CN106124766A - Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi - Google Patents
Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi Download PDFInfo
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Abstract
The invention discloses and a kind of use the method for carbendazim content in carbendazim detection of specific antibody edible fungi.It is by 2 aminobenzimidazole stirring and dissolving in acetonitrile, add succinic anhydride, 40 DEG C of stirring reaction 4h, filtering reacting solution obtains filtering residue, being rinsed twice with the acetonitrile of 40 DEG C by filtering residue, obtain crude product, crude product is further through column chromatography purification, draw through thin layer chromatography and Mass Spectrometric Identification, it is thus achieved that product is 2 succinyl benzimidazoles.Then at 2 amino of 2 aminobenzimidazoles, the spacerarm of 4 carbon of coupling, as hapten, then obtains artificial antigen by active ester method coupling carrier albumen；Secondly, specific polyclonal antibody is obtained by animal immune, purification antiserum；Finally, utilize simplicity, quick, sensitive direct competive ELISA method carbendazim in quantitative edible fungi that this antibody sets up, provide important technological means for the monitoring of carbendazim content in edible fungi.
Description
The present invention arrives " 12 " national science and technology supporting plan project (2014BAD04B03)；National Natural Science is young
Fund project (31301487)；The subsidy of Tianjin science and technology plan item (142CDGNC00098).
Technical field
The invention belongs to pesticide molecule compound immunochemistry and retention analysis field；Relate to haptenic synthesis, people
Work antigen and the preparation of polyclonal antibody and the foundation of direct competive ELISA method.Specifically a kind of carbendazim antibody and
Application in edible fungi residue detection.
Background technology
Carbendazim belongs to benzimidazole antifungal, due to its efficiently, low toxicity, the feature of interior suction and be widely used in
In agricultural production.Although, it belongs to lower toxicity pesticide, but considers residual period and the bioconcentration of its longer (more than 20 day)
Harm to health, is necessary to its consumption and the restriction of residual and monitoring.And, some countries establish the most respectively
The carbendazim detection method of standard, and formulated corresponding residue limits.Such as, Environmental Protection Agency USA (EPA) sets up
Play the content standard of carbendazim in orange juice residue, for 80ug/kg；The standard of European Union is 200ug/kg；And Australia requires not
More than 10ug/kg.European Union specifies that carbendazim is 0.1mg/kg at the MRLS of fresh mushroom apoplexy due to endogenous wind；Korea S, Britain and China's regulation
Carbendazim MRLS in edible fungi is 1mg/kg；Japan is 3mg/kg.
At present, in quantitative food, the method for carbendazim is mainly instrumental method, such as, and liquid chromatograph (Singh, 2004), gas
Phase chromatograph (Farber, 1993) and LC-MS (Zamorn, 2004) etc..Although along with instrument and equipment improves and experiment
The optimization of condition, these instrumental method accuracy and detection limit (by mg/kg to ug/kg) are become better and better, but there is also and much ask
Topic, such as complex pretreatment, instrument cost is high, and automaticity is low, accordingly, it would be desirable to the masterful technique type talent, and the many consumptions of step
Time, be not suitable for applying to the monitoring of great amount of samples.And in recent years, some novel immunochemical analyses methods, owing to it is simple
Pretreatment process and detecting step quick, easy to learn, be progressively established and extensively apply.But about the carbendazim in edible fungi
ELISA detection there is not yet document report.It is quick, clever that the present invention is exactly the one utilizing the specific antibody of carbendazim to set up
Immune analysis method quick, simple.
Summary of the invention
It is an object of the invention to, by connecting succinic anhydride acquisition hapten at the amino of 2-aminobenzimidazole, utilize
Active ester method prepares artificial antigen, and final immunity obtains the polyclonal antibody of carbendazim, and divides with this immunity setting up carbendazim
Analysis method.
For achieving the above object, the invention discloses following technology contents:
A kind of use the method for carbendazim content in carbendazim detection of specific antibody edible fungi, it is characterised in that include following step
Rapid:
(1) being coated: antibody pH=9.6,0.01M carbonic acid buffer is configured to the liquid that is coated of 5ug/mL, the every hole of ELISA Plate adds
100uL is coated liquid, hatches 12h at 4 DEG C, dries, washes 3 times with PBST；
(2) sample-adding: blank well adds 100uLPBS；Control (control) hole and add the enzyme-labelled antigen of 50uLPBS and 50uL concentration
(diluting 150 times with PBS)；The every hole of other experimental port adds 50uL mark product or sample, then adds (the PBS dilution 150 of 50uL enzyme-labelled antigen
Times), ELISA Plate agitator is hatched 0.5h, dries, wash 5 times with PBST；
(3) colour developing: colour developing uses 14.6mL substrate A and 0.45mL substrate B, the two mixes in use, and every hole adds 150uL, keeps away
After light stands 15min, every hole adds 50uL1.25M sulphuric acid and terminates reaction, and microplate reader reads OD value；Wherein substrate A is: sodium acetate
8.2g；Powder-beta-dextrin 2.5g；Carbamide peroxide 0.4290g；Citric acid 3.16g, distilled water is settled to 1000mL, and 4 DEG C standby；Substrate
B: tetramethyl benzidine 250mg, DMSO are settled to 25mL, room temperature keeps in Dark Place.Wherein PBS is: disodium hydrogen phosphate
13.76g；Sodium chloride 9g；Sodium dihydrogen phosphate 1.38g, distilled water is settled to 1000mL, and 4 DEG C standby；PBST is: PBS+0.05%
Tween 20.
The present invention further discloses monitoring method application in terms of carbendazim content detection in edible fungi.Wherein
Described edible fungi carbendazim content detection refers to: first, utilizes this antibody to set up carbendazim mark by direct competive ELISA method
The standard curve of quasi-product, then, utilizes the carbendazim in this standard curve liquid to be measured of the sample quantitatively after pre-treatment, i.e. sets up
Carbendazim direct competive ELISA detection method.Edible fungi of the present invention includes: Lentinus Edodes, Pleurotus ostreatus, Pleurotus eryngii, Agaricus Bisporus.
More detailed description of the present invention is as follows:
(1) preparation method of polyclonal antibody, comprises the following steps:
Immune animal is that (at 3 months monthly ages, male, body weight 1.5 kg, Tianjin biomedical engineering institute cultivates Japan large rabbit
) through dorsal sc multi-point injection immunogen, every rabbit immunizing dose first is 2 mg, halves later, immune every two weeks
Once, from the beginning of third time immunity, after the most immune one week, from hard of hearing arterial blood drawing, blood elder generation set at room temperature 15 min, so
Rear 3500 r/min are centrifuged 10 min, take supernatant i.e. antiserum, the Hydrazoic acid,sodium salt adding 0.1%, 4 DEG C of storages, measure antiserum effect
Valency, after the 5th immunity, antiserum titre reaches high value 1:90000, purifies anti-with Protein A-Sepharose 4B
Serum, eluent through PBS 72 h, adds 0.1% Hydrazoic acid,sodium salt, measures antibody concentration, and 4 DEG C standby.
(2) antibody described in is applied to the immune detection of carbendazim content in edible fungi and refers to: first, utilizes this antibody to lead to
Cross direct competive ELISA method and set up the standard curve of carbendazim standard product, then, utilize this standard curve quantitatively after pre-treatment
Sample liquid to be measured in carbendazim, i.e. set up the direct competive ELISA detection method of carbendazim.
(3) sample (edible fungi) pre-treating method, comprises the following steps: weigh 2 g (fragrant through the edible fungi of homogenate pre-cooling
Mushroom, Pleurotus ostreatus, Pleurotus eryngii, Agaricus Bisporus) sample in 15mL centrifuge tube, add 2mL acetonitrile, shake 2 min energetically, centrifugal
(6000r/min, 4 DEG C) 3min, take supernatant 1mL in brown vial 20 DEG C standby, dilute 40 times i.e. with 1*PBS buffer
ELISA detection can be carried out.
(4) direct competive ELISA method, comprises the following steps:
1) being coated: antibody 0.01M carbonic acid buffer (pH=9.6) is configured to the liquid that is coated of 5ug/mL, the every hole of ELISA Plate adds
100uL is coated liquid, hatches 12h at 4 DEG C, dries, washes 3 times with PBST；
2) sample-adding: blank well adds 100uLPBS；Control hole adds the enzyme-labelled antigen of the optimal concentration of 50uLPBS and 50uL and (uses
PBS dilutes 150 times)；The every hole of other experimental port adds 50uL mark product or sample, then adds 50uL enzyme-labelled antigen (PBS dilutes 150 times).
ELISA Plate agitator is hatched 30min, dries, wash 5 times with PBST；
3) colour developing: colour developing uses substrate A and substrate B, and the two mixes (14.6mL substrate A+0.45mL substrate B), often in use
Hole adds 150uL, and after lucifuge stands 15min, every hole adds 50uL1.25M sulphuric acid and terminates reaction, and microplate reader reads OD value.
Carbendazim antibody disclosed by the invention and the application in edible fungi residue detection thereof are compared with prior art had
Have has the active effect that
(1) present invention through one the simple condensation reaction of step synthesize carbendazim analog as hapten, this hapten
Highlighting the antigenic determinant of carbendazim, therefore, the immunogen induction obtained through coupling carrier albumen produces the anti-of high-affinity
Body, and by the cross reaction result of other analogs from the point of view of, antibody has the highest specificity.
(2) present invention antibody with high specificity based on carbendazim, can set up its immune analysis method, such as enzyme linked immunological
Adsorption test, radioimmunology etc..And the present invention establishes the direct competive ELISA detection method of carbendazim.The method is passed through
Enzyme-labelled antigen and mark product competition binding antibody response, establish the standard curve of carbendazim.
(3) hapten synthesis of the present invention reaction is simple, and synthesis material easily obtains, and by-product is few, and productivity is high.The antibody obtained
There are good sensitivity, specificity and stability, the foundation of immune analysis method and test kit can well be applied to and grind
Send out.Utilize its direct competive ELISA method set up easy, quick, sensitive, and sample pre-treatments step is simple, whole detection
Time completes within 1.5h, precise and high efficiency, and the response time is 30min.Therefore, sensitive is quickly the outstanding feature of this method.
Therefore, it is adaptable to the on-the-spot preliminary examination of a large amount of samples.In a word, this antibody and method have good application prospect.
Accompanying drawing illustrates:
Fig. 1 is carbendazim canonical plotting；
Fig. 2 is hapten structural characterization--mass spectrum；
Fig. 3 is carbendazim analog structural formula；
Fig. 4 is for adding recovery experiment result curve.
Detailed description of the invention:
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is
Method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and the model of the unrestricted present invention
Enclosing, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of bright spirit and scope, the various changes or the change that carry out the material component in these embodiments and consumption also belong to
In protection scope of the present invention.Raw material, reagent used in the present invention are commercially available.
Embodiment 1
(1) the haptenic synthesis of carbendazim
In 250mL beaker by 0.5g2-aminobenzimidazole stirring and dissolving in 100mL acetonitrile, add 0.564g succinic acid
Acid anhydride, after 40 DEG C of stirring reaction 4h, has the precipitate of a large amount of white to separate out, and filtering reacting solution obtains filtering residue, by filtering residue with 40 DEG C
Acetonitrile rinse twice, remove remain in unreacted raw material in filtering residue, obtain crude product.Crude product is further through column chromatography
(flowing is ethyl acetate mutually: normal hexane=1:3, v/v) purification, draws through thin layer chromatography and Mass Spectrometric Identification, it is thus achieved that product is 2-amber
Amber acyl benzimidazole (white powder), Fig. 2 is shown in its structural analysis.
(2) the immunogenic preparation of carbendazim
In the brown vial of 4mL by 18.6 mg hapten stirring and dissolving in 0.9 mL DMF (DMF),
It is added dropwise over 13.8 mgNHS and the mixed solution of 24.7 mgDCC dissolved with 100uLDMF again, reaction is stirred at room temperature overnight, 4
DEG C stand 2h, be centrifuged off white precipitate, Aspirate supernatant (Acibenzolar liquid) 4 DEG C saves backup.In the brown vial of 10mL,
Weigh 45 mgKLH to be dissolved in 9 mL 0.066mol dipotassium hydrogen phosphate (pH=9.0) solution, at 0 DEG C, be slowly added to 80uL and live
Changing ester liquid, at 4 DEG C, stirring reaction is overnight, with the PBS 3 days of pH=7.4, changes 3 dialysis solution every day, measures volume, adds
0.1% sodium azide ,-20 DEG C standby.
(3) preparation of carbendazim enzyme-labelled antigen
In the brown vial of 4mL by 18.6 mg hapten stirring and dissolving in 0.9 mL DMF (DMF),
It is added dropwise over 13.8 mgNHS and the mixed solution of 24.7 mgDCC dissolved with 100uLDMF again, reaction is stirred at room temperature overnight, 4
DEG C stand 2h, be centrifuged off white precipitate, Aspirate supernatant (Acibenzolar liquid) 4 DEG C saves backup.In the brown vial of 10mL,
Weigh 10 mgHRP to be dissolved in 2 mL 0.066mol dipotassium hydrogen phosphate (pH=9.0) solution, at 0 DEG C, be slowly added to 45 uL
Acibenzolar liquid, at 4 DEG C, stirring reaction is overnight, with the PBS 3 days of pH=7.4, changes 3 dialysis solution every day, measures volume, adds
Isopyknic glycerol ,-20 DEG C standby.
(4) preparation of carbendazim antibody
Immune animal is that (at 3 months monthly ages, male, body weight 1.5 kg, Tianjin biomedical engineering institute cultivates Japan large rabbit
) through dorsal sc multi-point injection immunogen, every rabbit immunizing dose first is 2 mg, halves later, immune every two weeks
Once, from the beginning of third time immunity, after the most immune one week, from hard of hearing arterial blood drawing, blood elder generation set at room temperature 15 min, so
Rear 3500 r/min are centrifuged 10 min, take supernatant i.e. antiserum, the Hydrazoic acid,sodium salt adding 0.1%, 4 DEG C of storages, measure antiserum effect
Valency, during until its titer is higher, takes whole blood.Purifying antiserum with Protein A-Sepharose 4B, eluent is saturating through PBS
Analysing 72 h, add 0.1% Hydrazoic acid,sodium salt, measure antibody concentration, 4 DEG C standby.
Embodiment 2
1, direct competive ELISA method is utilized to set up the standard curve of carbendazim:
Utilizing square formation titrimetry optimization to draw, the optimal package amount of antibody is 5ug/mL, and enzyme-labelled antigen optimum diluting multiple is 150
Times, ion concentration conveniently and pH value are the 0.01MPBS of pH=7.4.Use the experiment condition optimized, to mark product concentration (ug/
Kg) being abscissa, suppression ratio (%) is vertical coordinate, Criterion curve, and result is shown in Fig. 1, specifically comprises the following steps that
(1) being coated: antibody 0.01M carbonic acid buffer (pH=9.6) is configured to the liquid that is coated of 5ug/mL, the every hole of ELISA Plate adds
100uL is coated liquid, hatches 12h at 4 DEG C, dries, washes 3 times with PBST；
(2) sample-adding: blank well adds 100uLPBS；Control hole adds 50uLPBS and 50uL and dilutes the enzyme-labelled antigen of 150 times；Other
The every hole of experimental port adds the mark product of 50uL variable concentrations respectively, then adds the enzyme-labelled antigen of 50uL dilution 150 times.At ELISA Plate agitator
On hatch 30min, dry, wash 5 times with PBST；
(3) colour developing: colour developing uses substrate A and substrate B, and the two mixes (14.6mL substrate A+0.45mL substrate B), often in use
Hole adds 150uL, and after lucifuge stands 15min, every hole adds 50uL1.25M sulphuric acid and terminates reaction, and microplate reader reads OD value.
Wherein, carbendazim standard solution: with PBS liquid by (molten for the carbendazim standard solution that the concentration of purchase is 0.5g/kg
In methanol) be diluted to concentration be 0.042,0.128,0.64,1.6,3.2, the solution of 6.4ug/kg.
PBS(0.01M, pH=7.4): Na2HPO4·12H2O 13.76g；Nacl 9g；NaH2PO4·2H2O 1.38g, double
Steam water and be settled to 1000mL.
Substrate A: sodium acetate 8.2g；Powder-beta-dextrin 2.5g；Carbamide peroxide 0.4290g；Citric acid 3.16g, distilled water constant volume
To 1000mL, 4 DEG C standby.
Substrate B: tetramethyl benzidine 250mg, DMSO are settled to 25mL, room temperature keeps in Dark Place；
Suppression ratio:
In formula: ODcontrol mark product concentration is OD value when zero；
ODx mark product concentration is OD value during x；
The OD value of OD blank blank well.
2, antibody specificity
Select to belong to the benzene mushroom of benzimidazole, thiophanate, thiophanate-methyl, probenazole and their generation with carbendazim
Thank to thing 2-aminobenzimidazole, measure the IC of these analogs50Value and cross reacting rate (CR).
In addition to having certain cross reaction with benomyl, (that effective ingredient being because benomyl is exactly mesh to this antibody
Mark thing carbendazim), with other analogs almost without cross reaction, this well illustrates that object carbendazim is had by antibody
The highest specificity；
In formula: the mark product concentration that the suppression ratio of IC50 mark product is corresponding when being 50%
3, sample detection:
(1) sample pre-treatments
Weigh 2 g through homogenate pre-cooling edible fungi (Lentinus Edodes, Pleurotus ostreatus, Pleurotus eryngii) sample in 15mL centrifuge tube, add 2mL second
Nitrile, shakes 2 min energetically, centrifugal (6000r/min, 4 DEG C) 3min, take supernatant 1mL in brown vial 20 DEG C standby, use
1*PBS buffer dilutes 40 times can carry out ELISA detection.
(2) direct competive ELISA method detection sample
A, is coated: antibody 0.01M carbonic acid buffer (pH=9.6) is configured to the liquid that is coated of 5ug/mL, and the every hole of ELISA Plate adds
100uL is coated liquid, hatches 12h at 4 DEG C, dries, washes 3 times with PBST；
B, sample-adding: blank well adds 100uLPBS；Control hole adds 50uLPBS and 50uL and dilutes the enzyme-labelled antigen of 150 times；Other
The every hole of experimental port adds 50uL respectively and dilutes the sample extracting solution of 40 times, then adds the enzyme-labelled antigen of 50uL dilution 150 times.Each sample
Do 2 parallel, ELISA Plate agitator is hatched 30min, dry, wash 5 times with PBST；
C, colour developing: colour developing uses substrate A and substrate B, and the two mixes (14.6mL substrate A+0.45mL substrate B), often in use
Hole adds 150uL, and after lucifuge stands 15min, every hole adds 50uL1.25M sulphuric acid and terminates reaction, and microplate reader reads OD value.
Calculate the suppression ratio of each sample, and draw the carbendazim content of each sample according to carbendazim standard curve:
4, recovery experiment result is added
2 kinds of different samples, every kind of sample have been selected to add the carbendazim of 3 variable concentrations (0.5ppm, 1ppm, 2ppm) respectively,
Then processing through certain extracting method (Bin Guo et al., 2010) and obtain sample extracting solution, the sample extracting solution obtained is respectively
Detecting by HPLC method and direct competive ELISA method, wherein, in direct competive ELISA method, sample extracting solution need to first dilute with PBS liquid
1000 times are detected again, and two kinds of method testing results of same substrate carry out linear regression.
Result shows, the highest (the linearly dependent coefficient R of concordance of two kinds of method testing results2Value is 0.9822).Fully
Illustrate the accuracy of carbendazim direct competive ELISA detection method result in the edible fungi set up, the method is further described
The Quantitative Monitoring of carbendazim in food can be carried out, have the biggest practical value.
Claims (4)
1. one kind uses the method for carbendazim content in carbendazim detection of specific antibody edible fungi, it is characterised in that include following
Step:
(1) being coated: antibody pH=9.6,0.01M carbonic acid buffer is configured to the liquid that is coated of 5ug/mL, the every hole of ELISA Plate adds
100uL is coated liquid, hatches 12h at 4 DEG C, dries, washes 3 times with PBST；
(2) sample-adding: blank well adds 100uLPBS；Control hole adds 50uL PBS and the enzyme-labelled antigen of 50uL concentration；Other
The every hole of experimental port adds 50uL mark product or sample, then adds 50uL enzyme-labelled antigen (PBS dilutes 150 times), incubates on ELISA Plate agitator
Educate 0.5h, dry, wash 5 times with PBST；
(3) colour developing: colour developing uses 14.6mL substrate A and 0.45mL substrate B, the two mixes in use, and every hole adds 150uL, keeps away
After light stands 15min, every hole adds 50uL1.25M sulphuric acid and terminates reaction, and microplate reader reads OD value；Wherein substrate A is: sodium acetate
8.2g；Powder-beta-dextrin 2.5g；Carbamide peroxide 0.4290g；Citric acid 3.16g, distilled water is settled to 1000mL, and 4 DEG C standby；Substrate
B: tetramethyl benzidine 250mg, DMSO are settled to 25mL, room temperature keeps in Dark Place；The composition of described PBS is: disodium hydrogen phosphate
13.76g；Sodium chloride 9g；Sodium dihydrogen phosphate 1.38g, distilled water is settled to 1000mL, and 4 DEG C standby；The composition of PBST is: PBS
+ 0.05% tween 20.
2. the application in terms of carbendazim content detection in edible fungi of the monitoring method described in claim 1.
3. the application described in claim 2, wherein said edible fungi carbendazim content detection refers to: first, utilize this antibody
Set up the standard curve of carbendazim standard product by direct competive ELISA method, then, utilize this standard curve quantitatively through pre-treatment
After sample liquid to be measured in carbendazim, i.e. set up carbendazim direct competive ELISA detection method.
4. the application described in claim 2, described edible fungi includes: Lentinus Edodes, Pleurotus ostreatus, Pleurotus eryngii, Agaricus Bisporus.
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| CN109265404A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of carbendazim haptens and antigen |
| CN113203851A (en) * | 2021-04-28 | 2021-08-03 | 北京奥本生物技术有限公司 | ELisa method for detecting carbendazim and kit thereof |
| CN113514519A (en) * | 2021-04-15 | 2021-10-19 | 佛山职业技术学院 | Carbendazim electrochemical sensor and application thereof |
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| CN113203851B (en) * | 2021-04-28 | 2024-10-22 | 北京奥本生物技术有限公司 | ELisa method for detecting carbendazim and kit thereof |
| CN116008554A (en) * | 2023-02-06 | 2023-04-25 | 北京赛必达科技有限公司 | Test strip and method for detecting veterinary drug oxfendazole |
| CN116008554B (en) * | 2023-02-06 | 2023-08-08 | 北京赛必达科技有限公司 | Test strip and method for detecting veterinary drug oxfendazole |
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