CN107915774A - For detecting the monoclonal antibody and enzyme-linked immunoassay method and kit of zearalenone and its metabolite - Google Patents

For detecting the monoclonal antibody and enzyme-linked immunoassay method and kit of zearalenone and its metabolite Download PDF

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CN107915774A
CN107915774A CN201710615989.0A CN201710615989A CN107915774A CN 107915774 A CN107915774 A CN 107915774A CN 201710615989 A CN201710615989 A CN 201710615989A CN 107915774 A CN107915774 A CN 107915774A
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zearalenone
monoclonal antibody
metabolite
zen
kit
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CN107915774B (en
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彭大鹏
袁宗辉
董国良
王玉莲
潘源虎
陈冬梅
陶燕飞
刘振利
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Huazhong Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The invention discloses a kind of monoclonal antibody specific that can identify zearalenone and its metabolite, it is as secreted by hybridoma cell strain ZEN/6C2, the hybridoma cell strain ZEN/6C2, is deposited in China typical culture collection center, and preserving number is CCTCC NO:C201781.The invention also discloses the enzyme-linked immunoassay method and its kit of zearalenone and its metabolite in detection corn, pannage, edible animal tissue and milk.Monoclonal antibody sensitivity prepared by the present invention is higher, wide spectrum, and the accuracy of detection is high, and precision is good, have it is easy, quick, easy to operate etc. a little.

Description

For detecting the monoclonal antibody and enzyme-linked immunoassay method of zearalenone and its metabolite With kit
Technical field
The invention belongs to wild animal resources and immunological technique field, and in particular to one kind can identify zearalenone And its monoclonal antibody and enzyme-linked immunoassay method (ELISA) and kit of metabolite.
Background technology
Zearalenone (ZEN) is the cometabolism production of the fusarium fungus such as fusarium culmorum and Fusarium graminearum Thing, mainly pollutes the cereal such as corn, wheat, rice, barley and oat.Zearalenone has estrogen action, main to make For jenny reproductive system, cause reproductive capability abnormal or even dead, there is genotoxicity and carcinogenicity, immunotoxicity and Genotoxicity.European Union provides the maximum residue limit point of ZEN in undressed cereal (not including corn), bread and baby food Wei not 100,50.0,20.0 μ g kg-1.It is Chinese then regulation wheat and corn in ZEN maximum residue limit be 60.0 μ g kg-1.It is dynamic Thing intake zearalenone after, can be metabolized as α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearelone, and zearalenol is also existed in cereal.Since these metabolins have There is stronger estrogen active, therefore α-zearalanol was once widely used as cattle and sheep growth accelerator, but because it can influence human body life Systematic growth is grown, European Union forbids using it for animal-derived food, and China also forbids using it for food animal growth promoting function.
Method for detecting residue be accurate judgement residual whether exceeded important means, it is desirable to there is higher accuracy and standard Exactness.Method currently used for the detection of zearalenones mycotoxin mainly has instrumental method and immunology detection side Method.Instrumental method has the advantages that detection sensitivity is high, selectivity is good, separative efficiency is high and has a wide range of application, and disclosure satisfy that The testing requirements of most of mycotoxins., will to the technology of operating personnel but such method usually requires the instrument and equipment of costliness Ask higher, it is necessary to carry out the analysis of complexity, be more suitable for the confirmation detection to medicine and be not suitable for a large amount of screenings of sample.Exempt from Epidemiology detection method, especially ELISA method, have sensitive, special, simple, quick, stable and the spy such as are easily operated automatically Point, the defects of can overcoming Instrumental Analysis, be a kind of effective high-volume residual screening technique, there is huge development prospect. Although the zearalenones mycotoxin ELISA method of monoclonal antibody is currently based on it has been reported that but antibody sensitivity is low, wide spectrum Property it is poor, detection sample type it is single so that the application of such method is necessarily limited to.
The content of the invention
The purpose of the present invention is：
(1) a kind of monoclonal antibody of specific recognition zearalenone and its metabolite is provided；
(2) monoclonal antibody is provided and is preparing the ELISA reagent of detection zearalenone and its metabolite Application in box；
(3) monoclonal antibody is utilized, establishes a kind of ELISA side that can detect zearalenone and its metabolite Method；
(4) application of the enzyme-linked immunologic detecting kit in zearalenone and its metabolite detection is provided.
Above-mentioned purpose is achieved through the following technical solutions：
A kind of monoclonal antibody, can identify zearalenone and its metabolite, and it by preserving number is CCTCC NO that it, which is,: Secreted by the hybridoma cell strain ZEN/6C2 of C201781.
The metabolite is α-zearalenol, β-zearalenol, α-zearalanol, β-Gibberella zeae Alcohol, zearelone.
The hybridoma cell strain ZEN/6C2, is deposited in China typical culture collection center, preserving number CCTCC NO:C201781.It is with zearalenone (ZEN) and NH3Reaction generation haptens (7-NH2- ZEN), then by 7-NH2- ZEN is coupled to obtain conjugate (7-NH with bovine serum albumin(BSA) (BSA) by glutaraldehyde (GA) method2- ZEN-GA-BSA), should Conjugate is as being prepared after immunogen immune animal.The structural formula of the haptens is as follows：
The monoclonal antibody is in the enzyme linked immunological kit of detection zearalenone and its metabolite is prepared Application.
The kit of the monoclonal antibody is included, the kit is detection zearalenone and its metabolism production The enzyme linked immunological kit of thing.
Application of the kit in the detection of the non-diagnostic purpose of zearalenone and its metabolite.
A kind of enzyme-linked immunoassay method of the non-diagnostic purpose detection of zearalenone and its metabolite, including following step Suddenly：
(1) the following haptens of structural formula and chicken ovalbumin (OVA) coupling are obtained into coating antigen；
(2) it is CCTCC NO with preserving number:The hybridoma cell strain ZEN/6C2 of C201781 prepares monoclonal antibody；
(3) the coating primordial covering solid phase carrier of step (1) is used；
(4) processing and detection of corn, pannage, edible animal tissue such as pork, liver, kidney, milk sample.
The beneficial effects of the invention are as follows：
1. the present invention is when preparing monoclonal antibody, using zearalenone oxime as haptens, by haptens and carrier egg White coupling is used as immunogene, and the monoclonal antibody prepared by the immunogene can be specific while identifies zearalenone, α-jade Rice red mould enol, β-zearalenol, α-zearalanol, β-zearalanol and zearelone；
2. the enzyme linked immunological kit and method of the present invention are suitable for detection corn, pannage, edible animal tissue, ox The residual of zearalenone and its metabolite in milk, sensitivity, the accuracy of detection are high, and precision is good, Gibberella zeae alkene Ketone, α-zearalenol, β-zearalenol, α-zearalanol, the IC of β-zearalanol and zearelone50Value Respectively 114.0,127.4,290.4,114.9,205.6 and 257.1ng L-1, it is ELISA methods wide spectrum of the invention, sensitive Degree, accuracy are high, and precision is good；
3. detection method according to the present invention is simple, easy to operate, testing cost is low, operator is required low, and body is good for Health harm is relatively small.
Brief description of the drawings
Fig. 1 is the indirect competitive ELISA standard curve of zearalenone (ZEN).
Fig. 2 is the indirect competitive ELISA standard curve of α-zearalenol (α-ZEL).
Fig. 3 is the indirect competitive ELISA standard curve of β-zearalenol (β-ZEL).
Fig. 4 is the indirect competitive ELISA standard curve of α-zearalanol (α-ZAL).
Fig. 5 is the indirect competitive ELISA standard curve of β-zearalanol (β-ZAL).
Fig. 6 is the indirect competitive ELISA standard curve of zearelone (ZAN).
Embodiment
Below by embodiment, the invention will be further described, but does not limit the present invention.
The preparation of 1 immunogene of embodiment and coating antigen
1.1 immunogene 7-NH2The preparation of-ZEN-GA-BSA
Accurately weighing 2mg ZEN is dissolved in 1mL methanol, and ammonia is passed through under condition of ice bath, and reaction 1h is stirred at room temperature, adds 3mg Sodium cyanoborohydride, continues stirring reaction 3h.Nitrogen is dried up up to haptens 7-NH at room temperature2-ZEN。
Haptens is dissolved in 100 μ L DMF, 16.0mg BSA is weighed and is dissolved in 4mL PBS, both are slowly mixed to obtain A liquid. 40 μ L, 25% glutaraldehyde solutions are taken, 400 μ L are diluted to PBS, this is B liquid.B liquid is added dropwise to A liquid under the conditions of lucifuge, Reaction 9h is stirred at room temperature.Reaction solution is used to the PBS dialysis 5d, 5000 r min of pH=7.4 under the conditions of 4 DEG C-1Centrifuge 10min, Retain supernatant, up to 7-NH2- ZEN-GA-BSA, saves backup in -20 DEG C.Reaction is as follows：
The preparation of 1.2 coating antigen ZEN-CMO-OVA
2mg ZEN accurately are weighed, are dissolved in 1mL methanol, add half hydrochloride of 3mg carboxymethyls azanol and 5mg natrium carbonicum calcinatums, Stirring reaction 24h under room temperature, carries out nitrogen with nitrogen evaporator by resulting solution and blows, and 0.05mol L are added after drying-1Hydrochloric acid 2mL redissolves, and is extracted 3 times with isometric ethyl acetate, aqueous phase discarded, merges organic phase, carries out nitrogen and blows, and is obtained after drying faint yellow Oily residue, i.e. haptens ZEN-CMO.
1mg haptens (ZEN-CMO) is weighed, is dissolved in 400 μ L DMF, adds 1mg NHS and 2mg EDC, room temperature condition A liquid is stayed overnight to obtain in lower stirring reaction.Weigh 16.0mg OVA to be slowly dissolved in 4mL phosphate buffers (pH=7.4), this is B Liquid.Under condition of ice bath, A liquid is slowly added to B liquid dropwise, stirring reaction is overnight.Reaction solution is used into pH=7.4 under the conditions of 4 DEG C PBS dialysis 3d, 5000r min-110min is centrifuged, retains supernatant, up to ZEN-CMO-OVA, is saved backup in -20 DEG C.Instead Should be as follows：
The preparation of 2 monoclonal antibody of embodiment
2.1 animal immune
Utilize the immunogene (7-NH of preparation2- ZEN-GA-BSA) immunization Female Balb/C mouse (purchased from Hubei Province's disease it is pre- Anti- control centre's Experimental Animal Center).Immune programme is to take immunogene 7-NH2- ZEN-GA-BSA is respectively with protein content 100 μ g and 50 μ g make it produce specific serum with being injected after adjuvant mixed in equal amounts in Mice Body.
2.2 cell fusions and cloning
With reference to Yang Hanchun《Animal immunology》, using immunogen immune female Balb/C mouse, immune programme is：Exempt from basis After epidemic disease emulsifies immunogene with isometric Freund's complete adjuvant, in the subcutaneous multi-point injection of mouse back, later at interval of 2 weeks Booster immunization once, uses Freund's incomplete adjuvant emulsification instead.Most after fusion, first three day (is most better than after being immunized and rests and reorganizes January) abdomen Chamber is injected, and reinforced immunological, amount of antigen doubles, and is not added with adjuvant.
During fusion, the Balb/C mouse for last reinforced immunological of learning from else's experience one, eye socket sacrificed by exsanguination (collects serum, is the positive Serum), 5min disinfections are soaked in 75% alcohol.Sterile taking-up mouse spleen, isolates splenocyte, and freshly prepared SP2/0 myeloma cell's (SP2/0 myeloma cell comes from this laboratory) presses 1~2 × 107It is a and 108A immune spleen cell, its Ratio is 1:5~1:Between 10, it is all placed in 50mL centrifuge tubes, cell is resuspended with the RPMI-1640 basal liquids of 15mL, 1500r min-15min is centrifuged, washes cell 1 time.The gap of centrifugation is by the culture medium of warm bath, the water of warm bath, 50% poly- second of warm bath Glycol (PEG) etc. is put into super-clean bench.The blotting paper of sterilizing is then taken out, by the centrifugation equipped with myeloma cell and immune spleen cell After being emptied on pipe to the greatest extent, tip upside down on and water droplet is drained on blotting paper, tapping tube bottom loosens cell.Timer is opened, draws 0.8mL's PEG, holds the centrifuge tube equipped with cell mixing, places it in a moment in water-bath, PEG is slowly added drop-wise to cell mixing On, side edged is gently mixed, and is added in 1min, persistently stirs 10s.Then 10mL basic culture solutions are added, are slowly added along tube wall Onto fused cell, side edged gently shakes and (cannot blow and beat), and 5mL is added in 3min, 10mL is added in 5min, finally Basic culture solution is added to 40mL, after capping, overturns several times repeatedly, mixes cell.800r min-15min is centrifuged, and is abandoned Clearly.The HAT culture medium 5mL containing feeder cells are drawn, are slowly added to, and are gently agitated for, avoid piping and druming；Then it is the cell is slow It is added dropwise in the serum bottle containing feeder cells, after mixing by cell inoculation on Tissue Culture Plate, is dripped per hole two, be placed in training Support and cultivated in case.Single cell fusion can be inoculated with 5~7 piece of 96 orifice plate.Kind can also be lacked as needed, generally based on the cell number of SP2/0 Calculate, per hole inoculum concentration containing about 104Left and right SP2/0 cells.In 37 DEG C, 5%CO2Cultivated in incubator.
It is calculated as 0d on the day of fusion, preceding 3d tries not kinetocyte plate, keeps incubator homeostasis.3d is mended per hole Add 1 drop HAT complete mediums；5d suctions out l/2 culture supernatants (100 μ L) per hole, adds 1 drop HT complete mediums；After L/2-3/4 culture supernatants ibid are sucked every 2d, HT complete mediums are changed to after 7d.
Cell colony length to be fused is screened to 1/4 or so of culture hole with the indirect ELISA method of foundation.With zero Medicine hole is compared, and medicine hole OD values repressed can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, 2~4 are selected The cell hole of only 1~2 single colony of a strong positive, cloning is carried out using limited dilution method.
By 3~4 time clonings, the monoclonal antibody of the anti-zearalenone of secretion and its metabolite is finally filtered out Hybridoma cell strain, applicant are named as ZEN/6C2, and are delivered on May 22nd, 2017 positioned at Wuhan City, Hubei Province force China typical culture collection center (CCTCC) preservation in Chinese university, deposit number are CCTCC NO: C201781.To this Cell line has carried out chromosome counting, the results show that the chromosome number average value of hybridoma is 100.7, meets The chromosome number of SP2/0 is 62~68, and splenocyte chromosome is 40, illustrates the really SP2/0 cells and spleen of fused cell The hybrid product of cell.By the cell line through Balb/C mouse are injected intraperitoneally, monoclonal antibody is produced.Using purchased from Thermo Asia of the mouse monoclonal antibody Rapid ELISA isotyping kit of Sxientific companies to the obtained monoclonal antibody of the present invention Type and light chain are identified, are as a result mouse IgG1Hypotype.
The foundation of embodiment 3ZEN racing ELISA detecting methods
The preparation of 3.1 reagents (reagent that the present embodiment uses is prepared in addition to another indicate using following methods)
Phosphate buffer：NaCl 8.0g, KH2PO40.2g, Na2HPO4·12H2O 2.9g, KCl 0.2g, add three steamings Water adjusts pH to 7.4 to 1000mL；
Coating buffer：Take Na2CO31.59g NaHCO32.93g, adds tri-distilled water to adjust pH value to 9.6 to 1000mL；
Cleaning solution：NaCl 8.0g, KH2PO40.2g, Na2HPO4·12H2O 2.9g, KCl 0.2g, Tween 20 0.5mL, adds tri-distilled water to adjust pH to 7.4 to 1000mL；
Confining liquid：Ovalbumin 10.00g is dissolved in 1000mL phosphate buffers；
Substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 160mg, tetrabutyl hydroboration ammonia 21mg, add 10mL dimethylacetamides amine solvent mixes；
Substrate solution B：Citric acid 13.70g, trisodium citrate 10.14g, carbamide peroxide 282.00mg, adds tri-distilled water extremely 1000mL；
Substrate cocktail：By A liquid and B liquid by volume 1:100 mix to obtain the final product, now with the current；
Terminate liquid：2mol L-1Sulfuric acid solution.
3.2 coating original contents and antibody working concentration primarily determine that
Coating original content and antibody working concentration are primarily determined that according to square formation titration.Select the ZEN- of above-mentioned synthesis CMO-OVA is diluted to 4,2,1,0.5,0.25,0.125,0.0625 μ g mL as coating antigen with coating buffer-17 concentration, 96 hole elisa Plates, sequentially add from the 1st to the 7th row, and 4 DEG C overnight；Washing 3 times, pats dry, and adds 250 μ L of confining liquid, 37 DEG C of closings 60min；Washing 3 times, pats dry, and it is diluted dilute to sequentially add 100 μ L phosphate buffers in the 1st row to the 8th row of ELISA Plate The monoclonal antibody that multiple is 4000,8000,16000,32000,64000,128000,256000,512000 is released, 37 DEG C incubate 30min is educated, washs 3 times, pats dry；Each hole adds 1:The sheep anti-mouse igg of the diluted HRP marks of 5000 times of phosphate buffers resists Body (abbreviation secondary antibody, signified secondary antibody is the sheep anti-mouse igg antibody of HRP marks below, purchased from Wuhan Fei Yuan Science and Technology Ltd.s) 100 μ L, 37 DEG C of incubation 30min, wash 5 times, pat dry；Each hole adds 100 μ L Substrate cocktails, and lucifuge colour developing 15min, adds 50 μ L terminate liquids, OD value (OD values) is measured with automatic microplate reader at 450nm wavelength, OD values is selected close to 2.0, with adjacent holes The corresponding antigen coat concentration in the different more significant hole of OD value differences and antibody dilution combination, the results are shown in Table 1.
The result shows that the coating concentration for primarily determining that coating antigen ZEN-CMO-OVA is 1 μ g mL-1Or 0.5 μ g mL-1, resist Body running concentration is 1:64000 or 1:32000.
1 monoclonal antibody square formation of table titrates
3.3 optimal coating original contents and antibody working concentration determine
The coating concentration of the coating antigen ZEN-CMO-OVA primarily determined that with square formation titration, i.e., 1,0.5 μ g mL-12 concentration Coated elisa plate.ZEN standard solution is diluted to 0 with phosphate buffer, 50,100,200,400,800ng L-16 it is dense Degree, 1 is diluted to by monoclonal antibody respectively with phosphate buffer:64000 and 1:32000, to zero hole for adding 50 μ L PBS With in the medicine hole of 50 μ L ZEN standard solution respectively plus 50 μ L monoclonal antibodies carry out indirect competitive ELISA measure.With ZEN standards Solution concentration makees abscissa to numerical value, with the ratio (B/B of the OD values in medicine hole and " zero " hole OD values0) painted as ordinate Suppression curve processed, selects " 0 " hole OD values close to ZEN concentration (IC during the 2.0, suppression of generation 50%50) junior's conduct coating concentration. To be most preferably coated with original content coated elisa plate, ZEN standard solution is diluted to 0,50,100,200,400,800ng L-16 Concentration, by antibody to select concentration 1:64000 equal difference set dilution factor, are added separately to the ZEN standards of zero hole and series concentration Indirect competitive ELISA is carried out in the medicine hole of solution, suppression curve is drawn and calculates IC50Value.Select " 0 " hole OD values close to 2.0, IC50Junior is as optimum antibody working concentration.It the results are shown in Table 2,3.
The optimal coating antigen concentration optimization of table 2
3 optimum antibody dilution factor of table optimizes
Antibody dilution (1:X) 0 hole OD values IC50It is worth (ng L-1)
56000 2.181 174.55
60000 2.06 149.13
64000 1.998 139.38
68000 1.893 124.89
72000 1.869 116.89
The result shows that most preferably coating concentration is 1 μ g mL-1, optimum antibody dilution factor is 1:64000.
The foundation of 3.4 standard curves
By ZEN standard solution phosphate buffered saline into 0,10,50,250,1250,6250ng L-16 concentration, Each concentration repeats 5 holes, is measured according to indirect competitive ELISA method, replication 5 times.Using ZEN solution concentrations to numerical value as Abscissa, B/B0 draw standard curve for ordinate and calculate IC50.It is fitted using ELISA Calc softwares, as a result as schemed Shown in 1-6.The regression equation of standard curve is y=(A-D)/[1+ (x/C)^B]+D, A=0.97351, B=7.47822, C= 2.03041 D=0.07020, R2=0.99977, IC50It is worth for 114.0 ± 5.0ng L-1(n=5), the range of linearity for 10~ 6250ng L-1。
3.5 cross reactions are tested
By α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearelone With phosphate buffered saline into debita spissitudo, the IC of each medicine is measured with the ELISA method of foundation50Value, each medicine 3 Multiple holes, using monoclonal antibody to the cross reacting rate of zearalenone as 100%, according to formula 1 calculate monoclonal antibody pair The cross reacting rate of zearalenone and its 5 kinds of metabolins, the results are shown in Table 4.
4 monoclonal antibody of table is to zearalenone and its 5 kinds of metabolin cross reacting rates
The result shows that monoclonal antibody is to zearalenone, α-zearalenol, β-zearalenol, α-jade Rice red mould alcohol, β-zearalanol and zearelone have higher cross reaction, can detect 6 kinds of mycotoxins at the same time.
The assembling of embodiment 4ELISA kits
4.1 ELISA kits of the present invention are made of following part：
1) it is coated with the solid phase carrier (ELISA Plate) of coating antigen ZEN-CMO-OVA；
2) 6 bottles of ZEN standard solution, concentration is respectively 0,10,50,250,1250,6250ng L-1；
3) monoclonal antibody of hybridoma ZEN/6C2 secretions；
4) the sheep anti-mouse igg antibody working solution of horseradish peroxidase (HRP) mark；
5) concentrated phosphoric acid salt buffer：NaCl 80.0g, KH2PO42.0g, Na2HPO4·12H2O 29.0g, KCl 2.0g, adds distilled water to 1000mL；
6) concentrated cleaning solution：NaCl 80.0g, KH2PO42.0g, Na2HPO4·12H2O 29.0g, KCl 2.0g, Tween 20 5mL, add tri-distilled water to 1000mL；
7) substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 160mg, tetrabutyl hydroboration ammonia 21mg, add 10mL dimethylacetamides amine solvent mixes；
8) substrate solution B：Citric acid 13.70g, trisodium citrate 10.14g, carbamide peroxide 282.00mg, adds tri-distilled water extremely 1000mL；
9) terminate liquid：2mol L-1Sulfuric acid solution.
The preparation of 4.2 ELISA Plates
ZEN-CMO-OVA is diluted to 1 μ g mL with coating buffer-1, 100 μ L are added per hole, 4 DEG C are overnight, coating buffer of inclining, 250 μ L cleaning solutions are added per hole to wash 3 times, are patted dry, and 250 μ L of confining liquid are then added per hole, 37 DEG C of incubation 120min, incline Liquid in hole, cleaning solution are washed 3 times, patted dry, and are preserved with masking foil vacuum sealing.
The mensuration program of 5 enzyme linked immunological kit of embodiment
The preparation of 5.1 reagents
1) sample diluting liquid：Used after the concentrated phosphoric acid salt buffer provided in kit is diluted 10 times with tri-distilled water.
2) cleaning solution：Used after the cleaning solution provided in kit is diluted 10 times with tri-distilled water.
3) Substrate cocktail：According to each institute's expense, the substrate solution A of preparation and substrate solution B is pressed into volume 1:100 is mixed It is even, it is now with the current.
5.2 sample pre-treatments
1) sample pre-treatments of feed and corn：Pig mixed fodder is screened with corn mill into powder, and with 40- mesh mesh screen. The sample after 2.00 ± 0.02g screenings is taken to add 10mL methanol-waters (7 in 50mL centrifuge tubes:3, v/v), be vortexed concussion 5min, is filtered using Whatman NO.1 filter paper.1mL filtrates are taken in 10mL EP pipes, with PBS (0.01mol L-1, pH 7.4) filtrate is diluted to 4mL, if liquid is still muddy, is filtered again with filter paper, clear filtrate is detected for ELISA.
2) sample pre-treatments of edible animal tissue：To the fine homogenate of fresh pork (liver, kidney) tissue.Take 2.00 ± Sample after 0.02g processing adds 10mL methanol-waters (7 in 50mL centrifuge tubes:3, v/v), be vortexed concussion 5min, warp 4000r min-1Centrifuge 10min.1mL supernatants are taken in 10mL EP pipes, with PBS (0.01mol L-1, pH 7.4) and by filtrate 3mL is diluted to, adds 1mL n-hexane vortex oscillation degreasings, 5min is stood, sucks upper-layer fat layer, layer supernatant liquid is removed and uses Detected in ELISA.
3) pre-treatment of milk sample：2mL Fresh Milks are taken in 10mL EP pipes, through 4000r min under the conditions of 4 DEG C-1From Heart 15min, sucks upper-layer fat layer, takes intermediate layer emulsion PBS (0.01mol L-1, pH 7.4) dilution 10 times after be used for Elisa assay.
5.3 determination step
1) it is loaded：50 μ of zearalenone series concentration standard solution or sample extracting solution is added into ELISA Plate micropore L, then adds 50 μ L of monoclonal antibody working solution, is placed in wet box, 37 DEG C of constant-temperature incubation 30min；
2) wash：The liquid in hole is poured out, adding 250 μ L of cleaning solution in every hole washs 3 times and pat dry；
3) the sheep anti-mouse igg antibody working solution of horseradish peroxidase (HRP) mark is added：Horseradish peroxidating is added in per hole The 100 μ L of sheep anti-mouse igg antibody working solution of thing enzyme (HRP) mark, are placed in wet box, 37 DEG C of constant-temperature incubation 30min；
4) wash：The liquid in hole is poured out, 250 μ L of cleaning solution is added in every hole, washs 3 times and pat dry；
5) substrate is added：100 μ L of Substrate cocktail are added in per hole, are placed in wet box, 37 DEG C of constant-temperature incubation 15min；
6) terminate liquid is added：50 μ L of terminate liquid are added in per hole；
7) measure：The OD value (OD values) in every hole is measured at 450nm with microplate reader.
5.4 results judge
Standard curve：
With the standard items OD values divided by " zero " hole OD values (B/B measured0) it is ordinate, zearalenone (ZEN) is dense Degree makees standard curve to numerical value for abscissa, and carries out linear regression, provides regression equation.
The concentration of ZEN calculates in corn, pannage, edible animal tissue and milk：
The inhibiting rate (the OD values divided by " zero " hole OD values of the sample obtained) of sample is calculated, substitutes into the recurrence of standard curve Equation calculation toxin concentration.
Zearalenone metabolite concentration calculates in corn, pannage, edible animal tissue and milk：
The inhibiting rate (the OD values divided by " zero " hole OD values of the sample obtained) of sample is calculated, substitutes into the recurrence of standard curve In equation, zearalenone metabolite concentration is converted to according to formula 2.
The sensitivity of 6 ELISA method of the present invention of embodiment, precision, accuracy test
The sensitivity test of 6.1 kits of the present invention
By 20 part of 0 μ g L-1The detection OD values of standard solution substitute into standard curve, calculate 20 parts of blank determination concentration, obtain Average value and standard deviation, Z values (being shown in Table 5) are calculated according to formula Z=C+3SD.The sensitivity of the ELISA method is 11.3 ng L-1.Minimum detection limit (LOD) is determined by following steps：The OD values of 20 parts of blank tissue samples are measured, according to returning for standard curve Return equation calculation to go out corresponding ZEN concentration, then calculate the average value of ZEN concentrationWith standard deviation (SD), according to formulaCalculate the LOD in sample；Quantitative limitZEN and its 5 kinds of metabolins are not same LOD and LOQ in product refer to table 6.
The sensitivity of 5 kit of table
The test limit and quantitative limit of zearalenone and its 5 kinds of metabolins in the different samples of table 6
The precision test of 6.2 kits of the present invention
ZEN standard items are diluted to 0,10,50,250,1250,6250ng L-16 concentration, per 5 repetitions of concentration, are pressed According to indirect competitive ELISA method replication 5 times, go out each concentration ZEN standard solution using the regression equation calculation of standard curve Measured value, in computing board between plate the coefficient of variation, the results are shown in Table 7.
The interior coefficient of variation between plate of the plate of 7 standard curve of table
The accuracy of 6.3 kits of the present invention, repetitive test
The zearalenone of three concentration and its 5 kinds of metabolin standard items are added in 6 kinds of samples respectively, it is added The rate of recovery is between 62.9%~113.6%, batch interior and interassay coefficient of variation<15%；Specific measurement result is shown in Table 8~13.
TIANZHU XINGNAO Capsul in 8 corn of table
TIANZHU XINGNAO Capsul in 9 pannage of table
TIANZHU XINGNAO Capsul in 10 pork of table
TIANZHU XINGNAO Capsul in 11 pig liver of table
TIANZHU XINGNAO Capsul in 12 Ren sus domestica of table
TIANZHU XINGNAO Capsul in 13 milk of table

Claims (8)

1. a kind of monoclonal antibody that can identify zearalenone and its metabolite, it by preserving number is CCTCC NO that it, which is,: Secreted by the hybridoma cell strain ZEN/6C2 of C201781.
2. monoclonal antibody as claimed in claim 1, it is characterised in that：The metabolite for α-zearalenol, β- Zearalenol, α-zearalanol, β-zearalanol, zearelone.
3. monoclonal antibody as claimed in claim 1 or 2, it is characterised in that：The hybridoma cell strain ZEN/6C2, it is Using what is be prepared after haptens and carrier protein couplet as immunogene, the structural formula of the haptens is as follows：
4. the monoclonal antibody described in claim 1 or 2 is exempted from preparation detection zearalenone and its enzyme-linked of metabolite Application in epidemic disease kit.
5. include the kit of monoclonal antibody described in claim 1.
6. kit according to claim 5, which is detect zearalenone and its metabolite enzyme-linked Immune reagent kit.
7. application of the kit in zearalenone and its non-diagnostic purpose detection of metabolite described in claim 5.
8. a kind of zearalenone and its enzyme-linked immunoassay method of the non-diagnostic purpose detection of metabolite, it is characterised in that including Following steps：
(1) the following haptens of structural formula and chicken ovalbumin are coupled to obtain coating antigen；
(2) it is CCTCC NO with preserving number:The hybridoma cell strain ZEN/6C2 of C201781 prepares monoclonal antibody；
(3) the coating primordial covering solid phase carrier of step (1) is used；
(4) processing and detection of corn, pannage, edible animal tissue such as pork, liver, kidney, milk sample.
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CN111175509A (en) * 2020-02-21 2020-05-19 福州大学 ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN
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CN113474467A (en) * 2018-12-06 2021-10-01 艾尔柏股份公司 Biomarkers
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CN111175509A (en) * 2020-02-21 2020-05-19 福州大学 ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN
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CN114214288B (en) * 2021-12-24 2023-09-01 江南大学 Zearalenone monoclonal antibody hybridoma cell strain and application thereof

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