CN103157439A - Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof - Google Patents
Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof Download PDFInfo
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- CN103157439A CN103157439A CN201110410435XA CN201110410435A CN103157439A CN 103157439 A CN103157439 A CN 103157439A CN 201110410435X A CN201110410435X A CN 201110410435XA CN 201110410435 A CN201110410435 A CN 201110410435A CN 103157439 A CN103157439 A CN 103157439A
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- zol
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- zearalenone
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Abstract
The invention discloses an immunosorbent comprising a solid-phase carrier and an antibody coupled with the solid-phase carrier. The antibody comprises a monoclonal antibody secreted by a hybridoma cell line CGMCC NO.5504 and a monoclonal antibody secreted by a hybridoma cell line CGMCC NO.5505. The invention also provides a kit comprising the immunosorbent or an immunoaffinity column. Also, the invention provides applications of the immunosorbent, immunoaffinity column, and kit in detecting aflatoxins, sterigmatocystin, zearalenone congeners and ochratoxin A. The invention specifically provides separation and detection methods. According to the invention, the specific monoclonal antibodies with stable performances are developed, such that simultaneous purification and detection of 6 aflatoxins, sterigmatocystin, 6 zearalenone congeners and ochratoxin A are realized.
Description
Technical field
The present invention relates to a kind of immunosorbent, and immune affinity column and the kit of this immunosorbent is housed and they are at the purifying AFB
1, B
2, G
1, G
2, M
1, M
2, the application in sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
Background technology
Aflatoxin is one group of similar secondary metabolite of structure that aspergillus flavus and aspergillus parasiticus etc. produce, be one group take two compounds of cumarin as basic structure of muttering of barking.Separated at present identifying 12 kinds, especially AFB
1, B
2, G
1, G
2, M
1And M
2Be strong polluter, extensively be present in foodstuff grain and feed and processed goods thereof, the strongest at more than 200 kind of at present known mycotoxin Poisoning, to pollute frequency the highest.Aflatoxin has induced mutation, Immunosuppression and carcinogenic effect.The target organ of aflatoxin effect is mainly liver, is acknowledged as hepatocarcinogen matter, contains low-level aflatoxin food even eat, and long-term edible its liver of people also will suffer damage.The international cancer center is decided to be a class carcinogen with it.
Zearalenone claims again the F-2 toxin, and at first it separate from the corn that head blight is arranged and obtain.The toxigenic bacterium of zearalenone is mainly the bacterial strain of Fusarium, as Fusarium graminearum and fusarium tricinctum.Zearalenone ex hoc genus anne owner will pollute the cereal such as corn, wheat, rice, barley, millet and oat, and wherein, the positive rate of corn is 45%, and the highest toxic amount can reach 2909mg/kg; The recall rate of wheat is 20%, and toxic amount is 0.364-11.05mg/kg.The heat resistance of zearalenone ex hoc genus anne thing is stronger, processes 1h under 110 ℃ and is just destroyed fully.Zearalenone ex hoc genus anne thing has estrogen action, and Main Function can make domestic animal, poultry and experiment mice produce the hyperfunction disease of female hormone in reproductive system.The edible food that contains zearalenone ex hoc genus anne thing of gravidic animal (comprising the people) can cause miscarriage, stillborn foetus and monster.Edible contain the various wheaten food that Gibberella saubinetii flour makes and also can cause the poisoning symptom of central nervous system, as feel sick, feel cold, headache, mind depression and incoordination etc.
Sterigmatocystin is by a kind of mycotoxin with carcinogenicity of the generations such as aspergillus versicolor, aspergillus nidulans, belongs to the aflatoxin precursor substance on chemical constitution, can form adduct with DNA.The pollution in grain and feed all over the world of aspergillus and sterigmatocystin is very common.Domestic scholars research finds, in China's various places grain the pollution of sterigmatocystin very general, wherein the most obvious with wheat, pollution rate reaches as high as 100%.Sterigmatocystin may be closely related with the generation of cancer of the stomach, hepatodynia.
Ochratoxin is by a kind of mould nephrotoxin of Aspergillus ochraceus and the mould generation of pure green cyan, can be divided into two types of A and B, and the toxicity of ochratoxin A is larger.Ochratoxin Aspergillus ochraceus under the low temperature of 4 ℃ can produce the ochratoxin with toxic action concentration.The ochratoxin A of animal absorption 1ppm body weight dosage can be lethal at 5-6 days.Common pathology is that tubular epithelial injury and enteron aisle lymph body of gland are downright bad.The daily ration that contains 1ppm concentration ochratoxin of feeding can cause that animal polydipsia, frequent micturition, growth retardation and efficiency of feed utilization reduced in 3 months; The daily ration that the content of feeding is low to moderate 200ppb can detect injury of kidney several weeks.Other clinical symptoms also have diarrhoea, apocleisis and dehydration.Sometimes clinical symptoms are not obvious, and in the poisoning area that is local epidemic disease of ochratoxin, animal only observable pathology when butchering is that kidney is pale, hard.
At present, the detection method of aflatoxin, sterigmatocystin, zearalenone congener and ochratoxin A mainly contains TLC, ELISA (ELISA), immunoaffinity chromatography-liquid chromatography, immunoaffinity chromatography-fluorimetry etc.Wherein, TLC will contact a large amount of standard items, is unfavorable for experimenter's health, and sensitivity is very low.ELISA is only applicable to qualitative detection, is easy to occur false positive and false-negative phenomenon.And immunoaffinity chromatography-liquid chromatography can detect the content of the aflatoxin in many commodity quantitatively.Because the method sensitivity is special, and do not use poisonous solvent such as chloroform and carrene etc., use more and more extensive.
The key of immunoaffinity chromatography method is to select a kind of efficient special antibody, but, utilize the interpolation rate of recovery of the methods such as liquid-liquid extraction of the prior art, SPE, heating evaporation concentrate all lower, follow-up detecting step is caused very strong matrix effect, and do not have in prior art can the pending matrix of Simultaneous purification in 6 kinds of aflatoxin (B
1, B
2, G
1, G
2, M
1And M
2), the immune adsorbing medium of 6 kinds of zearalenone congeners (α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON), sterigmatocystin and ochratoxin A, obviously can improve the efficient of detection and can Simultaneous purification analyze the Various Diseases toxin.
Therefore, the task of top priority, to prepare stable performance, special immunosorbent, and set up a kind of economy, quick, accurately, safety, and can 6 kinds of aflatoxin of Simultaneous purification, the method for 6 kinds of zearalenones, sterigmatocystin and ochratoxin As.
Summary of the invention
The purpose of this invention is to provide a kind of immunosorbent, and immune affinity column (1AC) and kit that this immunosorbent is housed, and their application in purifying and detection zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A, with at purifying and detection AFB
1, B
2, G
1, G
2, M
1, M
2, the application in zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A, and specifically provide a kind of purifying AFB
1, B
2, G
1, G
2, M
1, M
2, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A method, and a kind of detection AFB
1, B
2, G
1, G
2, M
1, M
2, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A method.
a first aspect of the present invention is to provide a kind of immunosorbent, it is characterized in that, this immunosorbent comprise solid phase carrier and with the antibody of this solid phase carrier coupling, described antibody comprises the monoclonal antibody that obtained by hybridoma cell strain CGMCC NO.5504 (being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on November 24th, 2011) secretion and the monoclonal antibody that is obtained by hybridoma cell strain CGMCC NO.5505 (being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 24th, 2011) secretion.
A second aspect of the present invention is to provide the immune affinity column that is mounted with above-mentioned immunosorbent.
A third aspect of the present invention is to provide the kit that contains above-mentioned immunosorbent or above-mentioned immune affinity column, preferably, also comprise eluent in described kit and/or preserve liquid, described eluent is preferably at least a in methyl alcohol, acetonitrile and acetone, and described preservation liquid is preferably the NaN that contains the 0.001-0.1 % by weight
3The PBS buffer solution.
A fourth aspect of the present invention is to provide above-mentioned immunosorbent, above-mentioned immune affinity column or mentioned reagent box at purifying and the application at least a in detecting zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
A fifth aspect of the present invention is to provide above-mentioned immunosorbent, above-mentioned immune affinity column or mentioned reagent box at purifying and detects AFB
1, B
2, G
1, G
2, M
1, M
2, the application at least a in sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
A sixth aspect of the present invention is to provide a kind of purifying AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, the method comprises, makes and contains AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A fluid sample by above-mentioned immune affinity column, make at least part of AFB in described fluid sample
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A be adsorbed on this immune affinity column, then with eluent with described at least part of AFB
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A elute.
A seventh aspect of the present invention is to provide a kind of detection AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, the method comprises,
(1) according to above-mentioned purifying AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, obtain liquid to be measured, described liquid to be measured is with described at least part of AFB with eluent
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A elute and the solution that obtains;
(2) make described liquid to be measured enter detection system, and by relatively carrying out qualitative or quantitative detection with standard items.
The present invention is by developing stable performance, special monoclonal antibody, realized Simultaneous purification and detection to 6 kinds of aflatoxin, sterigmatocystin, 6 kinds of zearalenones and ochratoxin A.Result by embodiment can find out, in the detection to peanut sample and milk sample, adds the rate of recovery all between 80-100%, and RSD is all less than 5%.Show that method of the present invention satisfies fully to AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A purifying and detection the analysis requirement.
Description of drawings
Accompanying drawing is to be used to provide a further understanding of the present invention, and consists of the part of specification, is used from explanation the present invention with the following specific embodiment one, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is AFB
1, B
2, G
1, G
2, M
1And M
2The liquid chromatogram of standard items;
Fig. 2 is the liquid chromatogram of sterigmatocystin standard items;
Fig. 3 is the liquid chromatogram of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON standard items;
Fig. 4 is the liquid chromatogram of ochratoxin A standard items.
The specific embodiment
The invention provides a kind of immunosorbent, it is characterized in that, this immunosorbent comprise solid phase carrier and with the antibody of this solid phase carrier coupling, described antibody comprises the monoclonal antibody that is obtained by hybridoma cell strain CGMCCNO.5504 (antibody A) secretion and the monoclonal antibody that is obtained by hybridoma cell strain CGMCCNO.5505 (antibody B) secretion.
Preferably, described antibody also comprises the monoclonal antibody (antibody C) that is obtained by hybridoma cell strain CGMCC NO.5506 secretion.
Hybridoma cell strain ZEN-F09B10CGMCC NO.5504 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 24th, 2011, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature are anti-ZER cell strain of monoclonal antibody; Hybridoma cell strain OTA-E11B8 CGMCC NO.5505 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 24th, 2011, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature are anti-ochratoxin A cell strain of monoclonal antibody; Hybridoma cell strain Afla-D12A1CGMCC NO.5506 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 24th, 2011, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature are anti-sterigmatocystin aflatoxin cell strain of monoclonal antibody.
Wherein, in the present invention, described antibody A is anti-zearalenone congener α-ZER (α-zearalanol, α-zearalanol), α-ZOL (α-zearalenol, α-zearalenol), β-ZER (β-ZER, β-zearalanol), β-ZOL (β-zearalenol, β-zearalenol), ZAN (zearelone, zearalanone) and ZON (zearalenone, zearalenone) at least a monoclonal antibody.
In the present invention, the preparation method of hybridoma cell strain CGMCC NO.5504 and antibody A can be the preparation method of this area routine.Namely, at first with zearalenone congener haptens and carrier protein couplet, preparation is as immunogene, then this immunogene is carried out immunity to mouse, by splenocyte and myeloma cell being carried out Fusion of Cells and hybridoma cell clone screening obtain required hybridoma cell strain and by the monoclonal antibody of its secretion.Wherein, described carrier protein can be bovine serum albumin or ovalbumin.
Particularly, the method can comprise the following steps:
(1) preparation zearalenone immunogene
5mg zearalenone ZON is dissolved in the 4mL pyridine, adds the O-carboxymethyl azanol of 3mg, at room temperature stir 24h, vacuum drying is afterwards transferred pH to 8.0 after the water dissolving, uses ethyl acetate extraction 3 times, the dehydration crystallization.Crystal is dissolved in the solution that is made into 5mmol/L in dioxane, takes in the PBS that 30mg BSA is dissolved in 2mL, 0.05mol/L (pH 7.4).Under 4 ℃, to both mix, slowly drip wherein again the N-hydroxy succinic acid imines (NHS) of 3mg and be dissolved in the N of the 6mg in the solution of 0.5mL dioxane, N '-two hexamethylene carbodiimide (DCC), with gained mixture stirring reaction 24h under room temperature, dialysis, centrifugal, freezing standby.
(2) monoclonal antibody of preparation hybridoma and anti-zearalenone
Animal immune: immune animal is the female BALB/c mouse of 6-8 about age in week.With 5 mouse of zearalenone immunogen immune.Get appropriate zearalenone immunogene (100 μ g/ only) and add the equivalent Freund's complete adjuvant, make emulsifying agent and carry out immunity, adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain zearalenone is had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5504 of secretion zearalenone antibody, prepare monoclonal antibody.
Antibody B of the present invention is the monoclonal antibody of anti-ochratoxin A.
In the present invention, the preparation method of hybridoma cell strain CGMCC NO.5505 and antibody B can be the preparation method of this area routine.Namely, at first with ochratoxin A haptens and carrier protein couplet, preparation is as immunogene, then this immunogene is carried out immunity to mouse, by splenocyte and myeloma cell being carried out Fusion of Cells and hybridoma cell clone screening obtain required hybridoma cell strain and by the monoclonal antibody of its secretion.Wherein, described carrier protein can be bovine serum albumin or ovalbumin.
Particularly, the method can comprise the following steps:
(1) preparation ochratoxin A immunogene
2mg ochratoxin A (OTA) is joined in 500 μ L acetone, add 4mg CDI, obtain the first solution.8mg BSA is joined in the carbonate buffer solution of 3mL 0.1M pH9.6, obtain BSA solution.The first solution slowly is added drop-wise in BSA solution, room temperature reaction 2 days, after reaction, solution is under 4 ℃ of conditions, with PBS solution dialysis 3 days.
(2) monoclonal antibody of preparation hybridoma and anti-ochratoxin A
Animal immune: immune animal is the female BALB/c mouse of 6-8 about age in week.With 5 mouse of ochratoxin A immunogen immune.Get appropriate ochratoxin A immunogene (100 μ g/ only) and add the equivalent Freund's complete adjuvant, make emulsifying agent and carry out immunity, adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain ochratoxin A is had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5505 of secretion ochratoxin A antibody, prepare monoclonal antibody.
Antibody C is aspergillus flavus resisting toxin B
1, B
2, G
1, G
2, M
1, M
2, and at least a monoclonal antibody in sterigmatocystin.
Hybridoma cell strain CGMCC NO.5506 in the present invention and the preparation method of antibody C can be the preparation method of this area routine.Namely, at first with aflatoxin haptens and carrier protein couplet, preparation is as immunogene, then this immunogene is carried out immunity to mouse, by splenocyte and myeloma cell being carried out Fusion of Cells and hybridoma cell clone screening obtain required hybridoma cell strain and by the monoclonal antibody of its secretion.Wherein, described carrier protein can be bovine serum albumin or ovalbumin.
Particularly, the method can comprise the following steps:
(1) the preparation Aspergillus flavus toxin immuno is former
With the 4mg AFB
1Be dissolved in 2mL acetone, add the H of 40 μ L 10%
2SO
4, at 56 ℃ of stirring reaction 4h, after reacting the products therefrom evaporate to dryness, add 5mLH
2O with 25mL chloroform recovery twice, then uses 20mL H
2O washs organic layer, keeps organic layer, boils off organic solvent, obtains yellow solid product.
Get the 1.0mg yellow solid product, add wherein the BSA solution (dissolving 20mg BSA in 4mL PBS) of 2mL 0.5%, react 30min under 37 ℃; The NaHB that adds 100 μ L6.5mM
4, at 4 ℃ of reaction 30min; Add the HCl of 50 μ L 0.1N, remove excessive NaHB
4Under 4 ℃ of conditions, with PBS solution dialysis 3 days, make immunogene, i.e. AFB
1-BSA (Aflatoxin B
1-BSA).
(2) monoclonal antibody of preparation hybridoma and aspergillus flavus resisting toxin and sterigmatocystin
Animal immune: immune animal is the female BALB/c mouse of 6-8 about age in week.Use AFB
15 mouse of-BSA immunity.Get appropriate AFB
1-BSA (100 μ g/ only) adds the equivalent Freund's complete adjuvant, makes emulsifying agent and carries out immunity, and adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain aflatoxin and sterigmatocystin are had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5506 of secretion aflatoxin and sterigmatocystin antibody, prepare monoclonal antibody.
In the present invention, above-mentioned various immunogenes all can be selected for this area is conventional with the relative consumption of carrier protein, and for example, the mol ratio of various immunogenes and carrier protein is 2-8: 1.
Described solid phase carrier can be the various solid phase carriers that are applicable to carry out with antibody coupling of this area routine, include but not limited at least a in cellulose, sephadex, polyacrylamide gel, cellular glass, Ago-Gel and ultragel ACA22, be preferably the Ago-Gel of cyanogen bromide (CNBr) activation; The concentration of described Ago-Gel can be for the various selections of routine, as 4%.The Ago-Gel of this CNBr activation can be commercially available with dry powder form, as available from U.S. GE company, during use, adds acid solution to carry out swelling and get final product, and this acid treatment method is known to the skilled person, does not repeat them here.When using lyophilized form and contain the Ago-Gel of CNBr activation of additive, preferably wash away additive under the condition of low pH value (being 2-3 as the pH value), and then carry out coupling with antibody.
Described method with antibody A, antibody B and antibody C and solid phase carrier coupling can be the method for this area routine, and particularly, the method can comprise:
(1) use the coupling buffer lytic antibody, described antibody is antibody A, antibody B and antibody C, obtains first antibody solution,
(2) described first antibody solution is contacted with the solid phase carrier of activation and carries out coupling, obtain coupling liquid,
(3) solid phase carrier after coupling in coupling liquid is transferred in the Tris-HCl buffer solution that concentration is 0.05-1M standing, with the residual activity site on the solid phase carrier after coupling in sealing coupling liquid,
(4) solid phase carrier after standing with cleaning solution washing, removing the antibody of not coupling, the solid phase carrier after being washed,
(5) with the NaN that contains the 0.001-0.1 % by weight
3PBS buffer solution for cleaning washing after solid phase carrier.
There is no particular limitation to the usage ratio of described antibody and solid phase carrier and the relative consumption of antibody A, antibody B and antibody C in the present invention, usually, makes the saturated as far as possible coupling of described antibody and solid phase carrier, and therefore, described antibody generally is in excess in solid phase carrier.But those skilled in the art also can select the amount ratio of antibody and solid phase carrier as required.In the present invention, the weight ratio of described antibody A, antibody B and antibody C and solid phase carrier is preferably 1: 0.2-5: 0.2-5: 5-10.
Coupling buffer described in the present invention can be for the various selections of this area routine, as the NaHCO of 0.1-0.3M
3Solution, the pH value can be 8-8.5.
Coupling condition and coupling method can be the selection of routine, as, can be under 4-25 ℃ of condition, the method that adopts convertible (end-over-end) was mixed 1-24 hour antibody and solid phase carrier are full and uniform.
Described sealing and the method for removing the antibody of not coupling also can be the conventional method of this area.Wherein, described condition standing in the Tris-HCl of 0.05-1M buffer solution (pH8.0) can for, under 4-25 ℃, standing 2-4 hour.Cleaning solution in described solid phase carrier after standing with cleaning solution washing can be the buffer solution (as the acetate buffer of pH4.0 and the Tris-HCl buffer solution of pH8.0) of low, high two kinds of pH values, as long as the condition of described washing makes the antibody of not coupling substantially be removed, usually, according to washing at least 3 circulations with order low, that the buffer solution of high pH washs successively.
The amount of antibody that can be by measuring not coupling is calculated two kinds of coupling rates that antibody is total, and coupling rate computing formula is:
Formula I
The described measurement not method of the amount of the antibody of coupling can be the method for this area routine, as, by the protein concentration in the supernatant after the ultraviolet determination coupling.
Similarly, condition and the step of the condition of antibody A and antibody B and solid phase carrier coupling and step and above-mentioned three kinds of antibody are identical.
The invention provides the immune affinity column that is mounted with above-mentioned immunosorbent.The method that immunosorbent is prepared into immune affinity column is known to the skilled person.For example, with the dress of the solid phase carrier after above-mentioned cleaning post.The described NaN that contains the 0.001-0.1 % by weight
3PBS buffer solution (consisting of of PBS buffer solution contains potassium dihydrogen phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g in 1L solution) can be used as the preservation liquid of this immune affinity column.
The method of dress post for example can for: use to preserve the liquid prepared slarry, mix with 75% Ago-Gel and 25% ratio of preserving liquid.With successional operation impouring slurries in the post.Use a glass bar that leans on column wall to fill out column operation, will help to reduce the generation of bubble.After filling out post, close the opening of affinity column lower end, and take off the tip part of affinity column.Carefully operation adds and preserves liquid with the remaining part of filling affinity column, forms a meniscus that makes progress with the top at affinity column.The top sieve plate is inserted in affinity column with certain angle, and guaranteeing does not have air below sieve plate.Sieve plate is locked on stromal surface suitable position, opens the opening of affinity column below, cross post with the preservation liquid of the aseptic filtration of 5 times of bed volumes, and use and preserve liquid and preserve, so far, affinity column loaded and balance complete, can be for direct.
Can calculate the dynamic column capacity (every milliliter of immunosorbent or the bed volume obtained the maximum absorption to determinand) of immune affinity column and absolute column capacity (every milliliter of sessile antibody maximum binding capacity to determinand) according to the conventional method (as disclosed method in CN101059512A) of this area.Result shows that described immune affinity column of the present invention is to AFB
1, B
2, G
1, G
2, M
1, M
2Be respectively 310ng/mL and 860ng/mg with dynamic column capacity and the absolute column capacity of sterigmatocystin, this immune affinity column is respectively 580ng/mL and 1700ng/mg to dynamic column capacity and the absolute column capacity of zearalenone, and this immune affinity column is respectively 130ng/mL and 580ng/mg to dynamic column capacity and the absolute column capacity of ochratoxin A.Having used 5-10 later column capacity is that about the 60-90% of total column capacity, storage life is 1.5 years.
The invention provides the kit that contains above-mentioned immunosorbent or contain above-mentioned immune affinity column; Preferably, also comprise eluent in described kit and/or preserve liquid, described eluent is preferably at least a in methyl alcohol, acetonitrile and acetone, most preferably is hplc grade methanol.Described preservation liquid is preferably the NaN that contains the 0.001-0.1 % by weight
3The PBS buffer solution.
In addition, also can comprise box body, AFB in this kit
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A standard items, cleaning solution, sponge bracket at least a.Wherein, described cleaning solution can be phosphate buffer (being the solution that contains 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate dodecahydrate and 8.8g sodium chloride in 1L water) or water; Described sponge bracket is provided with hole and groove, and above-mentioned standard solution is housed in described groove, the reagent bottle of above-mentioned various solution is housed, and the IAC post that above-mentioned immunosorbent is housed.
When containing antibody A and antibody B, above-mentioned immunosorbent of the present invention, above-mentioned immune affinity column or mentioned reagent box can be applicable to purifying and detect at least a in zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
When containing antibody A, antibody B and antibody C, above-mentioned immunosorbent of the present invention, above-mentioned immune affinity column or mentioned reagent box can be applied to purifying and detect AFB
1, B
2, G
1, G
2, M
1, M
2, at least a in sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
Particularly, the invention provides a kind of purifying AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, the method comprises, makes and contains AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON and ochratoxin A fluid sample by the above-mentioned immune affinity column that contains antibody A, antibody B and antibody C, make at least part of AFB in described fluid sample
1, B
2, G
1, G
2, M
1, M
2, at least part of sterigmatocystin, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and at least part of ochratoxin A be adsorbed on this immune affinity column, then with eluent with described at least part of AFB
1, B
2, G
1, G
2, M
1, M
2, at least part of sterigmatocystin, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and at least part of ochratoxin A elute, described eluent is preferably at least a in methyl alcohol, acetonitrile and acetone, most preferably is Chromatographic Pure Methanol.
The method is washed immune affinity column with cleaning solution before can also being included in the eluent wash-out, to remove the fluid sample of the remnants of non-specific adsorption on immune affinity column.Described cleaning solution can be in water, phosphate buffer and 10% methyl alcohol at least a.
According to the present invention, the described AFB that contains
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A fluid sample can be any fluid sample that contains above-mentioned substance, be preferably at least a in the liquid extract of the liquid extract of liquid extract, biological sample of liquid extract, the feed of food and Chinese medicine.
Described food includes but not limited to cereal, condiment, dairy produce etc., and described cereal includes but not limited to peanut, corn, soybean and rice etc., and described biological sample includes but not limited to blood plasma, liver, kidney, lung and muscle etc.
The method of described food, feed, biological sample and Chinese medicine being made liquid extract can be the method for this area routine, for example, can comprise the following steps: take a certain amount of sample, after methanol aqueous solution extraction with 50-80%, filter with qualitative filter paper, filtrate is diluted with pure water, then filter with glass fiber filter paper.
The present invention also provides a kind of detection AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON and ochratoxin A method, the method comprises,
(1) according to above-mentioned purifying AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, obtain liquid to be measured, described liquid to be measured is with described at least part of AFB with eluent
1, B
2, G
1, G
2, M
1, M
2, at least part of sterigmatocystin, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and at least part of ochratoxin A elute and the solution that obtains;
(2) make described liquid to be measured enter detection system, and by relatively carrying out qualitative or quantitative detection with standard items.
There is no particular limitation for the concrete mode that detects in the present invention, can be first to collect to obtain liquid to be measured, and then manually make liquid to be measured carry out detection system, also can for be used in conjunction system, make liquid to be measured automatically enter detection system and detect.
Described detection system can for the various instruments for analyzing and testing of this area routine, preferably be used liquid chromatogram.
The condition of described analyzing and testing can be adjusted according to the difference of material to be detected, and the method for adjustment is known to the skilled person.For example, for AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A, chromatographic condition can for:
1. for AFB
1, B
2, G
1, G
2, M
1, M
2, mobile phase: methanol-water (volume of methanol/water is 45: 55); Detector: fluorescence detector, excitation wavelength are 360nm, and emission wavelength is 440nm; Chromatographic column: C-18 post (column length is 150mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m); Flow velocity: 0.8mL/min.
2. for sterigmatocystin, mobile phase: methanol-water (volume of methanol/water is 75: 25); Detector: UV-detector wavelength: 325nm; Chromatographic column: C-18 post (column length is 250mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m); Flow velocity: 0.8mL/min.
3. for zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, mobile phase: acetonitrile-water (volume ratio of acetonitrile/water is 60: 40); Detector: fluorescence detector, excitation wavelength are 274nm, and emission wavelength is 440nm; Chromatographic column: C-18 post (column length is 150mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m); Flow velocity: 0.8mL/min.
4. for ochratoxin A, mobile phase: acetonitrile: water: acetic acid (three's volume ratio is 49.5: 49.5: 1); Detector: fluorescence detector, excitation wavelength are 333nm, and emission wavelength is 477nm; Chromatographic column: C-18 post (column length is 250mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m); Flow velocity: 0.8mL/min.
The method of described " comparing with standard items " can be the conventional method of this area, for example, at first detect accurately being configured to certain density standard items by liquid chromatogram, determine the characteristic peak positions of these standard items and the relation between peak area (or peak height) and concentration.Then, under similarity condition, liquid to be measured is detected, by the comparison that goes out the peak position with standard items, confirm whether there be the material identical with standard items in liquid to be measured, by with the comparison of the peak area (or peak height) of standard items, determine the content of material identical with standard items in liquid to be measured.
The present invention will be described in more detail by following examples.
Embodiment 1
The present embodiment for the preparation of the zearalenone immunogene, Aspergillus flavus toxin immuno is former and the ochratoxin A immunogene
(1) 5mg zearalenone ZON is dissolved in the 4mL pyridine, adds the O-carboxymethyl azanol of 3mg, at room temperature stir 24h, vacuum drying is afterwards transferred pH to 8.0 after the water dissolving, uses ethyl acetate extraction 3 times, the dehydration crystallization.Crystal is dissolved in the solution that is made into 5mmol/L in dioxane, takes in the PBS that 30mg BSA is dissolved in 2mL, 0.05mol/L (pH 7.4).Under 4 ℃, to both mix, slowly drip wherein again the N-hydroxy succinic acid imines (NHS) of 3mg and be dissolved in the N of the 6mg in the solution of 0.5mL dioxane, N '-two hexamethylene carbodiimide (DCC), with gained mixture stirring reaction 24h under room temperature, dialysis, centrifugal, freezing standby.Obtain the zearalenone immunogene, i.e. ZON-BSA.
(2) with the 4mg AFB
1Be dissolved in 2mL acetone, add the H of 40 μ L 10%
2SO
4, at 56 ℃ of stirring reaction 4h; After reacting the products therefrom evaporate to dryness, add 5mL H
2O with 25mL chloroform recovery twice, then uses 20mL H
2O washs organic layer, keeps organic layer; Boil off organic solvent, get yellow solid product.
Get the described yellow solid product of 1.0mg, add wherein the BSA solution (being dissolved with 20mg BSA in 4mLPBS) of 2mL 0.5%, react 30min under 37 ℃; The NaHB that adds 100 μ L 6.5mM
4, 4 ℃ of reaction 30min; Add the HCl of 50 μ L 0.1N, remove excessive NaHB
4Under 4 ℃ of conditions, with PBS solution dialysis 3 days.Obtain Aspergillus flavus toxin immuno former, i.e. AFB
1-BSA.
(3) 2mg ochratoxin A (OTA) joins in 500 μ L acetone, adds 4mg CDI, obtains the first solution.8mg BSA is joined in the carbonate buffer solution of 3mL 0.1M pH9.6, obtain BSA solution.The first solution slowly is added drop-wise in BSA solution, room temperature reaction 2 days, after reaction, solution is under 4 ℃ of conditions, with PBS solution dialysis 3 days.Obtain the ochratoxin A immunogene, i.e. OTA-BSA.
Embodiment 2
The present embodiment is for the preparation of the monoclonal antibody (antibody A) of anti-zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and monoclonal antibody (antibody B) and the aspergillus flavus resisting toxin B of anti-ochratoxin A
1, B
2, G
1, G
2, M
1, M
2Monoclonal antibody (antibody C) with sterigmatocystin.
(1) animal immune: immune animal is the female BALB/c mouse of 6-8 about age in week.With 5 mouse of zearalenone immunogen immune.Get zearalenone immunogene (100 μ g/ only) and add the equivalent Freund's complete adjuvant, make emulsifying agent and carry out immunity, adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain zearalenone is had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5504 of secretion zearalenone antibody, prepare monoclonal antibody A.
(2) animal immune: immune animal is the female BALB/c mouse of 6-8 about age in week, with 5 mouse of ochratoxin A immunogen immune.Get ochratoxin A immunogene (100 μ g/ only) and add the equivalent Freund's complete adjuvant, make emulsifying agent and carry out immunity, adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain ochratoxin A is had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5505 of secretion ochratoxin A antibody, prepare monoclonal antibody B.
(1) animal immune: immune animal is about age in 6-8 week, female BALB/c mouse.With AFB1-3 mouse of BSA immunity.Get AFB1-BSA (100 μ g/ only) and add the equivalent Freund's complete adjuvant, make emulsifying agent and carry out immunity, adjuvant changes Freunds incomplete adjuvant into afterwards, and immunity is 6 times altogether, every 2 weeks of minor tick.Except being that the subcutaneous multi-point injection of nape section, all the other are lumbar injection for the first time.After finishing, immunity puts to death mouse, extracting spleen cell.
Fusion of Cells: splenocyte and hybridoma are carried out the Fusion of Cells test according to the ratio of 10: 1.
Hybridoma cell clone: adopt limiting dilution assay screening hybridoma, until obtain aflatoxin and sterigmatocystin are had the cell of good specific reaction.Filter out at last the hybridoma CGMCC NO.5506 of secretion aflatoxin and sterigmatocystin antibody, prepare monoclonal antibody C.
Embodiment 3
The present embodiment is for the preparation of immune affinity column
1, the preparation of Ago-Gel
Take required 1g with the Ago-Gel powder (be purchased the GE company from the U.S., every gram freeze-dried powder can form the swelling Ago-Gel of 3.5ml final volume) of CNBr activation, be dissolved in 1mM HCl.The Ago-Gel of swelling immediately is placed in fritted glass filter, with 1mM HCl washing 15min.
2, antibody coupling
(a) use coupling buffer (0.2M NaHCO
3, pH8.3) lytic antibody, antibody are antibody A, antibody B and antibody C, and wherein, the concentration of antibody A, antibody B and antibody C is 5.1mg/ml, and the antibody-solutions volume is 35ml, antibody-solutions is placed in ice bath temporary.With cover can add the above-mentioned coupling buffer that contains antibody in complete hermetic container at one, and add wherein rapidly the good Ago-Gel of step 1 washing.The lower above-mentioned mixture 2-4h of the abundant mixing of mode that adopts end-over-end of room temperature condition (20-25 ℃),
(b) calculate the coupling rate: the mixture that centrifugation step under 2,000rpm (a) obtains, centrifugal 1min is transferred to supernatant in new centrifuge tube to managing at the end Ago-Gel is centrifugal, measures the protein content of supernatant.
The total coupling rate that calculates antibody A, antibody B and antibody C by formula I is 99.8%, illustrates that coupling is very successful.Get centrifugal Ago-Gel to managing the end, use coupling buffer to wash, remove unnecessary antibody.
(c) sealing: shift solid phase carrier to 0.1M Tris-HCl buffer solution, standing 2-4h under room temperature condition is to seal the avtive spot of not coupling.
(d) remove unnecessary antibody in not coupling, the buffer solution with low, high two kinds of pH washs Ago-Gel successively, washs at least 3 circulations, and the use amount of every kind of buffer solution is at least 5 times of Ago-Gel volume.
Each wash cycle step: first use 0.1M acetic acid/sodium-acetate buffer (the pH value is 4.0) washing, then use again 0.1M Tris-HCl buffer solution (the pH value is 8.0) to wash.
(e) with the 0.01 % by weight NaN that contains of 5 times of Ago-Gel volumes
3PBS washing, and use and contain 0.01 % by weight NaN
3PBS solution (preservation liquid) preserve.
3, dress post
Use and preserve the liquid prepared slarry, mix with 75% Ago-Gel and 25% ratio of preserving liquid.With successional operation impouring slurries in the post.Use a glass bar that leans on column wall to fill out column operation, will help to reduce the generation of bubble.After filling out post, close the opening of affinity column lower end, and take off the tip part of affinity column.Carefully operation adds the remaining part of preserving liquid filling affinity column, forms a meniscus that makes progress with the top at affinity column.The top sieve plate is inserted in affinity column with certain angle, and guaranteeing does not have air below sieve plate.Sieve plate is locked on stromal surface suitable position, opens the opening of affinity column below, cross post with the preservation liquid of the aseptic filtration of 5 times of bed volumes, and use and preserve liquid and preserve, so far, affinity column loaded and balance complete, can be for direct.
Embodiment 4
The present embodiment is for detection of the AFB in peanut sample
1, B
2, G
1, G
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A; Detect the aflatoxin M in milk sample
1And M
2
1, utilize the AFB that adds in recovery experiment detection peanut sample
1, B
2, G
1, G
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
(1) adding respectively final concentration in the peanut sample is 10 μ g/kg, 20 μ g/kg, the AFB of three concentration gradients of 50 μ g/kg
1, B
2, G
1, G
2Adding final concentration is 10 μ g/kg, 200 μ g/kg, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and the ZON of three concentration gradients of 500 μ g/kg, adding final concentration is 10 μ g/kg, 20 μ g/kg, the sterigmatocystin of three concentration gradients of 50 μ g/kg, adding final concentration is 10 μ g/kg, 20 μ g/kg, the ochratoxin A of three concentration gradients of 50 μ g/kg.Five groups of parallel tests are done in each experiment.
(2) Aflatoxin in Peanut byHigh B
1, B
2, G
1, G
2, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A extraction and detection:
Accurately take peanut 50.0g through levigate (granularity is less than 2mm) in 250mL tool plug conical flask, add 5.0g sodium chloride and accurately add 100.0mL methanol-water (the methanol/water volume ratio is 8: 2), extract 2min with the homogenizer high-speed stirred, or shaking table concussion 30min.Quantitative filter paper filters, and accurately pipettes 10.0mL filtrate and adds the PBS solution dilution of 40.0mL pH7.0, filters 1～2 time with glass fiber filter paper, clarifies to filtrate.
The immune affinity column that embodiment 3 is made is connected under the 10.0mL glass syringe.Accurately pipette the 10.0mL sample extracting solution and inject this glass syringe, the air pressure pump is connected with glass syringe, regulate pressure make sample extracting solution with the flow velocity of about 6mL/min slowly by immune affinity column, until 2～3mL air passes through cylinder.With 10.0mL water wash pillar 2 times, discard whole effluxes, and make 2～3mL air pass through cylinder.Add 2.0mL hplc grade methanol wash-out, flow velocity is 1-2mL/min, collects whole eluents in teat glass, for detecting.
2, utilize the aflatoxin M that adds in recovery experiment detection milk sample
1And M
2
(1) adding respectively final concentration in the milk is 0.1 μ g/L, 0.5 μ g/L, the aflatoxin M of three concentration gradients of 1.0 μ g/L
1And M
2Five groups of parallel tests are done in each experiment.
(2) aflatoxin M in extraction and detection milk
1And M
2
Accurately measure 50ml milk, under the rotating speed more than 4000 rev/mins centrifugal 15 minutes.Remove skin on boiled milk, obtain defatted milk, the standby survey.The immune affinity column that embodiment 3 is made is connected under the 50.0mL glass syringe.Pipette the 50.0mL defatted milk and inject this glass syringe, the air pressure pump is connected with glass syringe, regulate pressure make defatted milk with the flow velocity of about 6mL/min slowly by immune affinity column, until 2～3mL air passes through cylinder.With 10.0mL water wash pillar 2 times, discard whole effluxes, and make 2～3mL air pass through cylinder.Add 2.0mL hplc grade methanol wash-out, flow velocity is 1-2mL/min, collects whole eluents in teat glass, for detecting.
3, high-efficient liquid phase chromatogram condition
AFB
1, B
2, G
1, G
2, M
1, M
2:
(a) mobile phase: methanol-water (the methanol/water volume ratio is 45: 55);
(b) detector: fluorescence detector, excitation wavelength are 360nm, and emission wavelength is 440nm;
(c) chromatographic column: C-18 post (column length is 150mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m);
(d) flow velocity: 0.8mL/min;
(e) photochemical derivatization system: photochemical derivatization pond (after being connected in chromatographic column, then leading to fluorescence detector).
Sterigmatocystin:
(a) mobile phase: methanol-water (the methanol/water volume ratio is 75: 25);
(b) detector: UV-detector, wavelength are 325nm;
(c) chromatographic column: C-18 post (column length is 250mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m);
(d) flow velocity: 0.8mL/min.
Zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON:
(a) mobile phase: acetonitrile-water (volume ratio of acetonitrile/water is 60: 40);
(b) detector: fluorescence detector, excitation wavelength are 274nm, and emission wavelength is 440nm;
(c) chromatographic column: C-18 post (column length is 150mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m);
(d) flow velocity: 0.8mL/min
Ochratoxin A:
(a) mobile phase: acetonitrile: water: acetic acid (three's volume ratio is 49.5: 49.5: 1);
(b) detector: fluorescence detector, excitation wavelength are 333nm, and emission wavelength is 477nm;
(c) chromatographic column: C-18 post (column length is 250mm, and internal diameter is 4.6mm, and the filler diameter is 5 μ m);
(d) flow velocity: 0.8mL/min.
4, quantitative
Draw 50 μ L standard operation liquid with injector and inject high performance liquid chromatograph, the response of bioassay standard solution under above-mentioned chromatographic condition (peak height or peak area) obtains AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A standard liquid high-efficient liquid phase chromatogram.Wherein, the spectrogram of aflatoxin is seen Fig. 1 (wherein, Afla represents aflatoxin), and the spectrogram of sterigmatocystin is seen Fig. 2, the spectrogram of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON is seen Fig. 3, and the spectrogram of ochratoxin A is seen Fig. 4.
Compare according to the spectrogram with standard items, to the AFB in peanut sample and milk sample
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A carry out quantitatively, measure through the AFB under immune affinity column absorption and wash-out
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A content, and calculate according to formula II and add the rate of recovery.
Testing result is as shown in table 1, table 2 and table 3.Wherein, be Aflatoxin in Peanut byHigh B shown in table 1
1, B
2, G
1, G
2The interpolation rate of recovery; Shown in table 2 is the interpolation rate of recovery of ochratoxin A in peanut; Shown in table 3 is the interpolation rate of recovery of sterigmatocystin in peanut; Shown in table 4 is the interpolation rate of recovery of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON in peanut; Shown in table 5 is aflatoxin M in milk
1And M
2The interpolation rate of recovery.
Table 1
Table 2
Table 3
Table 4
Table 5
Can be found out the AFB of peanut sample by testing result
1, B
2, G
1, G
2, M
1, M
2, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A the interpolation rate of recovery all between 80-100%, RSD is all less than 5%.Show that method of the present invention satisfies AFB fully
1, B
2, G
1, G
2, M
1, M
2, the analysis requirement that detects of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, sterigmatocystin and ochratoxin A.
All between 80-100%, RSD is all less than 5% for the interpolation rate of recovery of milk sample.Show that method of the present invention satisfies aflatoxin M fully
1And M
2The analysis requirement that detects.
Also can be found out by above-described embodiment, according to said method, the monoclonal antibody that the monoclonal antibody that is obtained by hybridoma cell strain CGMCCNO.5504 secretion and CGMCC NO.5505 secretion obtain also can be used jointly, is used for zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and at least a detection and purifying of ochratoxin A.
In addition, also can carry out any combination between various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. immunosorbent, it is characterized in that, this immunosorbent comprise solid phase carrier and with the antibody of this solid phase carrier coupling, described antibody comprises the monoclonal antibody that is obtained by hybridoma cell strain CGMCC NO.5504 secretion and the monoclonal antibody that is obtained by hybridoma cell strain CGMCC NO.5505 secretion.
2. immunosorbent according to claim 1, wherein, described antibody also comprises the monoclonal antibody that is obtained by hybridoma cell strain CGMCC NO.5506 secretion.
3. be mounted with the immune affinity column of claim 1 or 2 described immunosorbent.
4. contain the described immunosorbent of claim 1 or 2 or contain the kit of immune affinity column claimed in claim 3, preferably, also comprise eluent in described kit and/or preserve liquid, described eluent is preferably at least a in methyl alcohol, acetonitrile and acetone, and described preservation liquid is preferably the NaN that contains the 0.001-0.1 % by weight
3The PBS buffer solution.
5. immunosorbent claimed in claim 1, when quoting claim 1 immune affinity column claimed in claim 3 or when quoting claim 1 kit claimed in claim 4 at purifying and the application at least a in detecting zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
Immunosorbent claimed in claim 2, when quoting claim 2 immune affinity column claimed in claim 3 or when quoting claim 2 kit claimed in claim 4 at purifying and detect AFB
1, B
2, G
1, G
2, M
1, M
2, the application at least a in sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A.
7. purifying AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, the method comprises, makes and contains AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A fluid sample immune affinity column claimed in claim 3 during claim 2 by reference, make at least part of AFB in described fluid sample
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A be adsorbed on this immune affinity column, then with eluent with described at least part of AFB
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A elute.
8. method according to claim 7, wherein, described eluent is at least a in methyl alcohol, acetonitrile and acetone.
9. according to claim 7 or 8 described methods, wherein, the described AFB that contains
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A fluid sample be preferably at least a in the liquid extract of the liquid extract of the liquid extract of the liquid extract of food, feed, biological sample and Chinese medicine.
10. one kind is detected AFB
1, B
2, G
1, G
2, M
1, M
2, sterigmatocystin, zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A method, the method comprises,
(1) the described method of any one according to claim 7-9, obtain liquid to be measured, and described liquid to be measured is with described at least part of AFB with eluent
1, B
2, G
1, G
2, M
1, M
2, at least part of zearalenone congener α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON, at least part of sterigmatocystin and at least part of ochratoxin A elute and the solution that obtains;
(2) make described liquid to be measured enter detection system, and by relatively carrying out qualitative or quantitative detection with standard items.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103480340A (en) * | 2013-09-11 | 2014-01-01 | 广西壮族自治区粮油科学研究所 | Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof |
| CN103861569A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column |
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| CN103480340A (en) * | 2013-09-11 | 2014-01-01 | 广西壮族自治区粮油科学研究所 | Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof |
| CN104569382B (en) * | 2013-10-26 | 2016-09-21 | 北京勤邦生物技术有限公司 | A kind of immune affinity column detecting vomitoxin and application thereof |
| CN104569382A (en) * | 2013-10-26 | 2015-04-29 | 北京勤邦生物技术有限公司 | Immunoaffinity column for detecting vomitoxin and application of immunoaffinity column |
| CN103861569A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column |
| CN103861569B (en) * | 2014-03-28 | 2015-04-22 | 中国农业科学院油料作物研究所 | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column |
| CN104707362A (en) * | 2015-01-05 | 2015-06-17 | 重庆出入境检验检疫局检验检疫技术中心 | Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone |
| CN104707583A (en) * | 2015-01-05 | 2015-06-17 | 重庆出入境检验检疫局检验检疫技术中心 | Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof |
| CN107167591A (en) * | 2015-12-02 | 2017-09-15 | 南昌大学 | A kind of aflatoxin single domain heavy chain antibody affine in immunity sorbing material |
| CN108614054A (en) * | 2016-12-12 | 2018-10-02 | 北京农学院 | Preparation method based on the mycotoxin immune affinity column that polystyrene-divinylbenzene resin is carrier |
| CN107462641A (en) * | 2017-07-17 | 2017-12-12 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method that is a kind of while detecting ochratoxin A and zearalenone |
| CN109387592A (en) * | 2017-08-08 | 2019-02-26 | 中国科学院大连化学物理研究所 | A kind of fluorescence detector of integrated aflatoxin light derivatization device |
| CN109387592B (en) * | 2017-08-08 | 2021-06-04 | 中国科学院大连化学物理研究所 | Fluorescence detector integrated with aflatoxin light derivatization device |
| CN110618274A (en) * | 2019-09-04 | 2019-12-27 | 山东绿都生物科技有限公司 | Chimeric ELISA kit for simultaneously and quantitatively detecting total amount of aflatoxin and zearalenone in edible oil |
| CN110618274B (en) * | 2019-09-04 | 2022-09-20 | 山东绿都生物科技有限公司 | Chimeric ELISA kit for simultaneously and quantitatively detecting total amount of aflatoxin and zearalenone in edible oil |
| CN116165047A (en) * | 2023-04-22 | 2023-05-26 | 北京中检维康生物技术有限公司 | Immune magnetic bead kit for purifying aflatoxin, use method and application thereof |
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Effective date of registration: 20151230 Address after: Qingdao City, Shandong province 266002 qutangxia Road No. 70 Patentee after: Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine Patentee after: Inspection and Quarantine Technology Center, Beijing Entry-Exit Inspection and Q Patentee after: Beijing Zhong Jianwei health Bioisystech Co., Ltd Address before: Qingdao City, Shandong province 266002 qutangxia Road No. 70 Patentee before: Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine Patentee before: Inspection and Quarantine Technology Center, Beijing Entry-Exit Inspection and Q Patentee before: Clover Technology Group, Inc. |