CN101231291A - Kit and method for quantitative determination of Fumonisin B1 content in food by ELISA detection technique - Google Patents
Kit and method for quantitative determination of Fumonisin B1 content in food by ELISA detection technique Download PDFInfo
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- CN101231291A CN101231291A CNA2007100565596A CN200710056559A CN101231291A CN 101231291 A CN101231291 A CN 101231291A CN A2007100565596 A CNA2007100565596 A CN A2007100565596A CN 200710056559 A CN200710056559 A CN 200710056559A CN 101231291 A CN101231291 A CN 101231291A
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Abstract
The invention relates to a reagent box for quantificationally detecting the content of fumonisin B1 in food by adopting an enzymoimmunoassay technology and the detecting method thereof. The reagent box includes the steps that a fumonisin B1-KLH antigen is prepared; a polyclonal fumonisin B1 antibody is prepared; an enzyme labeling antigen is prepared; a coated plate is prepared, and a reagent is prepared. The invention mainly adopts the enzymoimmunoassay technology to detect the fumonisin B1, can realize the aim of quantificationally detecting the content of fumonisin B1 in food and alimentary crops; and the reagent box has simple structure, convenient usage, cheap price and high sensitivity; the detecting method has strong specificity, high sensitivity, good veracity, and the sensitivity can reach gamma/kg grade.
Description
Technical field
The invention belongs to food immuno analytical method field, relate to detection, especially a kind of kit and detection method thereof that adopts fumonisin B1 content in the enzyme linked immunosorbent detection technology detection by quantitative food the fumonisin B1 content in food and the cereal crops.
Background technology
Fumonisin (Fumonisins, FBs) be 1988 by South Africa scientist Gelderblom etc. (different name: the one group of analogue that is separated to MRC 826 cultures beading sickle spore F.moniliforme Sheldon) also claims fumonisins, rotten Ma Su etc. from F.verticilliodes first.This toxin is mainly produced by beading sickle spore and many births sickle spore (F.proliferatumMatsushinma), is to pollute the water-soluble secondary metabolite of corn.Laurent in 1989 etc. isolate the noxious material of two kinds of structural similarities from fumonisin, be named as fumonisin B respectively
1(FB
1) and fumonisin B
2(FB
2).Up to the present, found FB
1, FB
2, FB
3, FB
4, FA
1, FA
2, FC
1, FC
2, FC
3, FC
4, FQ and FP
1Etc. multiple derivant; Wherein, with FB
1Toxicity the strongest, level of pollution is the highest.
Fumonisin and crazy heifer disease virus, dioxin, Escherichia coli O-157 and five kinds of pollutants of propylene chlorohydrin are considered to the five big toxin that the world today jeopardizes human food's safety.Think that in the world fumonisin is a kind of mycotoxin with great health significance, formed another research focus after aflatoxin.The World Health Organization (WHO) also with fumonisin as one of several mycotoxins that needed in recent years preferentially to study.Many evidences show that fumonisin can cause the animal various diseases, but serious threat human and animal's health and food security also are considered to occurred frequently relevant with human esophagus cancer.1993, international cancer research institute (TheInternational Agency for Research on Cancer) was decided to be 2B group carcinogen (may be human carcinogenic substance) with fumonisin.
International cancer research institution (IARC) estimates in the toxicity of 1993 pairs of fumonisins, JECFA (the FAO/WHO food additives joint specialist council) determines that in calendar year 2001 the 56th meeting the acute and chronic action-less dose value of fumonisin is 20mg/kgbw, and with fumonisin B
1, B
2, B
3The PMTDI of (single or mixing) (tolerating intake tentative every day) is decided to be 2 μ g/kg.Because the data deficiency, CCFAC (food additives and pollutant code committee member) is the highest the limiting the quantity of of regulation fumonisin not as yet, and China does not have the limit standard of formulation about fumonisin in grain and the feed at present yet.Calendar year 2001 FDA (FDA) has issued for human edible corn and the highest directiveness bulletin of limiting the quantity of of corn product fumonisin, and the highest the limiting the quantity of of fumonisin is 2mg/kg in the human edible corn of regulation; In addition, the herding medical center (CVM) of FDA has also been issued the highest directiveness bulletin of limiting the quantity of of fumonisin in the animal feed simultaneously, stipulates that its scope of limiting the quantity of is 5-100mg/kg; Switzerland publilc health portion (Swiss Federal Office of Public Health) has also made regulation to fumonisin limiting the quantity of in corn, the FB that contains in the corn of doing for every gram
1And FB
2Total amount is less than 1mg; French committee for public health (French Council of Public Hygiene) then recommends the maximum in the cereal to limit the quantity of to be 3mg/kg.
The assay method of present fumonisin B1 has multiple, as: thin layer chromatography (TLC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC/MS), capillary electrophoresis (CE), liquid chromatography-mass spectrography (LC/MS) and enzymoimmunoassay methods such as (ELISA).Wherein, what research both at home and abroad and application were more is the HPLC method, and this is the designated analysis method of international AC association (AOAC), and is the most commonly used in the detection of fumonisin.But this method pre-treatment process is loaded down with trivial details, and complicated operation need carry out pre-column derivatization to fumonisin and handle when experiment, and need the professional to operate usually, the instrument and equipment costliness, testing process is consuming time and expense is higher, is unsuitable for the field quick detection of a large amount of samples; Enzymoimmunoassay ELISA is owing to its high specificity, and highly sensitive, accuracy is good, and is easy and simple to handle, and the advantages such as detection that are suitable for batch samples more and more are subjected to people's favor.Though the scientific research personnel of a lot of scientific research institutions and universities and colleges studies fumonisin B1 enzyme-linked immuno assay detection method, the sensitivity that detects and the stable aspect of kit also have certain distance from practical application.
Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) ultimate principle is: enzyme and antigen or antibody are combined with crosslinking chemical, corresponding antibodies or antigen generation idiosyncrasy in this kind enzyme-labelled antigen or antibody and the testing sample, and strong bonded; When adding the substrate of corresponding enzyme, substrate is generated the colour generation thing by enzymatic, can do qualitative or quantitative observation with the colour generation depth according to having or not of this colour generation thing.Because this technology is to be based upon on the basis of efficient catalytic effect of antigen-antibody reaction and enzyme, so this technology has characteristics such as detection sensitivity height, high specificity, accuracy are good.
Summary of the invention
But the objective of the invention is to overcome the deficiencies in the prior art and provide in the detection by quantitative food and cereal crops in the kit and the detection method thereof of fumonisin B1 content, this kit is simple in structure, easy to use, inexpensive, highly sensitive, and the sensitivity that this detection method detects can reach μ g/kg level.
The present invention solves its technical matters and is achieved through the following technical solutions:
Fumonisin B in a kind of employing enzyme linked immunosorbent detection technology detection by quantitative food
1The kit of content comprises reagent and 96 hole ELISA Plate, and the preparation of this kit may further comprise the steps:
(1) preparation fumonisin B
1-KLH antigen;
(2) preparation polyclone fumonisin B
1Antibody;
(3) preparation enzyme-labelled antigen;
(4) the preparation bag is by plate;
(5) reagent preparation.
Wherein, preparation fumonisin B
1The method of-KLH antigen is:
(1) high molecular weight protein keyhole hemocyanin KLH is dissolved with glutaraldehyde solution, 3～5 ℃ of continuous stirring 15～18h, the 45～50h that dialyses in damping fluid PBS is to remove too much glutaraldehyde;
(2) with fumonisin B
1Be dissolved in the methyl alcohol, dropwise join among the high molecular weight protein KLH that has activated, stirring at room reaction 1～2h is put in 3～5 ℃ of continuous stirring 15～20h then and spends the night, and forms mixed liquor;
(3) add sodium borohydride in mixed liquor, continuous stirring 3～5h forms reactant liquor to seal unreacted aldehyde radical;
(4) reactant liquor is packed into bag filter, 68～73h dialyses in damping fluid PBS;
(5) accurately measure the volume of protein conjugate solution and measure concentration, add 4 ℃ of preservations behind the Sodium azide.
And described glutaraldehyde solution is that 50% glutaraldehyde is dissolved among the 0.01mol/L damping fluid PBS its pH=7.4.
And the described method for preparing polyclone fumonisin B1 antibody is:
(1) fumonisin immunogene 1mg is dissolved among the NaCl, with Freund's complete adjuvant emulsification, makes Water-In-Oil antigen emulsifying agent;
(2) utilize new zealand white rabbit to carry out subcutaneous multi-point injection as immune animal, carry out initial immunity, carry out booster immunization after 14 days at initial immunity, immunogene dosage is 0.5mg, carry out emulsification with incomplete Freund, later on every 30 days left and right sides booster immunizations once, immunity is about 6 times altogether, and each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and affinity is measured;
(3) adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 3～5 ℃ behind the Sodium azide of gained antibody interpolation 0.1%W/V.
5. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: the described method for preparing enzyme-labelled antigen is:
(1) with horseradish peroxidase HRP adding 1mol/L sodium periodate solution and in room temperature activation 15～25min,, forms the horseradish peroxidase HRP of activation then to 1mM sodium-acetate buffer dialysed overnight;
(2) add fumonisin B among the HRP after activation
1, behind stirring at room reaction 1～3h, in reactant liquor, adding the 1mol/L sodium borohydride solution cessation reaction of 0.1mL, this course of reaction is carried out 0.5～1.5h under 3～5 ℃;
(3) all reactant liquors are put into the damping fluid PBS dialysis 70～73h of bag filter to 0.01mol/L.
And the described bag for preparing by the method for plate is:
Na with pH9.6
2CO
3-NaHCO
3Damping fluid with fumonisin B
1Antibody dilution be 1 μ g/100 μ L as coating buffer, the every hole of microwell plate adds 100 μ L, room temperature is placed and to be spent the night or 36～38 ℃ of constant temperature incubation 2～3h, the cleansing solution washing is removed coating buffer three times, adds the freeze-drying fluid-tight and closes 0.5～1.5 hour, after cleansing solution washs four times, freeze-drying, 3～5 ℃ of preservations.
And reagent formation in the described reagent box body and preparation method thereof is:
(1) damping fluid PBS: its formation comprises Na
2HPO
412H
2O, NaH
2PO
42H
2O and NaCl add the distilled water dilution, 1: 5 times of dilution in use;
(2) fumonisin B1 titer: with fumonisin B
1Dilute in the mother liquor of pure product preparation and obtain, the mother liquor solvent for use is a chromatographically pure methyl alcohol, and dilution is 5% methyl alcohol phosphate buffer, and totally six bottles, concentration is respectively: 10 μ g/L, 3.33 μ g/L, 1.11 μ g/L, 0.37 μ g/L, 0.123 μ g/L, 0.041 μ g/L;
(3) freeze-drying liquid: Tris is dissolved in the 100mL distilled water, and hydrochloric acid is regulated pH7.4, adds sucrose, lactose, bovine serum albumin and ascorbic acid;
(4) substrate A: it constitutes anhydrous sodium acetate, powder-beta-dextrin and carbamide peroxide, add distilled water dilution after, transfer about pH to 5.0 3～5 ℃ of preservations;
(5) substrate B: 50mg is dissolved in dimethyl sulfoxide (DMSO) 5mL;
(6) cleansing solution: the PBS aqueous solution that contains 0.05%Tween20;
(7) matrix masking agent: 0.5% fish glue from skin PBS solution;
(8) enzyme-labelled antigen.
And, get be coated with fumonisin B1 antibody the micropore bag by plate, add fumonisin B
1Standard or the sample handled well are in each micropore, add enzyme-labelled antigen (being fumonisin B1-HRP) again, concussion reaction 0.5～1.5h, the cleansing solution washing, mix substrate A and substrate B in advance as substrate solution and join in the micropore, every hole adds 1.25mol/L sulfuric acid stopped reaction behind reaction 0.4～0.6h, measures absorbance, the fumonisin B in the reference standard curve calculation sample
1Content.
And, described fumonisin B
1The disposal route of sample is: get each 8～12g of various grain samples, in various mark-on samples, add 30～50mL extract respectively, 200～300rpm oscillation extraction, 10～20min on mixer oscillator, leave standstill 10～20min after, get the part supernatant and get final product after with the dilution of matrix screening agent.
Description of drawings
Fig. 1 is fumonisin B of the present invention
1-ELISA canonical plotting;
Embodiment
Below in conjunction with embodiment the present invention is described, the scheme of embodiment described here does not limit the present invention; One of skill in the art can make improvements and change according to the solution of the present invention, and these improvement and variation all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.
One, preparation kit
1. fumonisin B
1The preparation of-KLH antigen
Take by weighing 10mg high molecular weight protein keyhole hemocyanin (KLH) (hereinafter to be referred as KLH), (50% glutaraldehyde is dissolved in 0.01mol/L PBS (damping fluid with the 10mL0.2% glutaraldehyde, hereinafter to be referred as PBS) in, pH 7.4) dissolving, 4 ℃ of continuous stirring 16h, dialysis 48h removes too much glutaraldehyde in 0.01mol/L PBS (pH 7.4).Get 1mg FB
1Be dissolved in the 0.4mL methyl alcohol, dropwise join among the KLH that has activated, stirring at room reaction 2h is put in 4 ℃ of continuous stirring then and spends the night (16h), adds the 12mg sodium borohydride at last in mixed liquor, and continuous stirring 4h is to seal unreacted aldehyde radical.The bag filter of at last reactant liquor being packed into, the 72h that dialyses in PBS is accurately measured the volume of protein conjugate solution then and is measured concentration, adds 4 ℃ of preservations behind the Sodium azide.
This fumonisin B
1Be a kind of micromolecule haptens, molecular weight is 721, has only reactionogenicity and non-immunogenicity, need with carrier protein couplet after just can make animal produce immune response.Amino on the 2nd carbon atom of fumonisin B1 is the optimal site of coupling, therefore can amino in the fumonisin B1 molecule and high molecular weight protein KLH be combined with glutaraldehyde method, carries out animal immune with synthetic fumonisin B1-KLH as immunogene.
2. the preparation of polyclone fumonisin B1 antibody
Immune animal is selected 3 of Japan large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are numbered respectively No. 1, No. 2 and No. 3.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
Immune programme for children: initial immunity: for improving the immunity of antigen, adopt the immunogene by Freund's complete adjuvant emulsification, dosage is 1mg (being dissolved in the NaCl of 1mL 0.9% and the Freund's complete adjuvant of 1mL).Booster immunization: carried out booster immunization on the 14th day behind the initial immunity, dosage is 0.5mg (being dissolved in the NaCl of 1mL 0.9% and the incomplete Freund of 1mL).Later on once, be total to immunity 6 times every 30 days booster immunizations.Since the 2nd booster immunization, each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and affinity is measured.Adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the Sodium azide of gained antibody interpolation 0.1% (W/V).
3. the preparation of enzyme-labelled antigen:
Accurately take by weighing 2.0mg HRP, add the 1mol/L sodium periodate solution and activate 20min, then to 1mM sodium-acetate buffer (pH 4.4) dialysed overnight in room temperature.Add 1.0mL fumonisin B among the HRP after activation
1(1mg fumonisin B
1Be dissolved in the carbonate buffer solution of 1mL pH 9.5), behind the stirring at room reaction 2h, in reactant liquor, add the sodium borohydride solution cessation reaction of 0.1mL 1mol/L, this process is carried out 1h under 4 ℃.At last all reactant liquors are put into the PBS dialysis 72h of bag filter to 0.01mol/L.
4. bag is by the preparation of plate
Fumonisin B
1Antibody sandwich is used pH9.6 Na in microwell plate
2CO
3-NaHCO
3Damping fluid be that 1 μ g/100 μ L is as coating buffer with fumonisin B1 antibody dilution, the every hole of microwell plate, 96 holes (or 48 holes) adds 100 μ L, room temperature is placed and to be spent the night or 37 ℃ of constant temperature incubations 2-3 hour, the cleansing solution washing is removed coating buffer three times, adding 200 μ L freeze-drying fluid-tights closed 1 hour, after the cleansing solution washing four times, freeze-drying, 4 ℃ of preservations.
5. the preparation of reagent
Damping fluid (PBS): Na
2HPO
412H
2O:68.8g, NaH
2PO
42H
2O:8.97g, NaCl:45g add distilled water to 1L, 1: 5 times of dilution during use;
Freeze-drying liquid: 0.121gTris is dissolved in the 100mL distilled water, and hydrochloric acid is regulated pH7.4, adds 1g sucrose, 1g lactose, 2g bovine serum albumin and 0.8g ascorbic acid.
Substrate A: anhydrous sodium acetate: 8.2g, powder-beta-dextrin: 2.5g, carbamide peroxide: 429mg adds distilled water to 1000mL, transfers pH to 5.0,4 ℃ of preservations;
Substrate B: 50mg is dissolved in dimethyl sulfoxide (DMSO) (DMSO) 5mL;
Cleansing solution: the PBS aqueous solution that contains 0.05%Tween20;
Stop buffer: 1.25mol/L sulfuric acid;
Fumonisin B1 titer, dilute in the mother liquor with the pure product preparation of fumonisin B1 and obtain, the mother liquor solvent for use is a chromatographically pure methyl alcohol, and dilution is 5% methyl alcohol phosphate buffer, totally six bottles, concentration is respectively: 10 μ g/L, 3.33 μ g/L, 1.11 μ g/L, 0.37 μ g/L, 0.123 μ g/L, 0.041 μ g/L.
Matrix masking agent: 0.5% fish glue from skin PBS solution
The reagent that kit provides:
1 * 96 orifice plate, (8 * 12 the hole can split) is coated with fumonisin B1 antibody;
6 * fumonisin B1 titer, the 1.0mL/ bottle, concentration of standard solution is: 10 μ g/L, 3.33 μ g/L, 1.11 μ g/L, 0.37 μ g/L, 0.123 μ g/L, 0.041 μ g/L;
1 * fumonisin B1-HRP, time spent PBS dilution in 1: 1000;
1 * substrate A, 15mL;
1 * substrate B, 1mL;
1 * cleansing solution, 30mL, the time spent is with distilled water dilution in 1: 5;
1 * damping fluid, 30mL;
1 * stop buffer, 5mL;
1 * matrix masking agent, 10mL.
Points for attention before measuring:
(1) uses before with all reagent recovery room temperatures (15-30 ℃);
(2) immediately all reagent are put back to 4 ℃ after the use;
(3) if the hyperchannel pipettor is used in the big suggestion of sample size.
Two, detect grain sample (is example with the corn)
Abundant powder essence is powdered respectively through comminutor for corn, 50 ℃ of oven dry in drying box, and-20 ℃ store for future use.Get each 10g of corn sample respectively, add the 40mL extract (75% methanol, V: V), 250rpm oscillation extraction 15min on mixer oscillator, leave standstill 10-20min after, get the part supernatant and be used for ELISA mensuration after with 15 times of dilutions of matrix screening agent.
In the mark-on test, wherein add the FB of variable concentrations respectively to sample
1, with the abundant mixing of mark-on sample, room temperature standing over night.All the other processing procedures are the same.
Three, detection method
Get and be coated with fumonisin B
1The freeze-drying lath of antibody, enzyme-added mark antigen and standard specimen (or sample extracting solution): every hole adds 100 μ L fumonisin B earlier
1Standard specimen solution or sample extracting solution add 100 μ L enzyme-labelled antigen solution, then not add FB
1The hole of standard specimen is a control wells.Room temperature reaction 1h washes plate 3 times with the PBST washing lotion then; In every hole, add 150 μ L substrate solutions, react 30min under the room temperature; Every hole adds 50 μ L stop buffers, cessation reaction; Under dual wavelength mode (450-650nm), read absorbance with microplate reader.In detection to corn sample, add recovery test at 250 μ g/kg and 500 μ g/kg levels respectively, its recovery is 103% and 86%.
Principle of work of the present invention is:
Adopt enzyme linked immunosorbent detection technology detection by quantitative fumonisin B1, the basis of its mensuration is the enzyme labeled immunoassay reaction, coated in microporous plate fumonisin B1 antibody, add fumonisin B1 standard or sample, add fumonisin B1 and horseradish peroxidase (HRP) connector again, free fumonisin B1 and fumonisin B1-HRP connector are competed fumonisin B1 antibody jointly, the fumonisin B1 that does not have to connect is washed and removes, add reaction substrate, reaction system colour developing under the HRP enzymatic, add and end to measure its absorbance with microplate reader behind the liquid, fumonisin B1 concentration in absorbance and the sample is inversely proportional to, and reference standard curve (Fig. 1) promptly can be determined the content of the fumonisin B1 in the sample.
The present invention mainly adopts the enzyme-linked immuno assay detection technique to detect fumonisin B1, can realize reaching the purpose of the detection by quantitative of the fumonisin B1 in the cereal crops in the food; And this kit is simple in structure, easy to use, inexpensive, highly sensitive; Its detection method high specificity, highly sensitive, accuracy is good, and its sensitivity can reach μ g/kg level.
Claims (9)
1. one kind is adopted fumonisin B in the enzyme linked immunosorbent detection technology detection by quantitative food
1The kit of content comprises reagent and 96 hole ELISA Plate, and it is characterized in that: the preparation of this kit may further comprise the steps:
(1) preparation fumonisin B
1-KLH antigen;
(2) preparation polyclone fumonisin B
1Antibody;
(3) preparation enzyme-labelled antigen;
(4) the preparation bag is by plate;
(5) reagent preparation.
2. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: preparation fumonisin B
1The method of-KLH antigen is:
(1) high molecular weight protein keyhole hemocyanin KLH is dissolved with glutaraldehyde solution, 3～5 ℃ of continuous stirring 15～18h, the 45～50h that dialyses in damping fluid PBS is to remove too much glutaraldehyde;
(2) with fumonisin B
1Be dissolved in the methyl alcohol, dropwise join among the high molecular weight protein KLH that has activated, stirring at room reaction 1～2h is put in 3～5 ℃ of continuous stirring 15～20h then and spends the night, and forms mixed liquor;
(3) add sodium borohydride in mixed liquor, continuous stirring 3～5h forms reactant liquor to seal unreacted aldehyde radical;
(4) reactant liquor is packed into bag filter, 68～73h dialyses in damping fluid PBS;
(5) accurately measure the volume of protein conjugate solution and measure concentration, add 4 ℃ of preservations behind the Sodium azide.
3. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 2
1The kit of content is characterized in that: described glutaraldehyde solution is that 50% glutaraldehyde is dissolved among the 0.01mol/L damping fluid PBS its pH=7.4.
4. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: the described method for preparing polyclone fumonisin B1 antibody is:
(1) fumonisin immunogene 1mg is dissolved among the NaCl, with Freund's complete adjuvant emulsification, makes Water-In-Oil antigen emulsifying agent;
(2) utilize new zealand white rabbit to carry out subcutaneous multi-point injection as immune animal, carry out initial immunity, carry out booster immunization after 14 days at initial immunity, immunogene dosage is 0.5mg, carry out emulsification with incomplete Freund, later on every 30 days left and right sides booster immunizations once, immunity is about 6 times altogether, and each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and affinity is measured;
(3) adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 3～5 ℃ behind the Sodium azide of gained antibody interpolation 0.1%W/V.
5. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: the described method for preparing enzyme-labelled antigen is:
(1) with horseradish peroxidase HRP adding 1mol/L sodium periodate solution and in room temperature activation 15～25min,, forms the horseradish peroxidase HRP of activation then to 1mM sodium-acetate buffer dialysed overnight;
(2) add fumonisin B among the HRP after activation
1, behind stirring at room reaction 1～3h, in reactant liquor, adding the 1mol/L sodium borohydride solution cessation reaction of 0.1mL, this course of reaction is carried out 0.5～1.5h under 3～5 ℃;
(3) all reactant liquors are put into the damping fluid PBS dialysis 70～73h of bag filter to 0.01mol/L.
6. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: the described bag for preparing by the method for plate is:
Na with pH9.6
2CO
3-NaHCO
3Damping fluid with fumonisin B
1Antibody dilution be 1 μ g/100 μ L as coating buffer, the every hole of microwell plate adds 100 μ L, room temperature is placed and to be spent the night or 36～38 ℃ of constant temperature incubation 2～3h, the cleansing solution washing is removed coating buffer three times, adds the freeze-drying fluid-tight and closes 0.5～1.5 hour, after cleansing solution washs four times, freeze-drying, 3～5 ℃ of preservations.
7. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 1
1The kit of content is characterized in that: reagent formation in the described reagent box body and preparation method thereof is:
(1) damping fluid PBS: its formation comprises Na
2HPO
412H
2O, NaH
2PO
42H
2O and NaCl add the distilled water dilution, 1: 5 times of dilution in use;
(2) fumonisin B1 titer: with fumonisin B
1Dilute in the mother liquor of pure product preparation and obtain, the mother liquor solvent for use is a chromatographically pure methyl alcohol, and dilution is 5% methyl alcohol phosphate buffer, and totally six bottles, concentration is respectively: 10 μ g/L, 3.33 μ g/L, 1.11 μ g/L, 0.37 μ g/L, 0.123 μ g/L, 0.041 μ g/L;
(3) freeze-drying liquid: Tris is dissolved in the 100mL distilled water, and hydrochloric acid is regulated pH7.4, adds sucrose, lactose, bovine serum albumin and ascorbic acid;
(4) substrate A: it constitutes anhydrous sodium acetate, powder-beta-dextrin and carbamide peroxide, add distilled water dilution after, transfer about pH to 5.0 3～5 ℃ of preservations;
(5) substrate B: 50mg is dissolved in dimethyl sulfoxide (DMSO) 5mL;
(6) cleansing solution: the PBS aqueous solution that contains 0.05%Tween20;
(7) matrix masking agent: 0.5% fish glue from skin PBS solution;
(8) enzyme-labelled antigen.
8. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food as claimed in claim 1
1The detection method of the kit of content is characterized in that: get be coated with fumonisin B1 antibody the micropore bag by plate, add fumonisin B
1Standard or the sample handled well are in each micropore, add enzyme-labelled antigen (being fumonisin B1-HRP) again, concussion reaction 0.5～1.5h, the cleansing solution washing, mix substrate A and substrate B in advance as substrate solution and join in the micropore, every hole adds 1.25mol/L sulfuric acid stopped reaction behind reaction 0.4～0.6h, measures absorbance, the fumonisin B in the reference standard curve calculation sample
1Content.
9. fumonisin B in the employing enzyme linked immunosorbent detection technology detection by quantitative food according to claim 8
1The detection method of the kit of content is characterized in that: described fumonisin B
1The disposal route of sample is: get each 8～12g of various grain samples, in various mark-on samples, add 30～50mL extract respectively, 200～300rpm oscillation extraction, 10～20min on mixer oscillator, leave standstill 10～20min after, get the part supernatant and get final product after with the dilution of matrix screening agent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100565596A CN101231291A (en) | 2007-01-25 | 2007-01-25 | Kit and method for quantitative determination of Fumonisin B1 content in food by ELISA detection technique |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100565596A CN101231291A (en) | 2007-01-25 | 2007-01-25 | Kit and method for quantitative determination of Fumonisin B1 content in food by ELISA detection technique |
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| CN101231291A true CN101231291A (en) | 2008-07-30 |
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| CN102095850A (en) * | 2011-01-26 | 2011-06-15 | 福建农林大学 | Reagent kit for detecting Fumonisin B1 of genetically engineered single-chain antibody and method thereof |
| CN102162813A (en) * | 2011-01-20 | 2011-08-24 | 福建农林大学 | Reagent kit and method for detecting T-2 toxin by using genetically engineered single-chain antibody |
| CN102478577A (en) * | 2010-11-30 | 2012-05-30 | 吉林大学 | Chemiluminescence kit for detecting Fumonisins and preparation method thereof |
| CN102520179A (en) * | 2011-12-07 | 2012-06-27 | 上海交通大学 | Quantitative detection method of fumonisins B1 |
| CN102080066B (en) * | 2009-11-26 | 2012-07-04 | 北京维德维康生物技术有限公司 | Method for detecting T-2 toxin and special reagent kit thereof |
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| CN104356208A (en) * | 2013-03-06 | 2015-02-18 | 南昌大学 | Dodecapeptide antigen mimic epitope for FB1 (fumonisins B1) and application of dodecapeptide antigen mimic epitope |
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| CN104356207A (en) * | 2013-03-06 | 2015-02-18 | 南昌大学 | Dodecapeptide antigen mimotope of FB1 (fumonisins B1) and application of dodecapeptide antigen mimotope |
| CN104076154A (en) * | 2013-03-28 | 2014-10-01 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof |
| CN103421743B (en) * | 2013-05-31 | 2015-01-28 | 华中农业大学 | Recombinant monoclonal antibodies H7 resisting fumonisins B1 |
| CN103421743A (en) * | 2013-05-31 | 2013-12-04 | 华中农业大学 | Recombinant monoclonal antibodies H7 resisting fumonisins B1 |
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Application publication date: 20080730 |