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CN103808938A - Method for detecting zearalenone - Google Patents

Method for detecting zearalenone Download PDF

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CN103808938A
CN103808938A CN201410098274.9A CN201410098274A CN103808938A CN 103808938 A CN103808938 A CN 103808938A CN 201410098274 A CN201410098274 A CN 201410098274A CN 103808938 A CN103808938 A CN 103808938A
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zen
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刘静波
陈晓飞
张燕
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Jilin University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones

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Abstract

本发明公开了一种检测玉米赤酶烯酮的方法，是采用Mannich法制备玉米赤酶烯酮的免疫抗原和检测抗原，免疫Balb/c小鼠，并获得相应的多抗血清，血清经过分离纯化后，间接ELISA检测其效价；然后将检测抗原包被于酶标板上，过夜、封闭，再加入标准毒素溶液、样品提取液，以及稀释后的多抗；反应后，加入羊抗鼠IgG-HRP，再加入底物溶液显色，终止后，在450nm处检测OD值，绘制相关标准曲线，进而求出待测样品中ZEN的含量，有益效果：更有效的利用参加反应的毒素分子，从而节省成本；整个过程简便、易于操作、毒素分子的利用率也相对较高；该检测方法操作简便，无需专业人员培训，便于实地生产应用。

The invention discloses a method for detecting zearalenone. The immune antigen and detection antigen of zearalenone are prepared by Mannich method, Balb/c mice are immunized, and corresponding polyantiserum is obtained, and the serum is separated After purification, indirect ELISA was used to detect its titer; then the detection antigen was coated on the microtiter plate, overnight, blocked, and then standard toxin solution, sample extract, and diluted polyclonal antibody were added; after the reaction, goat anti-mouse IgG-HRP, then add the substrate solution to develop the color, after the termination, detect the OD value at 450nm, draw the relevant standard curve, and then calculate the content of ZEN in the sample to be tested. Beneficial effect: more effective use of toxin molecules participating in the reaction , thereby saving costs; the whole process is simple, easy to operate, and the utilization rate of toxin molecules is relatively high; the detection method is easy to operate, does not require professional training, and is convenient for field production and application.

Description

A kind of method that detects the red enzyme ketenes of corn
Technical field
The invention belongs to a kind of method that detects mycotoxin, particularly a kind of method that detects the red enzyme ketenes of corn.
Background technology
Zearalenone (Zearalenone, ZEN) be a kind of steroids mycotoxin being produced by Fusarlum roseum and other sickle-like bacteria, be common in the cereal such as corn, wheat, rice, barley, millet and oat, it can cause the pathology of various cereal, there is similar estrogenic toxic action, can stimulate containing the breast cancer cell of estrogen receptor and increase, cause the reproductive problems that zoohormone level is too high and serious, also there is potential carcinogenicity.At present mainly contain thin-layered chromatography, high performance liquid chromatography, euzymelinked immunosorbent assay (ELISA) for detection of the method for the red enzyme ketenes of corn.Thin-layered chromatography is easy to detect, but detects limit for height, can not be well quantitative; High performance liquid chromatography detects accurately, detects low, but needs complicated sample pretreatment process, special instrument and the staff training of specialty; Aforesaid these methods all exist some shortcomings, can not measure effectively rapidly zearalenone contained in corresponding cereal.
Summary of the invention
The object of the invention is some problems that exist in the method for the red enzyme ketenes of existing detection corn and a kind of method that detects the red enzyme ketenes of corn providing.
The method of the red enzyme ketenes of detection corn of the present invention is to adopt immunizing antigen and the detectable antigens of Mannich legal system for the red enzyme ketenes of corn, immunity Balb/c mouse, and obtain corresponding polyvalent antibody, and serum is after separation and purification, and indirect ELISA detects it and tires; Then detectable antigens is coated in ELISA Plate, spends the night, seal, then add normaltoxin solution, sample extracting solution, and resisting after dilution more; After reaction, add sheep anti-mouse igg-HRP, then add substrate solution colour developing, after termination, detect OD value at 450nm place, draw relevant criterion curve, and then obtain the content of ZEN in testing sample, concrete steps are as described below:
The first step, prepare cationization albumen, mainly prepare cationization bovine serum albumin cBSA and cationization chicken egg white cOVA;
Second step, employing Mannich legal system are for immunizing antigen ZEN-cBSA and detectable antigens ZEN-cOVA;
The 3rd step, get healthy 6-8 week 4 of female Balb/c mouse, complete Freund's adjuvant emulsification pneumoretroperitoneum injection for initial immunity, every mouse immunizing dose is 100 μ g ZEN-cBSA, volume injected is 0.2mL; Afterwards every two weeks lumbar injections of booster immunization once, the emulsification full Freund's adjuvant that toos many or too much for use; Last immunity is used physiologic saline for substitute incomplete Freund's adjuvant, adopts tail vein injection, injected dose and identical several times above; Immunity 6 times altogether; Since the 3rd immunity, blood is got in each immunity docking in latter 10 days, and indirect ELISA detects mice serum and tires;
The 4th step, mouse be the 10th day after immunity the last time, plucks eyeball and get blood, puts to death mouse; The blood that obtains is first placed in 2h under room temperature, the then centrifugal 20min of 3500rpm under 4 ℃ of conditions, and centrifugal serum is afterwards frozen in the refrigerator of-20 ℃;
Serum after the 5th step, purifying are centrifugal, indirect ELISA is determined serum titer;
The 6th step, detectable antigens are coated in ELISA Plate, under 4 ℃ of conditions, spend the night;
The 7th step, use acetonitrile-water extract the toxin in sample to be checked, and the volume ratio of acetonitrile and water is 85:15;
The 8th step, detersive enzyme target, add confining liquid, reaction 2h;
The 9th step, washing, frozen ELISA Plate; Or add normaltoxin solution, sample extracting solution, add the serum supernatant after dilution;
The tenth step, washing, add two anti-sheep anti-mouse iggs-HRP reaction;
The 11 step, washing, add substrate solution colour developing;
The 12 step, termination, microplate reader detection reaction hole, 450nm place OD value.
In the immunizing antigen preparing in second step, ZEN is 10:1-20:1 with the scope of the molecule number ratio of cBSA.
In the 3rd step, initial immunity and the successful standard of booster immunization emulsification are that emulsion is a shape in water, can not spread rapidly, and the antigen dose of immunity is 100 μ g/.
The maximum dilution multiple in the positive hole of criterion of serum titer in the 5th step, and the criterion in positive hole is (A sample to be checked-A blank)/(A negative-A blank) >=2.1, wherein A treats the OD value of verify under 450nm condition.
In the 6th step, detectable antigens ZEN-cOVA uses the Na of 100mmol/L pH=9.6 2cO 3-NaHCO 3damping fluid is diluted to 2 μ g/mL.
The ELISA Plate of sealing in the 9th step can be directly used in and detect corn red enzyme ketenes, serum supernatant according to tiring/add after 2 multiple dilution, also can be frozen at-20 ℃, after being convenient to, detect at any time.
Beneficial effect of the present invention:
1, the albumen of toxin immunity and detection coupling is selected the albumen of cationization, be cationization bovine serum albumin (cBSA) and cationization chicken egg white (cOVA), cationization albumen compared with normal albumen can be in conjunction with more lps molecule, the lps molecule of reaction is participated in simultaneously more effective utilization, thereby saves cost;
2, coupling method is selected Mannich method, and unconventional active ester method and acid anhydrides method.Traditional method often needs first contratoxin molecule to carry out oximate, and oximate is often followed comparatively complicated organic reaction.Oximate process not only needs to introduce various poisonous and harmful reagent, process is loaded down with trivial details, and toxin likely loses in this process, affects the efficiency of later stage coupling.Mannich rule has been saved comparatively loaded down with trivial details oximate process, and whole process is easy, the utilization factor of easy operating, lps molecule is also relatively high;
3, the more traditional high-efficient liquid phase technique of this method, is easier to operation and high specificity; Thinner layer chromatography, detectability is low, is easy to quantitatively; After detectable antigens is coated in ELISA Plate, can be frozen, be convenient to the detection of later stage sample; And this detection method is easy and simple to handle, without professional's training, be convenient to production application on the spot.
Accompanying drawing explanation
Fig. 1 is polyvalent antibody indirect competitive ELISA curve map, and wherein horizontal ordinate is the logarithm of ZEN concentration, and ZEN concentration unit is ng/mL; Ordinate is the ratio that detects hole OD value B and blank well OD value B0.
Embodiment
The method of the red enzyme ketenes of detection corn of the present invention is to adopt immunizing antigen and the detectable antigens of Mannich legal system for the red enzyme ketenes of corn, immunity Balb/c mouse, and obtain corresponding polyvalent antibody, and serum is after separation and purification, and indirect ELISA detects it and tires; Then detectable antigens is coated in ELISA Plate, spends the night, seal, then add normaltoxin solution, sample extracting solution, and resisting after dilution more; After reaction, add sheep anti-mouse igg-HRP, then add substrate solution colour developing, after termination, detect OD value at 450nm place, draw relevant criterion curve, and then obtain the content of ZEN in testing sample, concrete steps are as described below:
The first step, prepare cationization albumen, mainly prepare cationization bovine serum albumin cBSA and cationization chicken egg white cOVA;
Second step, employing Mannich legal system are for immunizing antigen ZEN-cBSA and detectable antigens ZEN-cOVA;
The 3rd step, get healthy 6-8 week 4 of female Balb/c mouse, complete Freund's adjuvant emulsification pneumoretroperitoneum injection for initial immunity, every mouse immunizing dose is 100 μ g ZEN-cBSA, volume injected is 0.2mL; Afterwards every two weeks lumbar injections of booster immunization once, the emulsification full Freund's adjuvant that toos many or too much for use; Last immunity is used physiologic saline for substitute incomplete Freund's adjuvant, adopts tail vein injection, injected dose and identical several times above; Immunity 6 times altogether; Since the 3rd immunity, blood is got in each immunity docking in latter 10 days, and indirect ELISA detects mice serum and tires;
The 4th step, mouse be the 10th day after immunity the last time, plucks eyeball and get blood, puts to death mouse; The blood that obtains is first placed in 2h under room temperature, the then centrifugal 20min of 3500rpm under 4 ℃ of conditions, and centrifugal serum is afterwards frozen in the refrigerator of-20 ℃;
Serum after the 5th step, purifying are centrifugal, indirect ELISA is determined serum titer;
The 6th step, detectable antigens are coated in ELISA Plate, under 4 ℃ of conditions, spend the night;
The 7th step, use acetonitrile-water extract the toxin in sample to be checked, and the volume ratio of acetonitrile and water is 85:15;
The 8th step, detersive enzyme target, add confining liquid, reaction 2h;
The 9th step, washing, frozen ELISA Plate; Or add normaltoxin solution, sample extracting solution, add the serum supernatant after dilution;
The tenth step, washing, add two anti-sheep anti-mouse iggs-HRP reaction;
The 11 step, washing, add substrate solution colour developing;
The 12 step, termination, microplate reader detection reaction hole, 450nm place OD value.
In the immunizing antigen preparing in second step, ZEN is 10:1-20:1 with the scope of the molecule number ratio of cBSA.
In the 3rd step, initial immunity and the successful standard of booster immunization emulsification are that emulsion is a shape in water, can not spread rapidly, and the antigen dose of immunity is 100 μ g/.
The maximum dilution multiple in the positive hole of criterion of serum titer in the 5th step, and the criterion in positive hole is (A sample to be checked-A blank)/(A negative-A blank) >=2.1, wherein A treats the OD value of verify under 450nm condition.
In the 6th step, detectable antigens ZEN-cOVA uses the Na of 100mmol/L pH=9.6 2cO 3-NaHCO 3damping fluid is diluted to 2 μ g/mL.
The ELISA Plate of sealing in the 9th step can be directly used in and detect corn red enzyme ketenes, serum supernatant according to tiring/add after 2 multiple dilution, also can be frozen at-20 ℃, after being convenient to, detect at any time.
Specific implementation process is as described below:
One, prepare polyvalent antibody:
1, prepare cationization albumen:
Ethyl sulfonic acid (MES) solution (pH=4.8) of getting 670 μ L100mmol/L is placed in ice bath, adds the ethylenediamine (EDA) of same volume, and then regulating the pH value of mixed liquor with the dilute hydrochloric acid solution of 0.4mol/L is 4.8, is labeled as A liquid; Get 500 μ L MES damping fluids, add wherein 50mg bovine serum albumin (BSA) and 30mg1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), be labeled as B liquid; B liquid is joined to A liquid, room temperature lower magnetic force stirring reaction 2h; Add the acetum cessation reaction of 400 μ L2mol/L.The 72h that dialyses in distilled water, after cationization bovine serum albumin (cBSA) freeze drying of the desalination obtaining, is stored in-20 ℃, and same method is prepared cationization chicken egg white (cOVA);
2, prepare immunizing antigen and detectable antigens:
The preparation of immunizing antigen: the cBSA that weighs 600 μ g is dissolved in 500 μ L MES solution as conjugate, weighs 100 μ g ZEN and is dissolved in 50 μ L dimethyl formamides (DMF); ZEN solution is slowly joined in cBSA solution, be uniformly mixed gently liquid, then, to the formaldehyde that adds 50 μ L in mixed liquor, move into immediately in 37 ℃ of incubators stirring reaction 24h gently.Purify ZEN-cBSA bond with centrifugal filtration purification devices, repeat 2 times, after freeze-drying, be stored in the refrigerator of-20 ℃;
Same method selects cOVA to prepare detectable antigens as conjugate;
3, immune mouse: get 4 of all female Balb/c mouse of healthy 6-8, complete Freund's adjuvant emulsification pneumoretroperitoneum injection for initial immunity, every mouse immunizing dose is 100 μ g ZEN-cBSA, volume injected is 0.2mL; Afterwards every two weeks lumbar injections of booster immunization once, the emulsification full Freund's adjuvant that toos many or too much for use; Last immunity is used physiologic saline for substitute incomplete Freund's adjuvant, adopts tail vein injection, injected dose and identical several times above; Immunity six times altogether; From immunity for the third time, blood is got in each immunity docking in rear ten days, and indirect ELISA detects mice serum and tires;
4, after last immunity, the 10th day, pluck eyeball and get blood, put to death mouse; Under blood room temperature after static 2h, the centrifugal 20min of 3500rpm, serum is frozen under the environment of-20 ℃;
5, adopt caprylic acid method purified blood serum: acetic acid-sodium acetate buffer of the antiserum 60mmol/L of 4 times of volumes dilutes, be adjusted to pH4.5 with 100mmol/L NaOH; Slowly drip while stirring sad (25mL/L serum dilution), after adding, stir 30min; The centrifugal 30min of 10000rpm, collects supernatant; After filtered through gauze, add 10 × PBS by 1/10 volume, 5mol/L NaOH is adjusted to pH7.4; 4 ℃ of precoolings of supernatant, calculated population is long-pending, adds ammonium sulfate powder by 277g/L, and limit edged stirs, and adds rear continuation and stirs 30min; The centrifugal 15min of 5000rpm, supernatant discarded, collecting precipitation; Precipitation is dissolved with a small amount of dislysate, dialyses and changes dislysate twice; Collect supernatant, frozen under the condition of-20 ℃;
6, indirect ELISA detection supernatant is tired: the criterion in positive hole is (A sample to be checked-A blank)/(A negative-A blank) >=2.1, the maximum dilution multiple in positive hole is antibody titer, and wherein A treats the OD value of verify under 450nm condition.
Two, standard sample preparation:
1, cleansing solution: add 0.05% Tween-20 in the phosphate buffer that is 7.4 in 100mmol/L pH value;
2, confining liquid: the phosphate buffer that is 7.4 in 100mmol/L pH value adds 1% bovine serum albumin(BSA);
3, sample diluting liquid: add 10% methyl alcohol in the phosphate buffer that is 7.4 in 100mmol/L pH value;
4, detectable antigens dilution: add 0.05% bovine serum albumin(BSA) BSA in the phosphate buffer that is 7.4 in pH value;
5, stop buffer: 1mol/L hydrochloric acid solution;
6, extract: acetonitrile: water=85:15 (volume ratio);
7, substrate solution: the TMB Single Solution that selects Invitrogen company;
8, prepare the coated reaction plate of the red enzyme ketenes of corn antibody: with the carbonate buffer solution of 100mmol/L pH9.6, ZEN-cOVA being diluted is 2 μ g/mL, be added in 96 hole ELISA Plate, every hole 100 μ L, 4 ℃ of refrigerators are placed and are spent the night, discard coating buffer, full hole washing 3 times, every hole adds 200 μ L confining liquid sealings, be placed in 37 ℃ of 2h, abandon its confining liquid and will be coated with rearmounted-20 ℃ of freezing preservations of plate packing;
9, prepare the red enzyme ketenes of corn standard solution: be solvent preparation 5-400ng/ml(5,10,20,30,50,100,200,400 by sample diluting liquid) red enzyme ketenes standard serial solution.
Three, detect sample:
1, testing sample pre-treatment: take the 100ml conical flask that 10g pulverizes and the sample of 20 mesh sieves is placed in ground excessively, add acetonitrile-water (volume ratio 85:15) 40ml, the vibration 30min that jumps a queue, filters to be and treats sample measuring liquid;
2, reacting hole mark: 1-6 hole is the red enzyme ketenes of corn (ZEA) standard control hole, and No. 7 holes are blank hole, the another quantity of setting as required testing sample hole; All reacting holes are set Duplicate Samples;
3, add successively the standard solution configuring and treat sample measuring liquid according to mark, in 1-6 test tube, adding the red enzyme ketenes of corn standard solution 50 μ L, in No. 7 test tubes, adding 50 μ L sample diluting liquids, in all the other holes, adding respectively and treat sample measuring liquid 50 μ L;
4, in each hole, add respectively 50 μ L according to the how anti-supernatant of (tire/2) doubling dilution, vibration, mixes the reactant in each hole lightly;
5, ELISA Plate is placed in to 37 ℃ of constant temperature ovens and hatches 1h;
6, take out ELISA Plate, get rid of reactant liquor, pat dry, the washing of washing hydroful hole, after vibration 3min, gets rid of only, pats dry, and repeats to wash plate 3 times;
7, the every hole of ELISA Plate reacting hole adds the sheep anti-mouse igg-HRP of 1:5000 doubling dilution, is placed in 37 ℃ of constant temperature ovens and hatches 1h;
8, take out ELISA Plate, get rid of reactant liquor, pat dry, the washing of washing hydroful hole, after vibration 3min, gets rid of only, pats dry, and repeats to wash plate 5 times;
9, the every hole of ELISA Plate adds TMB solution 100 μ L, is placed in dark place room temperature reaction 20min;
10, take out ELISA Plate, every hole adds stop buffer 100 μ L, measures the OD value in each hole at 450nm place;
11, utilize GraphPad Prism5 software take the ratio (B/B0) of the OD value B in standard solution hole and the OD value B0 of blank well as functional value, take the concentration logarithm of standard solution as independent variable, make curve, ask regression equation, see accompanying drawing 1; Can obtain the concentration of the corresponding toxin of sample well according to equation;
12, the docs-effect (inhibition) carrying according to GraphPad Prism5 software carries out nonlinear regression and fitting to data, can obtain formula (1), wherein Y is the ratio of testing sample OD value and blank well OD value, and X is the concentration of ZEN in institute's test sample product, and unit is ng/mL; Can obtain formula (2) according to testing sample pretreatment process, wherein X is the concentration of ZEN in institute's test sample product, and unit is ng/mL, x is the content of ZEN in institute's test sample product, and unit is ng/g, the cumulative volume that V is extract, unit is mL, the quality that m is testing sample, and unit is g;
Y = 0.1856 + 0.8394 1 + 10 X - 2.094 - - - ( 1 )
x=20VX/m （2）
In conjunction with (1), (2) formula, can obtain:
x＝10 3.90-3X*20V/m
That is:
y＝10 3.90-3x*20V/m （3）
Wherein, x is the ratio of testing sample OD value and blank well OD value, the volume that V is extract, and unit is mL, the quality that m is testing sample, unit is g, and y is the content of ZEN in testing sample, and unit is ng/g.

Claims (6)

1.一种检测玉米赤酶烯酮的方法，具体步骤如下所述：1. A method for detecting zearalenone, the specific steps are as follows: 第一步、制备阳离子化蛋白，主要制备阳离子化牛血清蛋白cBSA和阳离子化鸡卵清蛋白cOVA；The first step is to prepare cationized protein, mainly to prepare cationized bovine serum albumin cBSA and cationized chicken ovalbumin cOVA; 第二步、采用Mannich法制备免疫抗原ZEN-cBSA和检测抗原ZEN-cOVA；The second step is to prepare the immune antigen ZEN-cBSA and the detection antigen ZEN-cOVA by Mannich method; 第三步、取健康的6-8周雌性Balb/c小鼠4只，初次免疫用完全弗氏佐剂乳化后腹腔注射，每只鼠免疫剂量为100μg ZEN-cBSA，注射体积为0.2mL；之后加强免疫每两周腹腔注射一次，乳化用不完全弗氏佐剂；最后一次免疫使用生理盐水代替不完全弗氏佐剂，采用尾静脉注射，注射剂量和前面几次相同；一共免疫6次；从第3次免疫开始，每次免疫后10天断尾取血，间接ELISA检测小鼠血清效价；Step 3: Take 4 healthy 6-8 week old female Balb/c mice and inject them intraperitoneally after initial immunization with complete Freund's adjuvant emulsification. The immune dose for each mouse is 100 μg ZEN-cBSA, and the injection volume is 0.2 mL; Afterwards, booster immunization was injected intraperitoneally once every two weeks, emulsified with incomplete Freund's adjuvant; the last immunization was performed with saline instead of incomplete Freund's adjuvant, and the tail vein was injected with the same dosage as the previous several times; a total of 6 immunizations ;From the third immunization, blood was collected by docking the tail 10 days after each immunization, and the serum titer of mice was detected by indirect ELISA; 第四步、小鼠在最后一次免疫后的第10天，摘眼球取血，处死小鼠；所获血液先置于室温下2h，然后在4℃条件下3500rpm离心20min，离心之后的血清冻存于-20℃的冰箱中；Step 4: On the 10th day after the last immunization, the mice were plucked from the eyeballs to take blood, and the mice were sacrificed; the obtained blood was first placed at room temperature for 2 hours, and then centrifuged at 3500rpm for 20 minutes at 4°C, and the serum after centrifugation was frozen. Store in a refrigerator at -20°C; 第五步、纯化离心后的血清，间接ELISA确定血清效价；The fifth step, purify the centrifuged serum, and determine the serum titer by indirect ELISA; 第六步、检测抗原包被于酶标板上，在4℃条件下过夜；The sixth step, the detection antigen is coated on the microtiter plate, overnight at 4°C; 第七步、使用乙腈-水提取待检样品中的毒素，乙腈与水的体积比为85:15；The seventh step, using acetonitrile-water to extract the toxin in the sample to be tested, the volume ratio of acetonitrile and water is 85:15; 第八步、洗涤酶标板，加入封闭液，反应2h；The eighth step, wash the microtiter plate, add blocking solution, and react for 2 hours; 第九步、洗涤，冻存酶标板；或者加入标准毒素溶液、样品提取液，添加稀释后的血清上清；The ninth step, wash and freeze the microplate; or add standard toxin solution, sample extract, and add diluted serum supernatant; 第十步、洗涤，加入二抗羊抗鼠IgG-HRP反应；The tenth step, washing, adding secondary antibody goat anti-mouse IgG-HRP reaction; 第十一步、洗涤，加入底物液显色；The eleventh step, washing, adding substrate solution for color development; 第十二步、终止，450nm处酶标仪检测反应孔OD值。The twelfth step, stop, detect the OD value of the reaction well with a microplate reader at 450 nm. 2.根据权利要求1所述的一种检测玉米赤酶烯酮的方法，其特征在于：所述的第二步中制备得到的免疫抗原中ZEN与cBSA的分子个数比的范围为10:1-20:1。2. a kind of method for detecting zearalenone according to claim 1, is characterized in that: the scope of the molecular number ratio of ZEN and cBSA in the immune antigen prepared in the described second step is 10: 1-20:1. 3.根据权利要求1所述的一种检测玉米赤酶烯酮的方法，其特征在于：所述的第三步中初次免疫和加强免疫乳化成功的标准是乳液在水中呈滴状，免疫的抗原剂量为100μg/只。3. a kind of method for detecting zearalenone according to claim 1, is characterized in that: in the described 3rd step, initial immunization and strengthening immune emulsification success standard is that emulsion is in drop shape in water, and immune The dose of antigen was 100 μg/monkey. 4.根据权利要求1所述的一种检测玉米赤酶烯酮的方法，其特征在于：所述的第五步中血清效价的判断标准为阳性孔的最大稀释倍数，而阳性孔的判断标准为（A待检样品-A空白）/（A阴性-A空白）>=2.1，其中A为待检孔在450nm条件下的OD值。4. a kind of method for detecting zearalenone according to claim 1, is characterized in that: the judging standard of serum titer in the described 5th step is the maximum dilution factor of positive hole, and the judgment of positive hole The standard is (A sample to be tested -A blank )/(A negative -A blank )>=2.1, where A is the OD value of the well to be tested under the condition of 450nm. 5.根据权利要求1所述的一种检测玉米赤酶烯酮的方法，其特征在于：所述的第六步中检测抗原ZEN-cOVA使用100mmol/L pH=9.6的Na2CO3-NaHCO3缓冲液稀释到2μg/mL。5. A method for detecting zearalenone according to claim 1, characterized in that: the detection of antigen ZEN-cOVA in the sixth step uses 100mmol/L Na 2 CO 3 -NaHCO with pH=9.6 3 buffer diluted to 2 μg/mL. 6.根据权利要求1所述的一种检测玉米赤酶烯酮的方法，其特征在于：所述第九步中的血清上清按照效价/2的倍数稀释后加入。6. A method for detecting zearalenone according to claim 1, characterized in that: the serum supernatant in the ninth step is added after being diluted according to the multiple of titer/2.
CN201410098274.9A 2014-03-16 2014-03-16 Method for detecting zearalenone Pending CN103808938A (en)

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