CN1766623A - Malachite green indirect competitive ELISA kit for detecting in the aquatic products - Google Patents
Malachite green indirect competitive ELISA kit for detecting in the aquatic products Download PDFInfo
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- CN1766623A CN1766623A CN 200510060995 CN200510060995A CN1766623A CN 1766623 A CN1766623 A CN 1766623A CN 200510060995 CN200510060995 CN 200510060995 CN 200510060995 A CN200510060995 A CN 200510060995A CN 1766623 A CN1766623 A CN 1766623A
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Abstract
The present invention is a leucomalachite green indirect competitive ELISA kit for detecting in a kind of aquatic products.The check-out console of kit is for wrapping by the removable 96 hole ELISA Plate of leucomalachite green and albumin conjugate, the monoclonal antibody of anti-leucomalachite green antibody for utilizing leucomalachite green and albuminous conjugate immune mouse to prepare, test sample is regulated the dilution of pH value back and is used for detecting earlier through extracting with ethyl acetate and cyclohexane after the even matter.The criterion of pattern detection is: the logarithm value with standard specimen concentration is a horizontal ordinate, and inhibiting rate is the regression curve of ordinate.Just can read the concentration of corresponding sample from curve according to the inhibiting rate of each sample.The sensitivity that this method detects is 0.0023 μ g/ml, and sensing range is 0.0016-1 μ g/ml.Kit of the present invention simple to operate can better satisfy the exigence that at present the aquatic products malachite green vestigial is detected, and is used for that customs is detected, food hygiene department is detected etc., is easy to apply on a large scale.
Description
Technical field
The invention belongs to biological technical field, relate to the fast diagnosis method and the utensil of a kind of medicine of aquatic product meat product, specifically is the malachite green indirect competitive ELISA kit for detecting
Background technology
(Leucomalachite green is that (malachite green, MG) main metabolic residue in vivo has carcinogenic danger to the mankind to malachite green LMG) to leucomalachite green.
It is green that malachite green is called aniline green, Victoria green or China again, it is a kind of green crystal with metallic luster, water-soluble, ethanol and methyl alcohol, the color of aqueous solution is blue-green (maximum absorption wavelength is 618nm), there was the people to find that malachite green and other dyes can destroy pathogen and not cause that the host damages, thereby makes these dyestuffs be used as antiseptic, trypanocides and other medical function as far back as 1913.From the appearance of sulfa drugs and other antibiotic, malachite green day by day fails as the application of antibiotic in animal husbandry.Yet in aquaculture because cheap and antibiotic, anti-mycotic efficiency is good, be widely used in to carry out water body disinfection and prevent the fish and water mildew.
Discovered in recent years malachite green particularly its metabolin has in the aquatic products body and significantly accumulates residual phenomena, and the residence time is also longer.Because its chemical functional group's triphenylmethane is a kind of carcinogen, so external some developed countries have announced to forbid that as European Union, the U.S. it uses in economic fish (except the aquarium fish) breeding process." the pollution-free food China suede huge legendary turtle crab " of China Ministry of Agriculture issue (NY5064-2001) and in " veterinary drug and other compound inventory of food animal forbidding " file (agriculture and animal husbandry is sent out [2002] No. 1) of Ministry of Agriculture's issue clearly stipulates to forbid malachite green in all edible tissues.
" the residual Supervisory Surveillance Program of poisonous and harmful substance in the 2000 annual Chinese exports animal derived foods " lists the monitoring of malachite green project in the eel in annual plan first, and continues into the present.Test confirms that malachite green 50% changes leucomalachite green into after 8 hours in animal body, and 84% is transformed into leucomalachite green after 24 hours, and is trapped in the tissue for a long time.Detect MG both at home and abroad and mainly adopt with chromatographic technique (HPLC) detection technique, though sensitive, quantitatively accurate, speed is slow during mass detection, and cost is higher relatively.With competitiveness enzyme linked immunosorbent assay (ELISA) is that the rapid screening method of representative is the immunology mainstream technology, fast, cost is low, can mass detection, still do not have this technology report both at home and abroad.
Summary of the invention
The purpose of this invention is to provide a kind of malachite green indirect competitive ELISA kit for detecting that uses anti-leucomalachite green monoclonal antibody to set up.
The object of the present invention is achieved like this: adopt leucomalachite green and albumin conjugate as antigen coated removable 96 hole ELISA Plate, add testing sample and anti-leucomalachite green monoclonal antibody then simultaneously, allow both compete antibody with the LMG in the test sample through pre-treatment.Specific as follows:
Be provided with the micropore check-out console in the detection kit of the present invention, the monoclonal antibody lyophilized products, ELIAS secondary antibody, sample diluting liquid, 10 * concentrated cleaning solution, colour developing thing A, colour developing liquid B, stop buffer and standard solution, the micropore check-out console is for wrapping by the removable 96 hole ELISA Plate of leucomalachite green and albumin conjugate (LMG-RSA or LMG-OVA), and standard solution is leucomalachite green (LMG) standard solution.
The best preparation scheme of micropore check-out console is: the 0.2M carbonate buffer solution with pH9.6 is made coating buffer; leucomalachite green and albumin conjugate (LMG-RSA or LMG-OVA) are diluted to 2 μ g/ml; add in the polystyrene micropore plate by 100 μ l/ holes; 4 ℃ of bags are spent the night; dry; contain 1% gelatin by the adding of 200 μ l/ holes; 37 ℃ of sealings of the phosphate buffer of PH7.4 2 hours; washing dries; add 20% sucrose phosphate buffer room temperature protection 3 hours again; after putting the hothouse drying, preserve in the packaging bag that contains drying agent of packing into.
Above-mentioned monoclonal antibody lyophilized products is an anti-leucomalachite green monoclonal antibody of utilizing leucomalachite green and albuminous conjugate immune mouse and the preparation of monoclonal antibody technology of preparing, can with the leucomalachite green specific bond.
Above-mentioned sample diluting liquid is the 0.01M phosphate buffer (pH7.4) that contains 0.05% Tween-20,10 * concentrated cleaning solution is the 0.1M phosphate buffer (pH7.4) that contains 0.5% Tween-20, colour developing thing A is o-phenylenediamine (OPD), colour developing liquid B is the citric acid-phosphate buffer that contains 0.5 ‰ hydrogen peroxide, the leucomalachite green monoclonal antibody is that the monoclonal antibody that 10 μ l purify adds 50 μ l cow's serum/pipe lyophilized products, stop buffer is the 2M sulfuric acid solution, being formulated as of LMG standard solution: accurately take by weighing LMG 10mg, with 0.1ml 0.1mol/L dissolving with hydrochloric acid, use ddH then earlier
2O is diluted to 10ml, is 0,1.6,8,40,200 with ddH2O preparation series concentration, the standard LMG solution of 1000ng/ml.
The trace routine of leucomalachite green indirect competitive ELISA kit for detecting of the present invention is:
(1) dilution of 10 * concentrated cleaning solution is cleansing solution for 10 times; (2) the even matter of sample to be checked is also extracted with ethyl acetate and cyclohexane, regulating pH value is that dilution in 1: 4 is done with dilution in about 7 backs; (3) in the part micropore, add 50 μ l standard solution respectively, adding the test sample solution that 50 μ l have finished pre-treatment in the micropore in addition, in a hole, add the control wells that 100 μ l cleansing solutions do not add monoclonal antibody; (4) except that control wells, in each micropore, add the suitably anti-leucomalachite green monoclonal antibody of dilution of 50 μ l again, hatched 120 minutes for 37 ℃, dry, every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (5) every hole adds the sheep anti mouse Ig-HRP of the dilution in 1: 1000 of 100 μ l, hatches 90 minutes for 37 ℃, dries, and every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (6) the liquid B that will develop the color moves among the colour developing thing A entirely, behind the dissolving mixing, adds 100 μ l colour developing liquid in every micropore, and 37 ℃ of lucifuges were hatched 10-15 minute; (7) add 50 μ l stop buffers, under the 490nm wavelength, read every hole absorbance value with microplate reader.
The above-mentioned specific practice of stating step (2) is: (a) get and shred sample and insert in the centrifuge tube, add 2mL adding ethyl acetate by every gram sample, fully smash even matter; (b) the centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; (c) mixed the extraction vortex 1 minute with equivalent cyclohexane and distilled water, get the cyclohexane layer, add 12.5 μ l by every gram sample and add 1M HCl, vortex 1 minute leaves standstill 30min; (d) add 2 times to the distilled water of HCl liquid measure, the centrifugal 10min of 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH 6.0～8.0 with 1N NaOH, carry out 4 times of dilutions with dilution after, get 50uL and analyze.
Malachite green indirect competitive ELISA kit for detecting of the present invention, its criterion that detects sample is: first basis of calculation sample inhibiting rate (B/BO), being ordinate then with the inhibiting rate, is horizontal ordinate with the logarithm value of standard specimen leucomalachite green concentration, does typical curve; Just can read the concentration of corresponding leucomalachite green from curve according to the B/BO value of each sample.
The good effect that the present invention is useful is: simple to operate, can better satisfy the exigence that at present the aquatic products malachite green vestigial is detected, be used for customs's detection, the detection of food hygiene department etc., be easy to apply on a large scale, have vast market prospect and bigger economical, societal benefits.
The present invention has following advantage:
(1) high specificity, the susceptibility height, security is good.Anti-leucomalachite green monoclonal antibody indirect competitive ELISA kit for detecting serves as forming of basis preparation with the monoclonal antibody of the pure antigen preparation of synthetic; Do not contain antibody, only combine, not with other antibiotic medicine of aquatic livestock and disinfectant generation cross reaction with corresponding leucomalachite green specific bond or with the malachite green part that contains same female ring structure at carrier protein.Therefore has very high specificity and susceptibility.
(2) easy and simple to handle quick.When using the malachite green indirect competitive ELISA kit for detecting to detect leucomalachite green, need not to join other reagent in addition, sample need not aseptic process, is the decidable testing result in 5-6 hour by the kit explanation.
(3) result judges image, accurate, reliable.The malachite green indirect competitive ELISA kit for detecting is with the qualitative demonstration testing result of the depth of colour developing, and promptly detecting the hole, orange colour colour developing to occur negative, is yellow or brown after the termination, and is colourless and light color is positive, and the result judges image.Adopt the machine-readable number of microplate reader quantitatively to show testing result, reduce subjectivity, accurately and reliably.
(4) cost is low, batch detection.But malachite green indirect competitive ELISA kit for detecting batch detection settles at one go, and is with low cost, small investment, instant effect.
Description of drawings
Fig. 1 suppresses curve for the LMG standard, and the vertical seat table among the figure is inhibiting rate=OD logarithm that (adding LMG)/the horizontal seat table of OD (not adding LMG) is the LMG solution concentration.
Fig. 2 is the light absorption value coefficient of variation (CV%) figure of the variable concentrations standard solution of test kit, and the vertical seat table among the figure is the coefficient of variation (CV%), and horizontal seat table is the logarithm of LMG solution concentration.
Embodiment
According to technical scheme of the present invention, be prepared as follows:
(1) preparation of leucomalachite green micropore check-out console
0.2M carbonate buffer solution with pH9.6 is made coating buffer; leucomalachite green and albumin conjugate (LMG-RSA or LMG-OVA) are diluted to 2 μ g/ml; add in the polystyrene micropore plate by 100 μ l/ holes; 4 ℃ of bags are spent the night; dry; the phosphate buffer that adds the 0.01M PH7.4 that contains 1% gelatin by 200 μ l/ holes sealed 2 hours for 37 ℃; washing dries; add 20% sucrose phosphate buffer room temperature protection 3 hours again; after putting the hothouse drying, preserve in the packaging bag that contains drying agent of packing into.
(2) sample diluting liquid, cleansing solution, monoclonal antibody dilution, the preparation of stop buffer
Sample or serum dilution are the 0.01M phosphate buffer (KH that contains 0.05% Tween-20
2PO
40.2g, Na
2HPO
412H
2O 2.9g, NaCl 8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again); 10 * concentrated cleaning solution is the 0.1M phosphate buffer (KH that contains 0.5% Tween-20
2PO
42g, Na
2HPO
412H
2O29g, NaCl 80g is settled to 1000ml, pH7.4 adds the 5ml Tween-20 again); Monoclonal antibody dilution: in the monoclonal antibody pipe, add 500 μ l dilutions, get the partial antibody dilution and dilute 200-500 more doubly; Stop buffer is the 2M sulfuric acid solution, promptly measures the 111.2ml concentrated sulphuric acid (18M) dilution and is settled to 1000ml.
(3) preparation of leucomalachite green standard solution
The preparation of LMG standard solution: accurately take by weighing LMG 10mg, with 0.1ml 0.1mol/L dissolving with hydrochloric acid, be diluted to 10ml with ddH20 then earlier.With ddH2O preparation series concentration is 0,0.32,1.6,8,40,200, the standard LMG solution of 1000ng/ml.
(4) preparation of colour developing liquid
Taking by weighing 2mg o-phenylenediamine (OPD) packs in the 6ml bottle as colour developing A; Getting PH5.0 phosphate-citrate salts damping fluid 5.9ml adds 3% hydrogen peroxide, 100 μ l and is mixed with colour developing liquid B; PH5.0 phosphate-citrate salts damping fluid preparation: Na
2HPO
412H
2O 1.84g, citric acid 0.467g adding distil water is to 50ml.
(5) sample pre-treatments:
The pre-treatment of flesh of fish sample: 1) get 4g and shredded sample and insert in the centrifuge tube, add 8mL ethyl acetate again, under homogenizer, fully smash; 2) centrifugal then 4000rpm/min 10min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; 3) mix the extraction vortex 1 minute with 1ml cyclohexane and 1ml distilled water, get the cyclohexane layer; Add 50 μ l 1M HCl, vortex 1 minute leaves standstill 30min; 4) add 0.1ml distilled water, centrifugal 8000rpm/min 10min discards the upper strata cyclohexane, drawing subnatant (if emulsion is arranged, after please earlier most of upper strata liquid-normal hexane being taken out, inserts glass tube in 80 ℃ of water again, water proof heated about 5～10 minutes, can improve the emulsification situation).Adjust to pH 6.0～8.0 with 1N NaOH, carry out 4 times of dilutions with dilution after, get 50uL and analyze.
(6) kit detecting operation program
Its trace routine is: (1) is cleansing solution for 10 times with the dilution of 10 * concentrated cleaning solution; (2) the even matter of flesh of fish sample to be checked is also extracted with ethyl acetate and cyclohexane, regulating pH value is that dilution in 1: 4 is done with dilution in about 7 backs, (3) in suitable micropore, add 50 μ l standard solution respectively, in other micropore, add 50 μ l and finished the sample solution of pre-treatment, in a hole, add control wells that 100 μ l cleansing solutions the do not add monoclonal antibody control wells when being used to measure the OD value.(4) except that control wells, in each micropore, add the suitably anti-leucomalachite green monoclonal antibody of dilution of 50 μ l again, hatched 120 minutes for 37 ℃, dry, every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (5) every hole adds the sheep anti mouse Ig-HRP of the dilution in 1: 1000 of 100 μ l, hatches 90 minutes for 37 ℃, dries; Every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (6) the liquid B that will develop the color moves in the colour developing thing A bottle entirely, behind the dissolving mixing, adds 100 μ l colour developing liquid in every micropore, and 37 ℃ of lucifuges were hatched 10-15 minute; (5) add 50 μ l stop buffers, under the 490nm wavelength, read every hole absorbance value (OD490nm value) with microplate reader.
(7) criterion as a result
B/BO value according to each sample just can be read corresponding leucomalachite green concentration from curve, multiply by corresponding extension rate again.
Be the reliability of checking effect of the present invention, carry out following evaluation:
(1) the LMG standard suppresses curve and sensitivity
Get the ELISA Plate of having sealed, the serial LMG standard solution for preparing (0,1.6,8,40,200,1000ppb) 50uL/ hole is mixed in the ELISA Plate hole with the monoclonal antibody working fluid 50uL/ hole of 20,000 times of dilutions, in a hole, add the control wells that 100 μ l cleansing solutions do not add monoclonal antibody, 37 ℃ in 37 ℃ of incubation 2h, wash plate; Add 1: 1000HRP enzyme mark sheep anti mouse (two is anti-), plate in 37 ℃ of incubation 1.5h, is washed in the 100uL/ hole; Every hole adds the low thing colour developing of freshly prepared OPD liquid 100uL, 37 ℃ of darkroom reaction 15min; Add 2mol/L H
2SO
4The reaction of 50ul color development stopping is with microplate reader interpretation under wavelength 490nm.With inhibiting rate I is ordinate, and the logarithm of LMG solution concentration is a horizontal ordinate, draws out typical curve.Inhibiting rate I=B/BO (B is sample OD value, and BO is blank OD value).Competitive ELISA result and correlation parameter see Table 1, and standard suppresses curve and sees Fig. 1
Table 1 indirect competitive ELISA result's correlation parameter
| LMG concentration of standard solution (μ g/ml) | LMG concentration logarithm | OD average (B) | Inhibiting rate (B/B0) | Standard deviation (SD) | The coefficient of variation (CV%) |
| 5 1 0.2 0.04 0.008 0.0016 0 | 0.7 0 -0.7 -1.4 -2.1 -2.8 | 0.012 0.077 0.424 0.699 0.825 0.943 1.096 | 0.011 0.070 0.388 0.638 0.754 0.860 1 | 0.00134 0.00732 0.02843 0.03147 0.03462 0.03163 0 | 12.48 10.394 7.3275 4.9301 4.5939 3.6798 0 |
The regression curve equation is Y=-0.0814X
2-0.5058X+0.0731, R
2=0.9979
Can see that from Fig. 1 the logarithm value of (B/Bo) and LMG concentration is the One-place 2-th Order curved line relation in the 0.0016-1 μ g/ml scope, related coefficient is r=0.9979, with the BO-3SD extrapolation method as calculated sensitivity be 0.0023 μ g/ml.
(2) repeatability as a result
Precision test at the LMG test kit repeats 6 times between batch, and the light absorption value coefficient of variation (CV%) of gained variable concentrations standard solution shows that this product has very high repeatability as shown in Figure 2:
(3) recovery is calculated
Be taken at adding leucomalachite green standard specimen in the blank flesh of fish of 4g, after extract is done suitably dilution, carry out ELISA and measure.Multiply by the content that corresponding extension rate is leucomalachite green the sample again according to OD value and B/BO value after typical curve checks in leucomalachite green content, calculate recovery rate sees Table 2 then.
The recovery of table 2 working sample
| Add LMG amount (μ g.g-1) in the sample | The amount of the LMG that measures | Recovery % |
| 250 125 12.5 2.5 | 265±12.4 120±5.5 11.5±0.8 2.8±0.20 | 106±4.85 96±4.4 92±6.4 112±6.0 |
Can find out recovery 92-112% from last table
(4) specificity as a result
Carry out specificity cross reaction test at following medicine utilization competitive ELISA, the result is as follows:
Table 3 leucomalachite green and several structure or act on the cross reaction of similar medicine
| Medicine | Cross reaction (%) |
| The blue chloramphenicol diethylstilbestrol of leucobase of malachite green malachite green paramagenta crystal violet methyl tetracycline | 100 36.56 ＜0.02 ＜0.06 ＜0.02 ＜0.02 ＜0.02 ＜0.02 |
Claims (6)
1, leucomalachite green indirect competitive ELISA kit for detecting, be provided with the micropore check-out console in the kit, the monoclonal antibody lyophilized products, ELIAS secondary antibody, sample diluting liquid, 10 * concentrated cleaning solution, colour developing thing A, colour developing liquid B, stop buffer and standard solution is characterized in that: the micropore check-out console is for wrapping by the removable 96 hole ELISA Plate of leucomalachite green and albumin conjugate (LMG-RSA or LMG-OVA), and standard solution is leucomalachite green (LMG) standard solution.
2; a kind of leucomalachite green indirect competitive ELISA kit for detecting according to claim 1; the best preparation scheme that it is characterized in that the micropore check-out console is: the 0.2M carbonate buffer solution with pH9.6 is made coating buffer; leucomalachite green and albumin conjugate (LMG-RSA or LMG-OVA) are diluted to 2 μ g/ml; add in the polystyrene micropore plate by 100 μ l/ holes; 4 ℃ of bags are spent the night; dry; contain 1% gelatin by the adding of 200 μ l/ holes; 37 ℃ of sealings of the phosphate buffer of PH7.4 2 hours; washing dries; added 20% sucrose phosphate buffer room temperature protection again 3 hours, put the hothouse drying after, preserve in the packaging bag that contains drying agent of packing into.
3, a kind of leucomalachite green indirect competitive ELISA kit for detecting according to claim 1, it is characterized in that: said monoclonal antibody lyophilized products is an anti-leucomalachite green monoclonal antibody of utilizing leucomalachite green and albuminous conjugate immune mouse and the preparation of monoclonal antibody technology of preparing, can with the leucomalachite green specific bond.
4, a kind of leucomalachite green indirect competitive ELISA kit for detecting according to claim 1, it is characterized in that: described sample diluting liquid is the 0.01M phosphate buffer (pH7.4) that contains 0.05% Tween-20,10 * concentrated cleaning solution is the 0.1M phosphate buffer (pH7.4) that contains 0.5% Tween-20, colour developing thing A is o-phenylenediamine (OPD), colour developing liquid B is the citric acid-phosphate buffer that contains 0.5 ‰ hydrogen peroxide, the leucomalachite green monoclonal antibody is that the monoclonal antibody that 10 μ l purify adds 50 μ l cow's serum/pipe lyophilized products, stop buffer is the 2M sulfuric acid solution, being formulated as of LMG standard solution: accurately take by weighing LMG 10mg, with 0.1ml 0.1mol/L dissolving with hydrochloric acid, use ddH then earlier
2O is diluted to 10ml, uses ddH
2O preparation series concentration is 0,1.6,8,40,200, the standard LMG solution of 1000ng/ml.
5, a kind of leucomalachite green indirect competitive ELISA kit for detecting according to claim 1, its trace routine is:
(1) dilution of 10 * concentrated cleaning solution is cleansing solution for 10 times; (2) the even matter of sample to be checked is also extracted with ethyl acetate and cyclohexane, regulating pH value is that dilution in 1: 4 is done with dilution in about 7 backs; (3) in the part micropore, add 50 μ l standard solution respectively, adding the test sample solution that 50 μ l have finished pre-treatment in the micropore in addition, in a hole, add the control wells that 100 μ l cleansing solutions do not add monoclonal antibody; (4) except that control wells, in each micropore, add the suitably anti-leucomalachite green monoclonal antibody of dilution of 50 μ l again, hatched 120 minutes for 37 ℃, dry, every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (5) every hole adds the sheep anti mouse Ig-HRP of the dilution in 1: 1000 of 100 μ l, hatches 90 minutes for 37 ℃, dries, and every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; (6) the liquid B that will develop the color moves among the colour developing thing A entirely, behind the dissolving mixing, adds 100 μ l colour developing liquid in every micropore, and 37 ℃ of lucifuges were hatched 10-15 minute; (7) add 50 μ l stop buffers, under the 490nm wavelength, read every hole absorbance value with microplate reader.
6, a kind of leucomalachite green indirect competitive ELISA kit for detecting according to claim 5, the specific practice of described step (2) is: (a) get and shred sample and insert in the centrifuge tube, add 2mL adding ethyl acetate by every gram sample, fully smash even matter; (b) the centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; (c) mixed the extraction vortex 1 minute with equivalent cyclohexane and distilled water, get the cyclohexane layer, add 12.5 μ l by every gram sample and add 1MHCl, vortex 1 minute leaves standstill 30min; (d) add 2 times to the distilled water of HCl liquid measure, the centrifugal 10min of 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH6.0～8.0 with 1N NaOH, carry out 4 times of dilutions with dilution after, get 50uL and analyze.
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| CN101226194B (en) * | 2008-01-18 | 2011-11-16 | 华南农业大学 | Malachite green vestigial ELISA detection kit and usage method thereof |
| CN101948531A (en) * | 2010-08-23 | 2011-01-19 | 上海海洋大学 | Complete antigen for detecting malachite green and preparation method thereof |
| CN102004099A (en) * | 2010-09-17 | 2011-04-06 | 大连理工大学 | Method for detecting malachite green through electrochemiluminescence |
| CN102353768A (en) * | 2011-07-07 | 2012-02-15 | 清华大学深圳研究生院 | Quantum dot based immunofluorescence detection method for malachite green and special kit |
| CN102353768B (en) * | 2011-07-07 | 2013-11-27 | 清华大学深圳研究生院 | Quantum dot based immunofluorescence detection method for malachite green and special kit |
| CN103288661A (en) * | 2012-03-03 | 2013-09-11 | 北京勤邦生物技术有限公司 | Preparation method and application of malachite green hapten |
| CN102661946A (en) * | 2012-04-11 | 2012-09-12 | 广州万联生物科技有限公司 | Malachite chemiluminescence ELISA detection method and kit |
| CN103304662A (en) * | 2013-04-27 | 2013-09-18 | 上海海洋大学 | Monoclonal antibody of leucomalachite green and preparation method and usage thereof |
| CN103728448A (en) * | 2013-12-31 | 2014-04-16 | 上海海洋大学 | Preparation method of horse radish peroxidase labeled leuco-malachite green immunogen |
| CN103983767A (en) * | 2014-05-06 | 2014-08-13 | 中国食品药品检定研究院 | Kit for detecting alpha-1,3Gal antigen and application thereof |
| CN103983767B (en) * | 2014-05-06 | 2016-01-06 | 中国食品药品检定研究院 | A kind of kit and application thereof detecting α-1,3Gal antigen |
| CN105277692A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay method for determination of malachite green in aquatic products |
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