CN105158458A - Method for detecting mycotoxin through combination of biotin-streptavidin and electrochemistry - Google Patents
Method for detecting mycotoxin through combination of biotin-streptavidin and electrochemistry Download PDFInfo
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- CN105158458A CN105158458A CN201510400407.8A CN201510400407A CN105158458A CN 105158458 A CN105158458 A CN 105158458A CN 201510400407 A CN201510400407 A CN 201510400407A CN 105158458 A CN105158458 A CN 105158458A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to the field of bioengineering. A purpose of the present invention is to provide a method for detecting mycotoxin through combination of biotin-streptavidin and electrochemistry. According to the method, on the basis of the conventional indirect competitive ELISA reaction, biotin-labeled mycotoxin monoclonal antibody is selected, alkaline phosphatase-labeled streptavidin is used to replace the traditional enzyme-labeled antibody, and the biotin-streptavidin signal amplification system is used to improve the detection sensitivity; nitrophenyl phosphoric acid is selected as the alkaline phosphatase catalytic substrate, the condition that the electrochemical properties of the product nitrophenol are different from the nitrophenyl phosphoric acid is used, and the final signal of the reaction can be collected through the electrochemical method; during the data processing, the content of the mycotoxin to be detected in the actual sample is calculated according to the standard curve. According to the present invention, the detection sensitivity on the substance to be detected can achieve the picogram-nanogram level, and the method has advantages of low cost, easy obtaining, high throughput detection, simpleness, economy, high sensitivity, easy operation, trace detection, and the like.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method that biotinstreptatin and galvanochemistry coupling detect mycotoxin
Background technology
Mycotoxin (Mycotoxins) is extensively present in the Fodder and food gone mouldy, and is the Small molecular secondary metabolite that mould produces.Because staple crops such as corn, barley, the wheat etc. in the whole world all can by mycotoxin contaminations, therefore the high-sensitivity detecting method setting up mycotoxin becomes the important content of countries in the world field of food safety, China is large agricultural country, and carrying out effective detection of mycotoxin and supervision is the importance keeping national economy and social development.Mycotoxin is of a great variety, and modal have ochratoxin A, vomitoxin, aflatoxin B1, fumonisin B1, zearalenone, Penicillium patulum toxin and T-2 toxin etc.Mycotoxin generally all has stronger toxicity, can cause the diseases such as cancer, offspring's distortion, sex premature.
Modal mycotoxin detection means is mass spectroscopy and euzymelinked immunosorbent assay (ELISA) (ELISA) mainly, the Sample pretreatment of mass spectroscopy due to complexity and the support of the expensive instrument of dependence, can not be used widely, and euzymelinked immunosorbent assay (ELISA) is due to simple to operate, result easily obtains, and has now become and has detected mycotoxin contamination a kind of method the most conventional.Enzyme linked immunological ratio juris is the combination based on antigen and specific antibody, different detection sample is different to the limit standard of mycotoxin, some special sample, as infant food, to the limitation of mycotoxin significantly lower than bread and cheese, and some toxicity of mycotoxins differs greatly, trace can cause the grievous injury of animal body, as ochratoxin, aflatoxin etc.Conventional euzymelinked immunosorbent assay (ELISA) carries out the amplification of detection signal only by enzyme labelled antibody, be limited by the restriction of the affinity of mycotoxin monoclonal antibody, and mycotoxin belongs to small haptens, itself does not possess immunogenicity, high-affinity antibody is difficult to obtain and cost is higher, present invention employs the method that biotinstreptatin signal amplifying system and galvanochemistry coupling detect mycotoxin, the sensitivity detected is improved greatly, adapt to the quick trace detection of sample, provide new platform for mycotoxin detects.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiency that existing enzyme-labeled immunity signal amplifies means, provides a kind of biotinstreptatin signal and amplifies the method detecting mycotoxin with galvanochemistry coupling.
For solving Related Technical Issues, solution of the present invention is: provide a kind of biotinstreptatin and galvanochemistry coupling to detect the method for mycotoxin, on the basis that conventional indirect competitive ELISA reacts, mycotoxin specific antibody is carried out biotin labeling, and replace traditional ELIAS secondary antibody with the Streptavidin of alkali phosphatase enzyme mark, the raising of detection sensitivity is carried out by biotinstreptatin signal amplifying system; Select Nitrophenyl phosphate (pNPP) as the catalytic substrate of alkaline phosphatase, because its product nitrophenol (pNP) has electrochemical properties, substrate nitro benzenephosphonic acid (pNPP) does not possess, the two can not form the mutual interference of signal, gathers the oxidation peak of final product by electrochemical method (screen printing electrode); During data processing, with the mycotoxin standard concentration logarithm of gradient dilution for horizontal ordinate, with the ratio of the positive control peak value of the oxidation peak of respective concentration and 0 standard items for ordinate, drawing standard suppresses curve; Finally, the content of mycotoxin to be checked in actual sample is calculated according to typical curve.
In the present invention, described mycotoxin to be checked is any one in ochratoxin, aflatoxin, zearalenone, fumonisin, vomitoxin.
In the present invention, the specific implementation step of the method comprises:
(1) utilize conventional polyvinyl 96 orifice plate (corning, 3590), mycotoxin coupled antigen is diluted by concentration with the best bag, and dilution is the carbonate buffer solution of pH=9.6; 100 μ l/ holes add, 4 DEG C are spent the night, and with containing after 0.01M phosphate buffer (PBST) washing of 0.05% (v/v) Tween20, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), 37 DEG C of closed 2h, dry for subsequent use after washing;
(2) after the biotin labeled mycotoxin monoclonal antibody mycotoxin standard items of 50 μ l gradient dilutions, sample to be checked and 50 μ l being diluted to optium concentration mixes, add 96 orifice plates respectively, each concentration do 3 parallel, and feminine gender and positive control are set simultaneously; Positive control is that 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeling mycotoxin monoclonal antibodies mix, and negative control is that 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeling mycotoxin monoclonal antibody dilutions mix; After 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(3) add the Streptavidin (0.5 μ g/ml) of alkali phosphatase enzyme mark, 100 μ l/ holes, after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(4) add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, after 37 DEG C of effect 10min, to be transferred to by solution in hole in EP pipe and to carry out mark;
(5) by EP pipe solution take out 80 μ l and be added on screen printing electrode, the oxidation peak current of assaying reaction product, sweep limit 0V-1.2V, amplitude is 80mV, pulse width 0.01s, sampling width 0.0025s, recurrence interval 0.1s;
(6) with the mycotoxin standard concentration logarithm of gradient dilution for horizontal ordinate, the peak point current of respective concentration and the ratio of 0 standard items positive control peak current are ordinate, drawing standard suppresses curve, according to typical curve, calculates the content of mycotoxin to be checked in actual sample.
In the present invention, the mycotoxin coupled antigen the best bag described in step (1), by the optium concentration of the biotin labeled mycotoxin monoclonal antibody described in concentration and step (2), is confirmed by following method:
Utilize 96 hole polyethylene boards (Corning, 3590), after carrying out gradient dilution, be added in plate with 100 μ l/ holes with the carbonate buffer solution of pH=9.6 (CBS) to mycotoxin coupled antigen, 4 DEG C of bags are spent the night, supernatant discarded, after washing unconjugated coupled antigen with PBST, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), at 37 DEG C of closed 2h, after washing, add the biotin labeling mycotoxin monoclonal antibody after gradient dilution, 100 μ l/ holes, after 37 DEG C of effect 30min, abandoning supernatant, wash away the antibody of non-specific binding, add the Streptavidin of the alkali phosphatase enzyme mark of 0.2 μ g/ml, 100 μ l/ holes), wash and be placed in thieving paper buckles after 37 DEG C of effect 30min and do, add the 10mM p-nitrophenyl phosphoric acid (pNPP) of now joining, 100 μ l/ holes, 37 DEG C of effect 10min, because product nitrophenol (pNP) has special absorption peak at 405nm place, after adding the termination of 2M NaOH by 50 μ l/ holes, microplate reader OD
405choose after reading some numerical value close to 1.0 mycotoxin coupled antigen bag by concentration and biotin labeling mycotoxin monoclonal antibody action concentration combination, draw conventional indirect ELISA typical curve respectively, concrete operation method is: 96 hole polyethylene board (Corning, 3590) bag is by mycotoxin coupled antigen, according to for subsequent use after operation Seal treatment described above, after washing is closed, get the mycotoxin standard items of 50 μ l gradient dilutions and 50 μ l dilute after biotin labeled mycotoxin monoclonal antibody carry out premix after add in reacting hole, 37 DEG C of effect 30min, and negative and positive control (negative control: add reacting hole after 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeled mycotoxin monoclonal antibody dilution mix are set simultaneously, positive control: add in reacting hole after 50 μ l mycotoxin standard dilutions and the biotin labeled mycotoxin monoclonal antibody of 50 μ l mix), wash after effect and be placed in thieving paper buckles and do, add the Streptavidin of the alkali phosphatase enzyme mark of 0.2 μ g/ml, 100 μ l/ holes, after 37 DEG C of effect 30min, PBST washing is placed on thieving paper buckle and does, add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, with 50 μ l after 37 DEG C of effect 10min, and 2M NaOH cessation reaction, microplate reader OD
405reading, with the mycotoxin standard concentration logarithm of gradient dilution for horizontal ordinate, with the absorbance ratio of correspondence (standard items OD
450-negative hole OD
450)/(positive hole OD
450-negative hole OD
450) for ordinate drawing standard suppresses curve, some typical curves of gained are analyzed, calculating half inhibiting rate and absorbance ratio are 0.5 (IC respectively
50) corresponding to mycotoxin standard concentration, the mycotoxin coupled antigen bag chosen corresponding to minimum is combined as optium concentration when detecting by with biotin labeling mycotoxin monoclonal antibody action concentration.
Inventive principle describes:
First the present invention sets up the ELISA system based on biotinstreptatin signal amplifying system, and optimizes and obtain best mycotoxin coupled antigen bag by the activity with biotin labeling mycotoxin monoclonal antibody.The substrate of alkaline phosphatase (ALP) adopts p-nitrophenyl phosphoric acid (pNPP), because colourless pNPP can be converted to yellow product p-nitrophenol (pNP) by alkaline phosphatase, this product has special absorption peak at 405nm wavelength place, can be used for setting up conventional enzyme-labeled immunity.Meanwhile, not catalyzed p-nitrophenyl phosphoric acid (pNPP) is without electrochemical activity, and product p-nitrophenol (pNP) then possesses, and in the detection method that this patent is set up, is detected by the signal of electrochemical method to product.What generally adopt in conventional ELISA is that the signal of enzyme labelled antibody amplifies means, detection means described in this patent adopts signal to strengthen the biotinstreptatin signal amplifying system of better effects if namely by selecting biotin labeled mycotoxin monoclonal antibody, the Streptavidin of alkali phosphatase enzyme mark is used to be combined with biotin labeled mycotoxin monoclonal antibody, select specific alkaline phosphatase substrate for enzymatic activity simultaneously, electrochemistry by enzymatic associated products final reaction signal of verifying measures, compare the method for conventional enzyme linked immunosorbent detection absorbance, the signal carrying out two steps altogether amplifies, greatly improve the sensitivity of detection, and this reaction all can be carried out in normal enzyme target, cost is low, signal detecting method is simple, for adapting to the quick of extensive sample, weigh to detect and new detection platform is provided.
Electrochemical signals numerical value in this test is all measured by the electrochemical workstation (CHI-832, occasion China, Shanghai) be connected with computer.
The present invention, on the basis of conventional indirect competitive ELISA, by biotinstreptatin signal amplifying system and galvanochemistry coupling, sets up the new method of mycotoxin trace detection.Principle is the biotin labeled mycotoxin monoclonal antibody specific of mycotoxin coupled antigen competitive binding of mycotoxin in measuring samples and bag quilt, after washing away non-specific binding, add the Streptavidin of alkali phosphatase enzyme mark, act on rear and wash, choose Nitrophenyl phosphate (pNPP) for alkaline phosphatase substrate for enzymatic activity, detect the redox peak of final product p-nitrophenol (pNP).The method adopts biotinstreptatin system to substitute conventional enzyme labelled antibody to carry out signal amplification and product p-nitrophenol (pNP) after utilizing alkaline phosphatase p-nitrophenyl phosphoric acid (pNPP) catalysis possesses this feature of electrochemical activity, the oxidation peak of solution after employing differential pulse method detection reaction, compare the reading absorbance of conventional method, greatly improve the sensitivity detected.Biotinstreptatin signal amplifying system combines with electrochemical signals assay method by this method, highly sensitive, and can accomplish high flux, the trace detection of extensive sample, and the qualitative and quantitative analysis for mycotoxin provides new detection platform.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the present invention fully utilizes biotinstreptatin signal amplifying system and electrochemical signals detection method, carries out trace detection to mycotoxin specific in sample.
(2) detection sensitivity that the present invention treats detection material can reach pik-nanogram levels; 96 orifice plates used in the present invention are conventional polyvinyl plate, low price, and cost is low, easily obtains, and can realize high flux detection.
(3) relative to background technology, the present invention has simply, economical, and high sensitivity is easy to operate, the advantages such as trace detection.
Accompanying drawing explanation
Fig. 1 is the course of reaction schematic diagram that alkaline phosphatase enzymatic p-nitrophenyl phosphoric acid (pNPP) generates p-nitrophenol (pNP).
Fig. 2 is the detection schematic diagram of end-product of the present invention; By screen printing electrode, final product is carried out to the detection of electrochemical signals, wherein, 1 is to electrode, and 2 is working electrode, and 3 is contrast electrode.
Embodiment
Inventive principle is introduced:
The present invention is by the basis of reacting at conventional indirect competitive ELISA, mycotoxin specific antibody is carried out biotin labeling, and replace traditional ELIAS secondary antibody with the Streptavidin of alkali phosphatase enzyme mark, the sensitivity detected is improved by biotinstreptatin signal amplifying system, and select Nitrophenyl phosphate (pNPP) as the catalytic substrate of alkaline phosphatase, its product nitrophenol (pNP) and Nitrophenyl phosphate (pNPP) difference electrochemical properties make the detection of final signal gather by electrochemical method, during data processing with the logarithm of various criterion product concentration for horizontal ordinate, the ratio of the peak point current of respective concentration and positive control (0 standard items) peak current is your ordinate, drawing standard suppresses curve, according to typical curve, thus the content of mycotoxin to be checked in calculating actual sample
By the method, can the high flux of realize target material, fast, the object of trace detection.
In the present invention, 96 orifice plates that immune response is carried out are conventional polyethylene board, mycotoxin coupled antigen can be prepared voluntarily according to testing requirement, biotin labeled mycotoxin monoclonal antibody is for providing for oneself or commercial (gel electrophoresis qualification, heavy chain light chain is clear, and accounts for more than 90% of total protein concentration) basic subscript remember.The Streptavidin of alkali phosphatase enzyme mark is that commercialization is buied.
Following embodiment can make the technician of this professional skill field more fully understand the present invention, but does not limit the present invention in any way.
Embodiment 1: the quick detection of ochratoxin OTA
Following program, for Aspergillus ochraceus OTA and biotin labeled ochratoxin monoclonal antibody Anti-OTA-Bio, is set forth biotinstreptatin signal and is amplified the method detecting ochratoxin OTA with galvanochemistry coupling:
(1) conventional polyvinyl 96 orifice plate (corning, 3590), ochratoxin coupled antigen OTA-BSA is diluted to 0.325 μ g/ml, and dilution is the carbonate buffer solution of pH=9.6; 100 μ l/ holes add, 4 DEG C are spent the night, with containing after 0.01M phosphate buffer (PBST) washing of 0.05% (v/v) Tween20, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), 37 DEG C of closed 2h, dry for subsequent use after washing;
(2) getting the ochratoxin OTA standard items of 50 μ l gradient dilutions and sample to be checked and 50 μ l concentration is join in reacting hole after the biotin labeled ochratoxin monoclonal antibody Anti-OTA-Bio premix of 0.125 μ g/ml, each standard concentration and sample standard deviation to be checked do 3 parallel holes, 37 DEG C of effect 30min, and negative and positive control (negative control: add reacting hole after 50 μ l ochratoxin OTA standard dilutions and 50 μ l biotin labeled ochratoxin monoclonal antibody Anti-OTA-Bio dilution mix are set; Positive control: add in reacting hole after 50 μ l ochratoxin OTA standard dilutions and the biotin labeled ochratoxin monoclonal antibody Anti-OTA-Bio of 50 μ l mix), after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(3) add the Streptavidin (0.2 μ g/ml, 100 μ l/ holes) of alkali phosphatase enzyme mark, after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(4) add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, after 37 DEG C of effect 10min, to be transferred to by solution in hole in EP pipe and to carry out mark;
(5) by EP pipe solution take out 80 μ l and be added on screen printing electrode, the oxidation peak current of assaying reaction product, sweep limit 0V-1.2V, amplitude is 80mV, pulse width 0.01s, sampling width 0.0025s, recurrence interval 0.1s;
(6) with the ochratoxin OTA standard concentration logarithm of gradient dilution for horizontal ordinate, the peak point current of respective concentration and the ratio of 0 standard items positive control peak current are ordinate, drawing standard suppresses curve, according to typical curve, calculate the content of ochratoxin OTA to be checked in actual sample.
In this detection method, step (1) is wrapped by concentration and biotin labeled ochratoxin toxin monoclone antibody Anti-OTA-Bio activity with the ochratoxin OTA coupled antigen OTA-BSA the best described in (2), is confirmed by following method:
96 hole polyethylene board (Corning, 3590), ochratoxin coupled antigen OTA-BSA (3mg/ml) is carried out gradient dilution (carbonate buffer solution, pH=9.6, CBS), concentration is followed successively by 3, 1.5, 0.75, 0.325, 0.1625, 0.08125 μ g/ml, 100 μ l/ holes are added in plate, 4 DEG C of bags are spent the night, supernatant discarded, after PBST washing, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), 37 DEG C of closed 2h, after washing, add the mould ochratoxin monoclonal antibody Anti-OTA-Bio (1mg/ml) of the biotin labeling after gradient dilution, concentration is followed successively by 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 μ g/ml, 100 μ l/ holes, after 37 DEG C of effect 30min, abandoning supernatant also washes away the antibody of non-specific binding, add Streptavidin (the 0.2 μ g/ml of alkali phosphatase enzyme mark, 100 μ l/ holes), wash and be placed in thieving paper buckles after 37 DEG C of effect 30min and do, add 10mM p-nitrophenyl phosphoric acid (pNPP) (100 μ l/ hole) the 37 DEG C effect 10min now joined, because product nitrophenol (pNP) has special absorption peak at 405nm place, after 2M NaOH (50 μ l/ hole) stops, microplate reader OD
405reading, choose numerical value close to about 1.0 antigen coated concentration and biotin labelled antibodies combine, conventional indirect ELISA typical curve is drawn by with biotin labeling ochratoxin monoclonal antibody Anti-OYA-Bio activity respectively according to the ochratoxin coupled antigen OTA-BSA bag of this condition, that is: 96 hole polyethylene board (Corning, 3590) bag is by ochratoxin OTA coupled antigen OTA-BSA and according to for subsequent use after aforesaid operations Seal treatment, after washing is closed, get the mycotoxin standard items of 50 μ l gradient dilutions and 50 μ l dilute after biotin labeled mycotoxin monoclonal antibody carry out premix after add in reacting hole, 37 DEG C of effect 30min, and negative and positive control (negative control: add reacting hole after 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeled mycotoxin monoclonal antibody dilution mix are set simultaneously, positive control: add in reacting hole after 50 μ l mycotoxin standard dilutions and the biotin labeled mycotoxin monoclonal antibody of 50 μ l mix), wash after effect and be placed in thieving paper buckles and do, add Streptavidin (the 0.2 μ g/ml of alkali phosphatase enzyme mark, 100 μ l/ holes), after 37 DEG C of effect 30min, PBST washing is placed on thieving paper buckle and does, add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, by 50 μ l2M NaOH cessation reactions after 37 DEG C of effect 10min, and microplate reader OD
405reading, using the ochratoxin standard items OTA log concentration of gradient dilution as horizontal ordinate, with the absorbance ratio of correspondence (standard items OD
450-negative hole OD
450)/(positive hole OD
450-negative hole OD
450) for ordinate drawing standard suppresses curve, some typical curves of gained are analyzed, calculating half inhibiting rate and absorbance ratio are 0.5 (IC respectively
50) corresponding to mycotoxin standard concentration, choose ochratoxin coupled antigen OTA-BSA corresponding to minimum to wrap by concentration and biotin labeling ochratoxin OTA monoclonal antibody action concentration Anti-OTA-Bio as best of breed when detecting, finally determining that ochratoxin coupled antigen OTA-BSA wraps by concentration is 0.325 μ g/ml, and the activity of biotin labeled ochratoxin monoclonal antibody Anti-OTA-Bio is 0.125 μ g/ml.
We detect 37 parts of maize and soybean samples in market, result display wherein 12 increments is originally the ochratoxin OTA positive (content >=1.25 μ g/kg), (visit purchased from Germany with import reagent box and send out, article No. R1311) coincidence rate is 100%, and through quantitatively detecting, content is between 1.2 μ g/kg-13.5 μ g/kg, sensitivity is up to state standards (5 μ g/kg), and the fast qualitative that can be used for ochratoxin A in food and feed detects and quantitative test.
Embodiment 2: the quick detection of zearalenone ZEN
Following program, for zearalenone ZEN and biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio, is set forth biotinstreptatin signal and is amplified the method detecting zearalenone ZEN with galvanochemistry coupling:
(1) conventional polyvinyl 96 orifice plate (corning, 3590), zearalenone coupled antigen ZEN-BSA is diluted to 0.125 μ g/ml, and dilution is the carbonate buffer solution of pH=9.6; 100 μ l/ holes add, 4 DEG C are spent the night, with containing after 0.01M phosphate buffer (PBST) washing of 0.05% (v/v) Tween20, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), 37 DEG C of closed 2h, dry for subsequent use after washing;
(2) getting the zearalenone ZEN standard items of 50 μ l gradient dilutions and sample to be checked and 50 μ l concentration is join in reacting hole after the biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio premix of 0.1875 μ g/ml, each standard concentration and sample standard deviation to be checked do 3 parallel holes, 37 DEG C of effect 30min, and negative and positive control (negative control: add reacting hole after 50 μ l zearalenone ZEN standard dilutions and 50 μ l biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio dilution mix are set; Positive control: add in reacting hole after 50 μ l zearalenone ketenes ZEN standard dilutions and 50 μ l biotin labeling zearalenone monoclonal antibody Anti-ZEN-Bio mix), after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(3) add the Streptavidin (0.2 μ g/ml, 100 μ l/ holes) of alkali phosphatase enzyme mark, after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(4) add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, after 37 DEG C of effect 10min, to be transferred to by solution in hole in EP pipe and to carry out mark;
(5) by EP pipe solution take out 80 μ l and be added on screen printing electrode, the oxidation peak current of assaying reaction product, sweep limit 0V-1.2V, amplitude is 80mV, pulse width 0.01s, sampling width 0.0025s, recurrence interval 0.1s;
(6) with the zearalenone ZEN standard concentration logarithm of gradient dilution for horizontal ordinate, the peak point current of respective concentration and the ratio of 0 standard items positive control peak current are ordinate, drawing standard suppresses curve, and calculates the content of zearalenone ZEN to be checked in actual sample according to typical curve.
In this detection method, step (1) is wrapped by concentration and biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio activity with the zearalenone coupled antigen ZEN-BSA the best described in (2), is confirmed by following method:
96 hole polyethylene board (Corning, 3590), zearalenone coupled antigen ZEN-BSA (4mg/ml) is carried out gradient dilution (carbonate buffer solution, pH=9.6, CBS), concentration is followed successively by 2, 1, 0.5, 0.25, 0.125, 0.0625 μ g/ml, 100 μ l/ holes are added in plate, 4 DEG C of bags are spent the night, supernatant discarded, after PBST washing, add 200 μ l containing 5% (w/v) skimmed milk power (PBST configuration), 37 DEG C of closed 2h, after washing, add the biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio (3mg/ml) after gradient dilution, concentration is followed successively by 3, 1.5, 0.75, 0.375, 0.1875, 0.09375 μ g/ml, 100 μ l/ holes, after 37 DEG C of effect 30min, abandoning supernatant also washes away the antibody of non-specific binding, add Streptavidin (the 0.2 μ g/ml of alkali phosphatase enzyme mark, 100 μ l/ holes), wash and be placed in thieving paper buckles after 37 DEG C of effect 30min and do, add the 10mM p-nitrophenyl phosphoric acid (pNPP) (100 μ l/ hole) of now joining, 37 DEG C of effect 10min, because product nitrophenol (pNP) has special absorption peak at 405nm place, after 2M NaOH (50 μ l/ hole) stops, microplate reader OD
405reading, choose numerical value close to about 1.0 zearalenone coupled antigen ZEN-BSA bag combined by concentration and biotin labeling zearalenone monoclonal antibody Anti-ZEN-Bio activity, draw conventional indirect ELISA typical curve, that is: 96 hole polyethylene board (Corning, 3590) bag is by zearalenone coupled antigen ZEN-BSA and according to for subsequent use after aforesaid operations Seal treatment, after washing is closed, get the zearalenone standard items ZEN of 50 μ l gradient dilutions and 50 μ l dilute after biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio carry out premix after add in reacting hole, 37 DEG C of effect 30min, and negative and positive control (negative control: add reacting hole after 50 μ l zearalenone ZEN standard dilutions and 50 μ l biotin labeled zearalenone ZEN monoclonal antibody Anti-ZEN-Bio dilution mix are set simultaneously, positive control: add in reacting hole after 50 μ l zearalenone ZEN standard dilutions and 50 μ l biotin labeled zearalenone ZEN monoclonal antibody Anti-ZEN-Bio mix), wash after effect and be placed in thieving paper buckles and do, add Streptavidin (the 0.2 μ g/ml of alkali phosphatase enzyme mark, 100 μ l/ holes), after 37 DEG C of effect 30min, PBST washing is placed on thieving paper buckle and does, add the Nitrophenyl phosphate (pNPP) of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, by 50 μ l2M NaOH cessation reactions after 37 DEG C of effect 10min, and microplate reader OD
405reading, using zearalenone ZEN standard concentration logarithm as horizontal ordinate, with the absorbance ratio of correspondence (standard items OD
450-negative hole OD
450)/(positive hole OD
450-negative hole OD
450) for ordinate drawing standard suppresses curve, some typical curves of gained are analyzed, calculating half inhibiting rate and absorbance ratio are 0.5 (IC respectively
50) corresponding to zearalenone standard concentration, choose zearalenone coupled antigen ZEN-BSA corresponding to minimum to wrap by concentration and biotin labeling zearalenone ZEN monoclonal antibody action concentration Anti-ZEN-Bio as best of breed when detecting, finally determining that zearalenone coupled antigen ZEN-BSA wraps by concentration is 0.125 μ g/ml, and the activity of biotin labeled zearalenone monoclonal antibody Anti-ZEN-Bio is 0.1875 μ g/ml.
We detect 32 parts of corn samples in market, result display wherein 21 increments is originally the zearalenone ZEN positive (content >=1.75 μ g/kg), (visit purchased from Germany with import reagent box and send out, article No. R1401) coincidence rate is 100%, and through quantitatively detecting, content is between 5.7 μ g/kg-17.5 μ g/kg, sensitivity is up to state standards (50 μ g/kg), and the fast qualitative that can be used for zearalenone ZEN in food samples detects and quantitative test.
Special instruction:
Although only list two kinds of mycotoxins in embodiments of the invention, and the biotinstreptatin signal of correspondence amplifies the detection method with galvanochemistry coupling.Do not enumerate the particular content of all biological molecule and disclose few nucleic acid chains complementary separately.But mycotoxin and corresponding monoclonal antibody thereof belong to general knowledge as well known to those skilled in the art.The invention provides described biotinstreptatin signal amplifying system and electrochemical signals and gather the detection method of coupling, the technical ability that those skilled in the art can grasp according to it be drawn inferences about other cases from one instance, and realizes detecting other mycotoxin objects.
Be more than in conjunction with specific embodiments son the present invention is done further describe.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; under the premise without departing from the principles of the invention; the present invention and application thereof also have various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (4)
1. biotinstreptatin and galvanochemistry coupling detect the method for mycotoxin, it is characterized in that, on the basis that conventional indirect competitive ELISA reacts, select biotin labeled mycotoxin monoclonal antibody, and replace traditional enzyme labelled antibody with the Streptavidin of alkali phosphatase enzyme mark, the sensitivity detected is improved by biotinstreptatin signal amplifying system; And select Nitrophenyl phosphate as the catalytic substrate of alkaline phosphatase, utilize its product nitrophenol to have the electrochemical properties different from Nitrophenyl phosphate, the final signal reacted by electrochemical method collection; During data processing, with the mycotoxin standard concentration logarithm of gradient dilution for horizontal ordinate, with the ratio of the positive control peak current of the peak point current of respective concentration and 0 standard items for ordinate, after drawing standard suppresses curve, calculate the content of mycotoxin to be checked in actual sample according to typical curve.
2. method according to claim 1, is characterized in that, described mycotoxin to be checked is any one in ochratoxin, aflatoxin, zearalenone, fumonisin, vomitoxin.
3. method according to claim 1, is characterized in that, the specific implementation step of the method comprises:
(1) utilize conventional polyvinyl 96 orifice plate, mycotoxin coupled antigen is diluted by concentration with the best bag, and dilution is the carbonate buffer solution of pH=9.6; 100 μ l/ holes add, 4 DEG C are spent the night, and with containing after the 0.01M phosphate buffer washing of 0.05% (v/v) Tween20, add 5% (w/v) skimmed milk power of 200 μ l phosphate buffer configurations, 37 DEG C of closed 2h, dry for subsequent use after washing;
(2) get the mycotoxin standard items of 50 μ l gradient dilutions and sample to be checked and 50 μ l dilute after biotin labeled mycotoxin monoclonal antibody premix after join in reacting hole, each standard concentration and sample standard deviation to be checked do 3 parallel holes, 37 DEG C of effect 30min, and feminine gender and positive control are set, after 37 DEG C of effect 30min, phosphate buffer washing be placed on thieving paper buckle do for subsequent use;
Described negative control refers to: add reacting hole after 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeled mycotoxin monoclonal antibody dilution mix; Described positive control refers to: add in reacting hole after 50 μ l mycotoxin standard dilutions and the biotin labeled mycotoxin monoclonal antibody of 50 μ l mix;
(3) add the Streptavidin of 0.2 μ g/ml alkali phosphatase enzyme mark, 100 μ l/ holes, after 37 DEG C of effect 30min, PBST washing be placed on thieving paper buckle do for subsequent use;
(4) add the Nitrophenyl phosphate of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, after 37 DEG C of effect 10min, to be transferred to by solution in hole in EP pipe and to carry out mark;
(5) by EP pipe solution take out 80 μ l and be added on screen printing electrode, the oxidation peak current of assaying reaction product, sweep limit 0V-1.2V, amplitude is 80mV, pulse width 0.01s, sampling width 0.0025s, recurrence interval 0.1s;
(6) with gradient dilution mycotoxin standard concentration logarithm for horizontal ordinate, the peak point current of respective concentration and the ratio of 0 standard items positive control peak current are ordinate, drawing standard suppresses curve, and according to typical curve, calculates the content of mycotoxin to be checked in actual sample.
4. method according to claim 3, it is characterized in that, the mycotoxin coupled antigen the best bag described in step (1) is confirmed by following method by the optium concentration of the biotin labeled mycotoxin monoclonal antibody described in concentration and step (2):
Utilize 96 hole polyethylene boards, after carrying out gradient dilution, be added in plate with 100 μ l/ holes with the carbonate buffer solution of pH=9.6 to mycotoxin coupled antigen, 4 DEG C of bags are spent the night, supernatant discarded, after washing unconjugated coupled antigen with PBST, add 5% (w/v) skimmed milk power of 200 μ l phosphate buffer configurations, at 37 DEG C of closed 2h, after washing, add the biotin labeling mycotoxin monoclonal antibody after gradient dilution, 100 μ l/ holes, after 37 DEG C of effect 30min, abandoning supernatant, wash away the antibody of non-specific binding, add the Streptavidin of the alkali phosphatase enzyme mark of 0.2 μ g/ml, 100 μ l/ holes, wash and be placed in thieving paper buckles after 37 DEG C of effect 30min and do, add the 10mM p-nitrophenyl phosphoric acid of now joining, 100 μ l/ holes, 37 DEG C of effect 10min, because product nitrophenol has special absorption peak at 405nm place, after adding the termination of 2M NaOH by 50 μ l/ holes, microplate reader OD
405choose after reading some numerical value close to 1.0 mycotoxin coupled antigen bag by concentration and biotin labeling mycotoxin monoclonal antibody action concentration combination, draw conventional indirect ELISA typical curve respectively, concrete method of operating is: 96 hole polyethylene board bags are by mycotoxin coupled antigen, according to for subsequent use after aforesaid operations Seal treatment, after washing is closed, get the mycotoxin standard items of 50 μ l gradient dilutions and 50 μ l dilute after biotin labeled mycotoxin monoclonal antibody carry out premix after add in reacting hole, 37 DEG C of effect 30min, and feminine gender and positive control are set simultaneously, negative control: add reacting hole after 50 μ l mycotoxin standard dilutions and 50 μ l biotin labeled mycotoxin monoclonal antibody dilution mix, positive control: add in reacting hole after 50 μ l mycotoxin standard dilutions and the biotin labeled mycotoxin monoclonal antibody of 50 μ l mix, wash after effect and being placed in thieving paper buckle dry, adds the Streptavidin of the alkali phosphatase enzyme mark of 0.2 μ g/ml, 100 μ l/ holes, and after 37 DEG C of effect 30min, phosphate buffer washs to be placed on thieving paper buckles and does, add the Nitrophenyl phosphate of 10mM, dilution is the TrisHCl of 50mM, 0.1MNaCl, pH=8.5,100 μ l/ holes, with 50 μ l after 37 DEG C of effect 10min, and 2M NaOH cessation reaction, microplate reader OD
405reading, with the mycotoxin standard concentration logarithm of gradient dilution for horizontal ordinate, with the absorbance ratio of correspondence (standard items OD
450-negative hole OD
450)/(positive hole OD
450-negative hole OD
450) for ordinate drawing standard suppresses curve, some typical curves of gained are analyzed, calculating half inhibiting rate and absorbance ratio are 0.5 (IC respectively
50) corresponding to mycotoxin standard concentration, the mycotoxin coupled antigen bag chosen corresponding to minimum is combined as optium concentration when detecting by with biotin labeling mycotoxin monoclonal antibody action concentration.
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