CN103472228B - Malachite green vestigial single step chemiluminescence enzyme immunoassay detection method and kit - Google Patents
Malachite green vestigial single step chemiluminescence enzyme immunoassay detection method and kit Download PDFInfo
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Abstract
The invention discloses a kind of malachite green vestigial single step chemiluminescence enzyme immunoassay detection method and kit, this method combines indirect exzyme immunoassay and chemiluminescence, when not needing to prepare monoclonal antibody linked with peroxidase in addition, traditional two-step Chemiluminescence quantitative immunoassay is reduced to a step, and utilize 15 ~ 35% acetonitriles to the limited characteristic of high concentration leucomalachite green dissolving power, it can be used as sample dissolution liquid.This method is applied in animal derived food Malachite Green residue detection, has the features such as quick, easy, special, sensitive, accurate, sensing range is wide, more meets the requirement detected fast, has good application prospect.The IC of kit of the present invention
50be 0.45 μ g/L, the recovery is 86.37 ~ 116.84%, is 100% with the identical rate of standard detecting method, is suitable for trace analysis and the batch detection of malachite green.
Description
Technical field
The present invention relates to malachite green vestigial and detect analysis field, particularly relate to a kind of malachite green vestigial single step chemiluminescence enzyme immunoassay detection method and kit.
Background technology
Malachite green (MalachiteGreen, MG) molecular formula is C
23h
25n
2cl, belongs to triphenylmethane dye, the remarkable efficacy of Zeng Yinqi in prevention and therapy saprolegniasis, branchiomycosis, ich, fish-egg mould etc., and is widely used in fishery cultivating.Research in recent years finds, malachite green particularly its metabolic product has and significantly accumulates residual phenomena in aquatic animal body, and because MG has the harm such as Growth and reproduction ability, carcinogenicity making animal liver cell vacuolation, affect animal, serious threat is to aquatic animal and human health, so many countries such as the U.S., Canada, European Union all forbid using malachite green in aquatic products; European Union, Australia and New Zealand determine that the minimum law enforcement limitation of malachite green and leucomalachite green is 2 μ g/kg; China was also listed in 2002 " veterinary drug of food animal forbidding and compound inventory thereof " (Ministry of Agriculture announces No. 193), and list the monitoring of MG project in eel in annual plan first in " 2000 annual Chinese exports animal derived food poisonous and harmful substances remain Supervisory Surveillance Program ", and continue into the present.But owing to there is no the better substitute of malachite green at present, therefore some illegal retailers are still at illegal use, cause the malachite green vestigial event of exceeding standard to happen occasionally.
In view of the harm of malachite green, in succession making laws and forbid that it uses in food animal in countries in the world, and establishes many method for detecting residue, for detecting the residual quantity of malachite green.Conventional rationalization method (as high performance liquid chromatography, gas chromatography, LC-MS, thin-layer chromatography) and immunological method (as radioimmunology, fluoroimmunoassay, enzyme linked immunosorbent assay, electrochemical methods, chemiluminometry etc.).Wherein physico-chemical method has precision and the high advantage of accuracy, often be used as the detection method of final confirmation, and immunological method is because of features such as it is easy, quick, cheap, high sensitivity, high fluxs, compensate for the deficiency of physico-chemical method preferably, be applied to the detection of medicament residue in recent years more and more.
Test confirms, malachite green enters in animal body can be rapidly converted into leucomalachite green (LeucomalachiteGreen, LMG), in 24h, conversion ratio reaches 80%, and LMG is water insoluble, be difficult to excrete, release rate is slow, and the residence time reaches more than 100 days, and residual toxicity is higher than MG, being regarded as residual marker in a lot of country, is therefore that detected object more can illustrate the situation that MG remains in animal body with LMG.
Chinese patent application " a kind of chemiluminescence enzyme linked immune detection method of malachite green and kit " (application number 201210106196.3 publication date on September 12nd, 2013) adopts two-step chemiluminescent enzyme-linked immunosorbent immune detecting system and detection method to detect testing sample Malachite Green is residual, and operation steps is many, analysis time is long, sensing range is narrow.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of quick, easy, special, sensitive, accurate, malachite green vestigial single step chemiluminescence enzyme immunoassay detection method that sensing range is wide and kit.
Another technical matters that the present invention will solve provides the leucomalachite green monoclonal antibody that a species specificity is high, affinity is strong.
In order to solve the problems of the technologies described above, the present invention by the following technical solutions:
Malachite green vestigial single step chemiluminescence enzyme immunoassay detection method, comprises the following steps:
<1> process testing sample;
<2> using the conjugate (LMG-OVA) of haptens leucomalachite green and carrier protein ovalbumin as antigen coated in luminous solid phase carrier, after closing, add series standard sample successively or react through the testing sample of pre-treatment, ELIAS secondary antibody HRP-goat anti-mouse IgG and leucomalachite green monoclonal antibody, and then add chemical luminescence for liquid, measure the luminous value of series standard sample and testing sample;
The luminous value that <3> records with step <2> calculates inhibiting rate, drawing standard curve, and the content calculating testing sample Malachite Green according to the regression equation of typical curve and the inhibiting rate of testing sample.
Step <1> is undertaken by following operation: get 5g tissue sample and add 10mL acetonitrile ultrasonic oscillation 15min, the centrifugal 10min of 4000r/min, get supernatant and add 20g neutral alumina, concussion mixing 5min, the centrifugal 5min of 4000r/min, get supernatant to dry up in nitrogen, add 15 ~ 35% acetonitrile solutions and dissolve, for detecting;
Step <2> is undertaken by following operation: with coating buffer, antigen diluent is become 5 μ g/mL, and every hole adds 100 μ L, 4 DEG C hatch 10 ~ 16h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1 ~ 2h for 37 DEG C, and incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations; Chemiluminescent plate is taken out from 4 DEG C, treat that equalized temperature is to room temperature, according to every hole 50 μ L, series standard sample solution and testing sample are added Chemiluminescent plate, 3 holes are respectively established to repeat, then ELIAS secondary antibody HRP-goat anti-mouse IgG, leucomalachite green monoclonal antibody is added according to every hole 50 μ L successively, mixing, hatches 60min for 37 DEG C; Incline liquid, washing 5 times, patting dry with washing trigger; Add 100 μ L chemical luminescence for liquid in every hole, mixing, measures each hole luminous value as early as possible.
Confining liquid is the skimmed milk power of 5%; The dilution of leucomalachite green monoclonal antibody is 1.15 μ g/mL; The concentration of series standard sample solution is 0 μ g/L, 0.001 μ g/L, 0.01 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L; Coating buffer is the carbonate buffer solution of pH9.6.
Malachite green vestigial single step chemiluminescence enzyme immunoassay detection kit, comprises the Chemiluminescent plate, leucomalachite green series standard sample solution, leucomalachite green monoclonal antibody working fluid, concentrated cleaning solution, HRP-goat anti-mouse IgG (ELIAS secondary antibody) working fluid, the chemical luminescence for liquid that are coated with envelope antigen (LMG-OVA); Envelope antigen is the conjugate of leucomalachite green and carrier protein ovalbumin; Leucomalachite green series standard sample solution is 7 concentration gradients, is 0 μ g/L, 0.001 μ g/L, 0.01 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L respectively; Concentrated cleaning solution is by NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, Tween-20 0.50mL, adding water is settled to 100mL and makes; Chemical luminescence for liquid is from super quick ECL chemical luminescence reagent kit P0018--BeyoECLPlus.
The application of mentioned reagent box in animal derived food Malachite Green residue detection.
Leucomalachite green monoclonal antibody, by following operation preparation: using haptens leucomalachite green and the clear albuminous conjugate (LMG-BSA) of carrier proteins Bovine as immunogene, immunity female BAl BIc/c mouse in 6 week age, conventionally carry out the screening of Fusion of Cells and positive strain, continuous clone is involved in a criminal case 3 times to the positive cell filtered out, through qualification, frozen and after building strain, carry out the preparation of ascites, then by purifying ascites, the monoclonal antibody of energy specific recognition leucomalachite green is obtained.
Above-mentioned leucomalachite green monoclonal antibody, by following operation preparation:
<1> animal immune
By 6 of health week age female BAl BIc/c mouse by after fundamental immunity and 5 booster immunizations, impacts is carried out to the strongest the highest, competitive mouse of tiring immune;
<2> Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing eyeball of mouse bloodletting, collect blood, asepticly get spleen, cell-fusion techniques is utilized mouse immune splenocyte and Sp2/0 cell to be merged, cell suspension after fusion adds to and is covered with in 96 orifice plates of feeder cells in advance, adopts HAT Selective agar medium screening fused cell;
When Growth of Cells is to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatant, adds in the ELISA Plate of envelope antigen (LMG-OVA); Survey cells and supernatant with indirect elisa method to tire, limiting dilution assay is adopted to clone 3 times continuously to positive cell, until when positive rate is 100%, cell line is carried out that expansion is cultivated, qualification, frozen and build strain, carry out Continuous Cultivation to cell line after qualification to go down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations;
<3> cell cryopreservation and recovery
With cryopreserving liquid, the hybridoma being in exponential phase is made cell suspension, be sub-packed in cryopreservation tube, be placed in liquid nitrogen and preserve; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal segregation cryopreserving liquid, move in cell bottle and cultivate;
The preparation and purification of <4> ascites
Adopt in body and induce method, sterilizing paraffin oil is injected Balb/c mouse peritoneal in 8 week age, injects hybridoma after 7-14 days, after 7-10 days, collect ascites; After the ascites saturated ammonium sulfate method of collecting is pure at the beginning of carrying out, then be further purified with affinity chromatography, obtain leucomalachite green monoclonal antibody.
The present invention has inquired into first and has detected the residual single step chemiluminescence enzyme immune mechanism of leucomalachite green, inventor combines indirect exzyme immunoassay and chemiluminescence, when not needing to prepare monoclonal antibody linked with peroxidase in addition, traditional two-step Chemiluminescence quantitative immunoassay is reduced to a step, and utilize 15 ~ 35% acetonitriles to the limited characteristic of high concentration leucomalachite green dissolving power, it can be used as sample dissolution liquid, successfully establish single step malachite green chemiluminescence enzyme immunoassay detection method and kit.The linear detection range (0.001 ~ 100 μ g/L) of the present invention's " single stage method " than " two-step approach " and ELISA wide, lowest detectable limit (0.0016 μ g/L) reduces 10 ~ 100 times, more meets trace analysis and batch detection; In addition, 15 ~ 35% acetonitriles of the present invention's preparation are limited to high concentration malachite green dissolving power, when testing sample Malachite Green content exceed highest detection of the present invention limit 100 μ g/L time, there will be macroscopic muddiness, therefore, when detecting the sample of malachite green severe overweight (content exceed above-mentioned two kinds of method sensing ranges and lower than 100 μ g/L), without the need to diluting further sample and duplicate detection; And when Malachite green residues exceedes highest detection in limited time, then utilizing 15 ~ 35% acetonitrile sample dissolution to point out intuitively need dilute further to sample, and then detects.By above improvement, this law simplifies operation steps, decreases workload, reduce test error, shorten detection time, save testing cost, there is the features such as quick, special, sensitive, accurate, more meet the requirement detected fast, there is good application prospect.The IC of kit of the present invention
50be 0.45 μ g/L, the recovery is 86.37 ~ 116.84%, is 100% with the identical rate of standard detecting method, is suitable for trace analysis and the batch detection of malachite green.
Accompanying drawing explanation
Fig. 1 is the typical curve of embodiment 2 malachite green vestigial single step chemiluminescence enzyme immunoassay detection method.
In figure: X-axis is the logarithm value of malachite green gradient concentration standard model concentration; Y-axis is that the luminous value of each concentration malachite green standard model is divided by " 0 " concentration hole luminous value (RLU/RUL
0%).
Embodiment
The preparation of embodiment 1 leucomalachite green monoclonal antibody
1.1 animal immune
Using haptens leucomalachite green and the clear albuminous conjugate (LMG-BSA) of carrier proteins Bovine as immunogene, after fundamental immunity and 5 booster immunizations are carried out to the female BAl BIc/c mouse in 6 week age of health, measure serum titer and IC
50, choose the strongest the highest, competitive mouse of tiring and carry out impact immunity;
1.2 Fusion of Cells and colony screening
Impact immunity after 3 days, put to death after extracing eyeball of mouse bloodletting, collect blood, asepticly get spleen, cell-fusion techniques is utilized mouse immune splenocyte and Sp2/0 cell to be merged, cell suspension after fusion adds to and is covered with in 96 orifice plates of feeder cells in advance, adopts HAT Selective agar medium screening fused cell;
When Growth of Cells is to culture hole area 1/10, aseptic accurate absorption 100 μ L supernatant, adds in the ELISA Plate of envelope antigen LMG-OVA; Survey cells and supernatant with indirect elisa method to tire, limiting dilution assay is adopted to clone 3 times continuously to positive cell, until when positive rate is 100%, cell line is carried out that expansion is cultivated, qualification, frozen and build strain, carry out Continuous Cultivation to cell line after qualification to go down to posterity more than 2 months, detect its stability and antibody-secreting ability every 5 generations; 1.3 cell cryopreservations and recovery
With cryopreserving liquid, the hybridoma being in exponential phase is made cell suspension, be sub-packed in cryopreservation tube, be placed in liquid nitrogen and preserve; Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, centrifugal segregation cryopreserving liquid, move in cell bottle and cultivate;
The preparation and purification of 1.4 ascites
Adopt in body and induce method, sterilizing paraffin oil is injected Balb/c mouse peritoneal in 8 week age, injects hybridoma after 7-14 days, after 7-10 days, collect ascites; After the ascites saturated ammonium sulfate method of collecting is pure at the beginning of carrying out, then be further purified with affinity chromatography, through SDS-PAGE electroresis appraisal, confirm that the leucomalachite green monoclonal antibody 1H10 obtained reaches electrophoresis pure.The qualification of 1.5 monoclonal antibodies
1.5.1 Hybridoma Cell Culture supernatant and the purified antibodies mensuration of tiring
Adopt indirect ELISA method, what measure LMG-McAb in Hybridoma Cell Culture supernatant tires as 1:640, and the titer of ascites after affinitive layer purification reaches 1:5 × 10
5.
1.5.2 the mensuration of affinity
By non-competing ELISA, respectively with OD
450value is ordinate, with the concentration of LMG-McAb for horizontal ordinate, draws affine curve, and the affinity costant with the formula under the different antigen coated concentration of 1 calculating, the affinity costant (Ka) drawing leucomalachite green monoclonal antibody of averaging is 6.2 × 10
8l/mol.
1.5.3 the qualification of subclass
Adopting the immunoglobulin subclass detection kit of SouthernBiotech company of the U.S. to measure leucomalachite green monoclonal antibody 1H10 is IgG
1subclass, κ light chain.
The foundation of embodiment 2 malachite green vestigial single step chemiluminescence enzyme immunoassay detection method
The preparation of 2.1 related reagents
Carbonate buffer solution (pH9.6, i.e. coating buffer): accurately take Na
2cO
31.59g, NaHCO
32.93g, after ultrapure water dissolves, regulates pH to 9.6, is settled to 1000mL.
Cleansing solution (pH7.4): accurately take NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, after ultrapure water dissolves, regulate pH to 7.4, add Tween-20 0.50mL, be settled to 1000mL.
Phosphate buffer (PBS) (pH7.4): accurately take NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, after ultrapure water dissolves, regulate pH to 7.4, be settled to 1000mL.
Confining liquid: accurately take skimmed milk power 1.0g, adds 20mL phosphate buffer, stirs to dissolving completely.
The super quick ECL chemical luminescence reagent kit of BeyoECLPlus(, P0018,100mL): purchased from green skies biotechnology research institute.
The determination of 2.2 antigens, antibody dilution multiple
Adopt square formation titration determination antigen, antibody dilution multiple: by LMG-OVA(10mg/mL) wrap by Chemiluminescent plate according to after 1:2000,1:4000,1:6000 and 1:8000 dilution, and dilute leucomalachite green monoclonal antibody (4.6mg/mL) with 1:1000,1:2000,1:3000 and 1:4000, react, select reaction normal curve IC
50(concentration of medicine during 50% inhibiting rate, the sensitivity of namely reacting) minimum condition is as the antigen coated condition of the best.1:2000,1:4000 is respectively, i.e. 5 μ g/mL, 1.15 μ g/mL by the optimum diluting multiple of table 1 result determination envelope antigen and monoclonal antibody.
The determination of table 1 antigen, antibody dilution multiple
The determination of 2.3 confining liquids
With the antigen coated condition bag of the best by Chemiluminescent plate, using the PBS of 20mM, 5% skimmed milk power, 1% BSA, 5% hyclone as closed material, select reaction normal curve IC
50minimum, as reaction sealed liquid.Determine that the skimmed milk power of 5% is best by table 2 result.
The determination of table 2 confining liquid
| 10mM PBS | 5%Skim milk | 1%BSA | 1%OVA | |
| IC 50 | 56.829 | 3.067 | 3.367 | 23.211 |
2.4 acetonitrile concentrations are on the impact of reaction
Acetonitrile deionized water is diluted to 0%, 15% ~ 35%, 40% ~ 60%, 70% ~ 100%, and prepare 10ppbLMG standard items respectively, react, marked the ratio of product RLU by the 10ppb standard items and 0 of more each reaction, select the minimum acetonitrile solution of ratio to be the solvent preparing LMG standard items.From table 3, when use 15 ~ 35% acetontrile LMG standard items react, RLU
10/ RLU
0ratio is minimum, illustrates that 15 ~ 35% acetonitrile solutions can improve the susceptibility of reaction, and supposition is caused by the impact of organic solvent on albumino reaction.In addition, respectively with above concentration acetonitrile solution preparation 100ppbLMG standard items, find when dissolving more than 100ppbLMG standard items with 15 ~ 35% acetonitrile solutions, there will be muddiness, form suspension, this also just can point out tester need dilute further sample intuitively, disappears until muddy.
Table 3 acetonitrile concentration is on the impact of reaction
| 0% acetonitrile | 15% ~ 35% acetonitrile | 40% ~ 60% acetonitrile | 70% ~ 100% acetonitrile | |
| RLU 10/RLU 0 | 63.65% | 33.15% | 78.38% | 87.33% |
The determination of 2.5 competitive reaction times
Under aforementioned fixed top condition, select 15min, 30min, 60min and 90min as the competitive reaction time respectively, with reaction normal curve IC
50as Judging index, the R of combined standard curve simultaneously
2value, RLUmax/IC
50select the competitive reaction time.Determine that 60min is the best competitive reaction time by table 4 result.
The determination of table 4 competitive reaction time
| Formula | R 2 | IC 50（μg/L） | RLUmax/IC 50 | |
| 20min | y=-27.434x+74.484 | 0.9595 | 7.81 | 57002 |
| 45min | y=-27.489x+68.523 | 0.9843 | 4.72 | 169551 |
| 60min | y=-16.174x+48.742 | 0.9775 | 0.836 | 2122784 |
| 90min | y=-27.081x+81.071 | 0.9876 | 14.040 | 90321 |
The foundation of 2.6 typical curves, IC
50and lowest detectable limit
In optimal conditions, leucomalachite green is made into 0.001 ~ 100 μ g/L series concentration, each concentration 3 hole is repeated, and detects.With the logarithm value of standard model solution concentration for horizontal ordinate, with the luminous value of each concentration leucomalachite green standard items divided by " 0 " concentration hole luminous value (RLU/RUL
0%) be ordinate, drawing standard curve.As shown in Figure 1, the regression equation of typical curve is y=-10.462x+46.389, R
2=0.9829, curve presents good linear relationship between 0.001 ~ 100 μ g/L, and lowest detectable limit is 0.016 μ g/L, IC
50be 0.45 μ g/L.
2.7 specificitys of the present invention
With analytical review method of the present invention, LMG analogue methylene blue and some common antibiotics (chloromycetin, diethylstilbestrol, sulfadimidine, ceftiofur sodium) are detected, measure IC
50, according to formula 2, calculate the cross reacting rate of each medicine.From table 5, the cross reacting rate of the method that the present invention sets up and common antibiotics is all less than 0.01%, shows that this law specificity is better.
Cross reacting rate=IC
50(leucomalachite green) IC50 (other drug) × lO0% (formula 2)
Table 5 specific assay result of the present invention
| Compound | Cross reacting rate (%) |
| Leucomalachite green | 100 |
| Methylene blue | <0.01 |
| Chloromycetin | <0.01 |
| Diethylstilbestrol | <0.01 |
| Sulfadimidine | <0.01 |
| Ceftiofur sodium | <0.01 |
2.8 accuracy of the present invention
Recovery experiment is carried out with basic, normal, high three concentration (0.1 μ g/L, 1 μ g/L, 10 μ g/L), each sample sets 3 holes and repeats, and measures its values of chemiluminescence, the mean value obtained is substituted into the regression equation of typical curve, obtain respective standard sample concentration value, calculate the recovery.Determine that the recovery of the present invention is between 86.37 ~ 116.84% by table 6 result, show that this law has reliable accuracy.
The measurement result of table 6 accuracy of the present invention
2.9 with the comparing of two-step Chemiluminescence quantitative immunoassay
React with single step of the present invention and conventional two-step method respectively under optimum reaction condition, calculate respective IC
50value and RLUmax/IC
50value, from table 7, the two index is close, in single stage method, standard items antigen or testing sample, LMG-McAb and ELIAS secondary antibody HRP-goat anti-mouse IgG are joined is coated with in the ELISA Plate of LMG-OVA simultaneously, comparatively two-step method, and single stage method decreases operation steps, shorten experimental period and be about 1.5h, more meet the requirement detected fast.
The comparative result of table 7 single step and two-step Chemiluminescence quantitative immunoassay
| Single step | Two-step | |
| IC 50（μg/L） | 0.45 | 0.4650 |
| RLUmax/IC 50 | 1165539 | 1009487 |
The development of embodiment 3 malachite green vestigial single step chemiluminescence enzyme immunoassay detection kit
According to the result of study of embodiment 1 and 2, assembling malachite green vestigial single step chemiluminescence enzyme immunoassay detection kit.
3.1 kit compositions
A is coated with the Chemiluminescent plate of envelope antigen (LMG-OVA);
B leucomalachite green series standard sample solution: 0 μ g/L, 0.001 μ g/L, 0.01 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L;
C leucomalachite green monoclonal antibody 1H10 working fluid;
DHRP-goat anti-mouse IgG working fluid;
E concentrated cleaning solution: NaCl8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o2.90g, KCl0.20g, Tween-20 0.50mL, add water and be settled to 100mL;
F chemical luminescence for liquid A liquid, B liquid (super quick ECL chemical luminescence reagent kit P0018--BeyoECLPlus);
The bag quilt of 3.2 Chemiluminescent plates
With coating buffer, antigen diluent is become 5 μ g/mL, every hole adds 100 μ L, 4 DEG C hatch 10 ~ 16h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then every hole adds 350 μ L confining liquids, and hatch 1h for 37 DEG C, incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations.
The application of embodiment 4 malachite green vestigial single step chemiluminescence enzyme immunoassay detection kit
The preparation of 4.1 reagent
A cleansing solution: use after the concentrated cleaning solution ultrapure water provided in kit is diluted 10 times.
B chemical luminescence for liquid: before interpolation chemical luminescence for liquid, chemical luminescence for liquid A liquid, B liquid are mixed according to 1:1.
The pre-treatment of 4.2 tissue samples
A gets 5g tissue sample in 50mL centrifuge tube;
B adds 10mL acetonitrile ultrasonic oscillation and extracts the centrifugal 10min of 15min, 4000r/min;
C gets supernatant and adds 20g neutral alumina, grease in adsorption sample, the centrifugal 5min of concussion mixing 5min, 4000r/min;
D gets supernatant and dries up in nitrogen, adds 15 ~ 35% acetonitrile solutions and dissolves, for detecting.
4.3 testing process
Chemiluminescent plate takes out by A from 4 DEG C, treats that equalized temperature is to room temperature;
Series standard sample solution and testing sample are added Chemiluminescent plate according to every hole 50 μ L by B, 3 holes are respectively established to repeat, then every hole adds 50 μ L ELIAS secondary antibody HRP-goat anti-mouse IgG, 50 μ L leucomalachite green monoclonal antibodies successively, and mixing, hatches 60min for 37 DEG C;
C inclines liquid, washing 5 times, patting dry with washing trigger;
Add 100 μ L chemical luminescence for liquid in the every hole of D, mixing, measures each hole luminous value as early as possible;
E is according to formula, and according to obtained sample luminous value, the inhibiting rate of calculation sample, and inhibiting rate is substituted into standard regressive method, calculates residual quantity; As sample once diluted, be then multiplied by extension rate, be the residual quantity of sample Malachite Green.
The detection of 4.4 samples
Gather totally 206 parts, commercially available shrimp, fish sample, carry out the pre-treatment of sample with reference to said method, then detect with kit of the present invention.Detect content more than 1 part of 2 μ g/ μ L, more than 4 parts of 1 μ g/ μ L, more than 1 part of 0.1 μ g/ μ L, all the other do not detect.
Embodiment 5 chemiluminescence Enzymoimmune reagent kit of the present invention compares with standard detecting method
Choose 10 parts of tissue samples, detect wherein leucomalachite green residual quantity with detection kit of the present invention and China's marine industry standard detecting method " the mensuration liquid phase chromatography of SC/T3021-2004 Malachite Green Residues in Aquatic Product " respectively, the results are shown in Table 8.Because kit lowest detectable limit of the present invention (0.0016 μ g/L) is far below industry standard detection method (4 μ g/L), so its sample that cannot detect can be detected.This result illustrates that the coincidence rate of two kinds of methods is 100%, and kit of the present invention has good effect.
The comparison of table 8 testing result
Claims (2)
1. a malachite green vestigial single step chemiluminescence enzyme immunoassay detection method, is characterized in that comprising the following steps:
<1> process testing sample;
<2> using the conjugate of haptens leucomalachite green and carrier protein ovalbumin as antigen coated in luminous solid phase carrier, after closing, add series standard sample successively or react through the testing sample of pre-treatment, ELIAS secondary antibody HRP-goat anti-mouse IgG and leucomalachite green monoclonal antibody, and then add chemical luminescence for liquid, measure the luminous value of series standard sample and testing sample;
The luminous value that <3> records with step <2> calculates inhibiting rate, drawing standard curve, and the content calculating testing sample Malachite Green according to the regression equation of typical curve and the inhibiting rate of testing sample;
Step <1> is undertaken by following operation: get 5g tissue sample and add 10mL acetonitrile ultrasonic oscillation 15min, the centrifugal 10min of 4000r/min, get supernatant and add 20g neutral alumina, concussion mixing 5min, the centrifugal 5min of 4000r/min, get supernatant to dry up in nitrogen, add 15 ~ 35% acetonitrile solutions and dissolve, for detecting;
Step <2> is undertaken by following operation: with coating buffer, antigen diluent is become 5 μ g/mL, and every hole adds 100 μ L, 4 DEG C hatch 10 ~ 16h after, incline coating buffer, with washing trigger washing chemistry luminous plaque 3 times, pats dry; Then, every hole adds 350 μ L confining liquids, hatches 1 ~ 2h for 37 DEG C, and incline deblocking liquid, washing 3 times, patting dry with washing trigger; After 37 DEG C of oven dry, use masking foil vacuum seal, 4 DEG C of preservations; Chemiluminescent plate is taken out from 4 DEG C, treat that equalized temperature is to room temperature, according to every hole 50 μ L, series standard sample solution and testing sample are added Chemiluminescent plate, 3 holes are respectively established to repeat, then ELIAS secondary antibody, leucomalachite green monoclonal antibody is added according to every hole 50 μ L successively, mixing, hatches 60min for 37 DEG C; Incline liquid, washing 5 times, patting dry with washing trigger; Add 100 μ L chemical luminescence for liquid in every hole, mixing, measures each hole luminous value as early as possible.
2. malachite green vestigial single step chemiluminescence enzyme immunoassay detection method according to claim 1, is characterized in that: described confining liquid is the skimmed milk power of 5%; The dilution of described leucomalachite green monoclonal antibody is 1.15 μ g/mL; The concentration of described series standard sample solution is 0 μ g/L, 0.001 μ g/L, 0.01 μ g/L, 0.1 μ g/L, 1 μ g/L, 10 μ g/L, 100 μ g/L; Described coating buffer is the carbonate buffer solution of pH9.6.
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| CN110954582B (en) * | 2019-12-30 | 2024-03-22 | 佛山职业技术学院 | Malachite green electrochemical sensor |
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Application publication date: 20131225 Assignee: Guangxi Boshu Agricultural Development Co.,Ltd. Assignor: GUANGXI VETERINARY Research Institute Contract record no.: X2023980044852 Denomination of invention: One Step Chemiluminescence Enzyme Immunoassay Detection Method and Kit for Malachite Green Residues Granted publication date: 20151125 License type: Common License Record date: 20231031 |
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