/L standard solution of average absorbance of the values
10.1.2 with MG concentrations of values for the X axis, 100 cent absorbance of the value
of the Y axis, draw standard curve. According to the sample percentage absorbency
values, which get corresponding points from curve, namely the abscissa denotes the
multi-goal MG concentration on the numerical curvature.the against several namely to
determine MG concentration C （ppb）
10.1.3 because the sample after diluted in advance, so according to the standard curve
gains from different concentration samples must again multiply the diluted times.
10.2 half quantitatively
10.1.1 visual half quantitative determination: first, choose an appropriate standard fluid
and samples with operation, according to the samples and standard substance
absorbency value the discretion of the judge is compared, sample chroma value is less
than or greater than standard values.
10.1.2 instrument half quantitative determination: first, choose an appropriate standard
fluid and samples with operation, according to the sample and standard color depth
comparison, judge sample chroma value is less than or greater than standard values.
11 Specificity
Physical cross reaction
MG 100%
Crystal Violet: 95%
Recessive malachite green: 0.1%
Hidden Crystal Violet: <0.1%
12 Kit parameters
This kit detection limit is 0.05 PPB
B0 absorbance of the optimal value should be greater than 1.0
Kit absorbency board inside error is less than 8%, board between error is less than 15%.
With this manual provided a tissue sample extraction method recovery is greater than
80%.
13 Analysis restriction
This kit testing positive for samples should use another method such as HPLC or GC/MS
to be verified.
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