CN102746403A - Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin - Google Patents

Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin Download PDF

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CN102746403A
CN102746403A CN2012101176172A CN201210117617A CN102746403A CN 102746403 A CN102746403 A CN 102746403A CN 2012101176172 A CN2012101176172 A CN 2012101176172A CN 201210117617 A CN201210117617 A CN 201210117617A CN 102746403 A CN102746403 A CN 102746403A
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afla
standard substance
toxins
concentration
elisa
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李培武
管笛
张奇
张文
丁小霞
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The present invention relates to a standard substance universal alternate for aflatoxin detection by using ELISA, a preparation method thereof, and an ELISA detection method for aflatoxin. The standard substance universal alternate is characterized in that: the standard substance universal alternate is a rabbit anti-mouse antibody capable of recognizing various mouse anti-aflatoxin monoclonal antibodies. The preparation method comprises: adopting a mouse anti-aflatoxin B1 monoclonal antibody, a mouse anti-aflatoxin M1 monoclonal antibody, and a mouse anti-aflatoxin G1 monoclonal antibody as a mixed antigen; adopting the mixed antigen to immunize New Zealand long ear white rabbits; sampling blood from carotid arteries; and carrying out affinity purification on serums to obtain the standard substance universal alternate. The standard substance universal alternate of the present invention is non-toxic and harmless, and can respectively replace various corresponding aflatoxin standard substances in ELISA detection methods for aflatoxin to quantitatively detect various corresponding aflatoxin concentrations in samples.

Description

Be used for the general surrogate of standard substance, its preparation method and Toxins, afla ELISA detection method that ELISA detects Toxins, afla
Technical field
The invention belongs to the Toxins, afla detection range, be specifically related to be used for the general surrogate of standard substance, its preparation method and the Toxins, afla ELISA detection method that ELISA detects Toxins, afla.
Background technology
Toxins, afla mainly be by Aspergillus flavus ( Aspergillus flavs), the Aspergillus parasiticus bacterium ( Aspergillus parasiticus) and collection honeybee aspergillus tubigensis ( Aspergillus nonius) the one group high poison and the strong carcinogenic secondary metabolite that produce, mainly contain AFB1 (Aflatoxin B 1, AFB 1), aflatoxin M 1 (Aflatoxin M 1, AFM 1), aflatoxin G 1 (Aflatoxin G 1, AFG 1).It extensively is present in various agricultural-food, food and the feed, and grain oil products such as severe contamination peanut, rice, corn, wheat greatly threaten people health and life security.Because Toxins, afla is on the rise to the mankind's potential threat, existing in the world more than 100 countries have carried out the strict requirement of limiting the quantity of to the content of Toxins, afla in the agricultural-food food.Therefore pressing for detection technique simple to operate, quick, high-throughout monitors, detects pollutent.That the immunology detection technology has is highly sensitive, sample pre-treatments simple, convenience operation.Wherein (Enzyme-Linked Immunosorbent Assay ELISA) has the advantage that the detection flux is high, the sample requirement is few to enzyme-linked immunosorbent assay, has been widely used in examination of aflatoxin in food and the agricultural-food.Owing to need competing reaction and typical curve during the ELISA standard measure, need to use a large amount of Toxins, afla standard substance in the testing process and carry out Quality Control, and detect the Toxins, afla standard substance that different Toxins, afla need be different.And the use meeting of these high malicious strong carcinogens brings great harm to operator and environment, and because of cost an arm and a leg, in short supply, often influence the carrying out of testing process.Therefore be badly in need of a kind of general surrogate of standard substance safe, the Toxins, afla of ELISA detection cheaply and can reduce the risk of testing process greatly, simplify and detect step.
Summary of the invention
Technical problem to be solved by this invention provides and is used for the general surrogate of standard substance, its preparation method and the Toxins, afla ELISA detection method that ELISA detects Toxins, afla.The general surrogate of these standard substance is nontoxic, can come each corresponding Toxins, afla concentration in the detection by quantitative sample through each the corresponding Toxins, afla standard substance that substitutes in the Toxins, afla ELISA detection method respectively.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
Be used for the general surrogate of standard substance that ELISA detects Toxins, afla, it is characterized in that: it is for discerning the rabbit anti-mouse antibody of each mouse source aspergillus flavus resisting toxin monoclone antibody.
Press such scheme, said aspergillus flavus resisting toxin monoclone antibody is preferably aspergillus flavus resisting toxin B1 monoclonal antibody, aspergillus flavus resisting toxin M1 monoclonal antibody and aspergillus flavus resisting toxin G1 monoclonal antibody.
Be used for the preparation method that ELISA detects the general surrogate of standard substance of Toxins, afla; It is characterized in that: it is as hybrid antigen with mouse source aspergillus flavus resisting toxin B1 monoclonal antibody, aspergillus flavus resisting toxin M1 monoclonal antibody and aspergillus flavus resisting toxin G1 monoclonal antibody; Through immune nz screech owl White Rabbit; Carotid artery is got blood, obtains behind the serum affinity purification.
Press such scheme, described aspergillus flavus resisting toxin B1 monoclonal antibody is for by deposit number being the aspergillus flavus resisting toxin B1 monoclonal antibody 3G1 of the hybridoma cell strain 3G1 secretion generation of CCTCC NO. C201014; Described aspergillus flavus resisting toxin M1 monoclonal antibody is to be the aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 of the hybridoma cell strain 2C9 secretion generation of CCTCC NO. C201018 by deposit number, and described aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 is for by deposit number being the aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 of the hybridoma cell strain 1C8 secretion generation of CCTCC NO. C201017; Said aspergillus flavus resisting toxin B1 monoclonal antibody 3G1, aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 and aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 make hybrid antigen for the equimolar amount proportioning.
Be used for ELISA and detect of the application of the general surrogate of standard substance of Toxins, afla in Toxins, afla ELISA detection.
Toxins, afla ELISA detection method, it is characterized in that: it may further comprise the steps:
(1) adopts conventional indirect competitive ELISA method, the general surrogate of standard substance is replaced each Toxins, afla standard substance, set up the general surrogate ELISA of each corresponding Toxins, afla standard substance standard analysis curvilinear equation a (x respectively 1, y 1),
---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate;
(2) adopt conventional indirect competitive ELISA method, measure the inhibiting rate of testing sample;
In the curvilinear equation of the corresponding substitution step of testing sample inhibiting rate (1) that (3) step (2) is obtained, the corresponding relation conversion equation b (x of the general surrogate concentration of the concentration results substitution of the general surrogate of the corresponding Toxins, afla standard substance of each that will calculate the then corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, calculate the concentration of each corresponding Toxins, afla in the testing sample.
Press such scheme, said step (3) is: the general surrogate elisa assay of each corresponding Toxins, afla standard substance of corresponding combination curvilinear equation a (x 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of corresponding Toxins, afla standard substance concentration with each with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, obtain: the curvilinear equation e (y that does the corresponding Toxins, afla concentration of inhibiting rate under the ELISA standard analysis curve at the general surrogate of standard substance with each 1, x 2),---y 1Be inhibiting rate, x 2Be each corresponding Toxins, afla concentration;
The above-mentioned curvilinear equation e of the testing sample inhibiting rate substitution (y that step (2) is obtained 1, x 2), calculate the concentration of each corresponding Toxins, afla in the testing sample.
Press such scheme, the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of the corresponding Toxins, afla standard substance concentration of each in the said step (3) with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, adopt following method to obtain:
1. adopt conventional indirect competitive ELISA method to set up the ELISA standard analysis curvilinear equation c (x of each corresponding Toxins, afla standard substance respectively 2, y 2),---x 2Be the concentration of each corresponding Toxins, afla standard substance, y 2Be inhibiting rate;
2. adopt conventional indirect competitive ELISA method, the general surrogate of standard substance is substituted each corresponding Toxins, afla standard substance, set up the general surrogate ELISA of each corresponding Toxins, afla standard substance standard analysis curvilinear equation d (x respectively 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate;
3. each corresponding Toxins, afla standard substance analytic curve equation c (x of corresponding combination 2, y 2) the general surrogate elisa assay of corresponding Toxins, afla standard substance with each curvilinear equation d (x 1, y 1), obtain the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance.
The corresponding relation conversion equation b (x of the general surrogate concentration of the said corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2) be invariable, in the subsequent detection experiment, need not to rebulid.
Press such scheme, 3. said step is: each corresponding Toxins, afla standard substance analytic curve equation c (x of corresponding combination 2, y 2),---x 2Be the concentration of each corresponding Toxins, afla standard substance, y 2Be inhibiting rate---the general surrogate elisa assay of corresponding Toxins, afla standard substance with each curvilinear equation d (x 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---, the general surrogate concentration of the pairing corresponding Toxins, afla standard substance with each of each corresponding Toxins, afla standard substance concentration of same inhibiting rate in the inhibiting rate scope of calculating 20%～80% is then with each corresponding Toxins, afla standard substance concentration x 2Be X-coordinate, the general surrogate concentration of each corresponding Toxins, afla standard substance x 1Be ordinate zou, map match respectively and obtain the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance.
Press such scheme, the measuring method of inhibiting rate is following in the described conventional indirect competitive ELISA method: get the micropore that encapsulates the corresponding Toxins, afla complete antigen of each Toxins, afla and encapsulate plate, add the corresponding aspergillus flavus resisting toxin monoclone antibody of each Toxins, afla; Add testing sample or each corresponding Toxins, afla standard substance or the general surrogate of standard substance then, after the reaction, the washings washing; It is anti-to add enzyme labelling sheep anti mouse two, carries out the labelled immune reaction, after the washings washing; Add colour developing liquid, add stop buffer after leave standstill the dark place, under 450 nm, measure absorbance value; According to treating that the gaging hole absorbance value is B, the absorbance value of control wells is B 0, the calculating inhibiting rate is B/B 0
Press such scheme, the general surrogate ELISA of said each corresponding Toxins, afla standard substance standard analysis curvilinear equation d (x 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---foundation in, the concentration laying scope of the general surrogate of each corresponding Toxins, afla standard substance is 0.4-100 μ g/mL.
Be measured as example with AFB1 in the testing sample, the ELISA typical case detection method of AFB1 is:
(1) the corresponding relation conversion equation b (x of the general surrogate concentration of AFB1 standard substance concentration and standard substance 1, x 2) foundation, x 1Be the concentration of the general surrogate of standard substance, x 2Concentration for the AFB1 standard substance;
(1.1) adopt conventional indirect competitive ELISA method to set up the ELISA standard analysis curvilinear equation c (x of AFB1 standard substance 2, y 2),---x 2Be the concentration of AFB1 standard substance, y 2Be inhibiting rate;
(1.2) adopt conventional indirect competitive ELISA method, the general surrogate of standard substance is replaced the AFB1 standard substance, set up the general surrogate ELISA of AFB1 corresponding standard article standard analysis curvilinear equation d (x 1, y 1),---x 1Be the concentration of the general surrogate of standard substance, y 1Be inhibiting rate;
(1.3) combine AFB1 standard substance analytic curve equation c (x 2, y 2) and the general surrogate elisa assay of its corresponding standard article curvilinear equation d (x 1, y 1), same inhibiting rate pairing AFB1 standard substance concentration and the general surrogate concentration of standard substance in the inhibiting rate scope of calculating 20%～80% are then with AFB1 standard substance concentration x 2Be X-coordinate, the general surrogate concentration of standard substance x 1Be ordinate zou, the mapping match and the corresponding relation conversion equation b (x of corresponding AFB1 standard substance concentration and the general surrogate concentration of standard substance 1, x 2),---x 1Be the concentration of the general surrogate of standard substance, x 2Be the AFB1 standard substance;
(2) adopt conventional indirect competitive ELISA method, the general surrogate of standard substance is replaced the AFB1 standard substance, set up the general surrogate ELISA of AFB1 corresponding standard article standard analysis curvilinear equation a (x 1, y 1),---x 1Be the concentration of the general surrogate of standard substance, y 1Be inhibiting rate;
(3) the general surrogate elisa assay of combined standard article curvilinear equation a (x 1, y 1),---x 1Be the concentration of the general surrogate of standard substance, y 1Be inhibiting rate---and the conversion equation b (x of AFB1 standard substance concentration and the general surrogate concentration of standard substance 1, x 2),---x 1Be the concentration of the general surrogate of standard substance, x 2Concentration for the AFB1 standard substance---, obtain being the curvilinear equation e (y of inhibiting rate and AFB1 concentration under the ELISA standard analysis curve at the general surrogate of standard substance 1, x 2),---y 1Be inhibiting rate, x 2Be AFB1 concentration;
(4) adopt conventional indirect competitive ELISA method, measure the inhibiting rate of testing sample;
(5) the above-mentioned curvilinear equation e of the testing sample inhibiting rate substitution (y that step (4) is obtained 1, x 2), calculate the concentration of AFB1 in the testing sample.
In testing sample in the detection first of AFB1, the general surrogate ELISA of the standard substance standard analysis curvilinear equation a (x in the step (2) 1, y 1) can directly take steps (1.2) the general surrogate ELISA of standard substance standard analysis curvilinear equation d (x of obtaining 1, y 1), need not repetition.In subsequent detection is analyzed; Because of the corresponding relation conversion equation of AFB1 standard substance concentration and the general surrogate concentration of standard substance is invariable, can directly adopt the AFB1 standard substance concentration set up in advance and the corresponding relation conversion equation b (x of the general surrogate concentration of standard substance 1, x 2), and need not to rebulid the ELISA standard analysis curvilinear equation c (x of AFB1 standard substance for the corresponding relation conversion equation that obtains AFB1 standard substance concentration and the general surrogate concentration of standard substance 2, y 2), having reduced the AFB1 standard substance and used link, the harm that the use of reduction AFB1 standard substance causes environment reduces and detects cost, and step simplifies the operation.The analyzing and testing of other Toxins, afla is also like this.
Traditional competitive ELISA quantitative model is based on the Toxins, afla that dissociates in the sample and combines the aspergillus flavus resisting toxin monoclone antibody with the fixedly Toxins, afla antigenic competition that encapsulates on enzyme plate; Free Toxins, afla in the sample is many more; Combine many more with the competition of Toxins, afla monoclonal antibody; Stay on the enzyme plate few more with immobilized antigen bonded aspergillus flavus resisting toxin monoclone antibody; The ELIAS secondary antibody that then can be combined on the aspergillus flavus resisting toxin monoclone antibody is also few more, the degree that finally develops the color more a little less than.
The operating mode that the present invention is based on is that the general surrogate of standard substance combines the aspergillus flavus resisting toxin monoclone antibody with the ELIAS secondary antibody competition; The general surrogate of standard substance is many more; The ELIAS secondary antibody that finally is attached on the aspergillus flavus resisting toxin monoclone antibody is just few more; Enzyme is few more during coupling reaction, the colour developing degree more a little less than.Hence one can see that; Exist positively related relation between general surrogate of standard substance and the Toxins, afla standard substance; As long as verify the corresponding relation of measuring between the two; Can the general surrogate of standard substance be substituted the Toxins, afla standard substance and carry out the calibration of elisa assay curve, detect aflatoxin content.Because the general surrogate of standard substance is that the non-marked two of Toxins, afla monoclonal antibody is anti-; It can be discerned each aspergillus flavus resisting toxin monoclone antibody and comprise aspergillus flavus resisting toxin B1 monoclonal antibody, aspergillus flavus resisting toxin M1 monoclonal antibody and aspergillus flavus resisting toxin G1 monoclonal antibody; Therefore the general surrogate of these standard substance can be applied to comprise AFB1, aflatoxin M 1 and aspergillus flavus resisting toxin G1 in the ELISA detection method based on each Toxins, afla to have versatility.
Beneficial effect of the present invention is:
(1) essence of the general surrogate of standard substance of this Toxins, afla is a kind of antibody, and nontoxic, harmless, cost is low;
(2) in Toxins, afla detects; Use the general surrogate of these standard substance and carry out the elisa assay calibration curve; Reduce hypertoxic strong each carcinogenic corresponding Toxins, afla standard substance and used link, helped protecting operator's health not receive the infringement of Toxins, afla standard substance; Reduce the harm that the use of each corresponding Toxins, afla standard substance causes environment; Reduce and detect cost;
(3) the ELISA detection method based on the general surrogate of these standard substance has versatility, can be used for detecting various Toxins, afla, like AFB1, G1 and M1.
Description of drawings
Each corresponding Toxins, afla standard substance ELISA standard analysis curve of Fig. 1 is among the figure :-■-AFB1;-▼-AFG1;-●-AFM1;
The general surrogate ELISA of each corresponding Toxins, afla standard substance of Fig. 2 standard analysis curve is among the figure :-■-mab:3G1;-▼-mab:2C9;-●-mab:1C8;
Corresponding relation curve between the general surrogate concentration of Fig. 3 AFB1 standard substance concentration and standard substance;
Corresponding relation curve between the general surrogate concentration of Fig. 4 aflatoxin G 1 standard substance concentration and standard substance;
Corresponding relation curve between Fig. 5 aflatoxin M 1 standard substance concentration and the general surrogate concentration of standard substance.
Embodiment
The preparation of the general surrogate of embodiment 1 standard substance
1. hybridoma cell strain 3G1, this cell strain has been preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, and the preservation address is, China, Wuhan, Wuhan University, deposit number is CCTCC NO. C201014.Its screening method, aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR and hypotype characteristic thereof are following:
(1) screening of hybridoma cell strain 3G1
1. antigen synthesizes and animal immune
Buying commercially available AFB1 standard substance, to carry out complete antigen synthetic, and concrete synthesis step is following: 4 mg AFB1 are dissolved in 2 mL acetone, add 40 μ L, 10% H 2SO 4, mixture is at 56 ℃ of stirring reaction 4 h; Behind the product evaporate to dryness, add the H of 5 mL 2O with 25 mL chloroform extraction twice, uses 20 mL H then 2O washs organic layer, keeps organic layer; Boil off organic solvent, get yellow solid product.Get 1.0 mg products, to wherein adding 2 mL, 0.5% BSA solution (37 ℃ of reaction 30 min of 4 mL PBS (phosphate buffered saline buffer, pH7.4) dissolving 20 mg BSA); Add 100 μ L, 6.5 mM NaBH 4, 4 ℃ of reaction 30 min; Add 50 μ L 0.1M HCl, remove excessive N aBH 4Under 4 ℃ of conditions, (phosphate buffered saline buffer, pH7.4) dialysis 3d removes AFB1 and AFB to PBS solution 2a, carrying out conventional UV scanning method at last and identify, qualification result shows and has prepared AFB1 complete A antigen FB 2a-BSA.
6 of purchase female BALB/c mouses in 6 ages in week, immunity is synthetic AFB1 complete A antigen FB voluntarily 2a-BSA.Immunity is with complete A antigen FB for the first time 2a-BSA and equivalent Fu Shi Freund's complete adjuvant mixing and emulsifying, the subcutaneous multi-point injection in mouse carotid back then.For the second time be immune to head and exempt to carry out after 4 weeks, adopt freund 's incomplete adjuvant and equal-volume complete A antigen FB 2a-BSA emulsification, the mouse peritoneal injection.Immunity for the third time and immunity 4 weeks of pitch time for the second time, immunization ways and dosage be with for the second time identical, carries out after being immune to immune for the third time 3 weeks the 4th time, and immunization ways and immunizing dose are immune identical with for the second time, and immunizing dose is every mouse 50 μ g at every turn.3 times each immunity one week of back, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum antibody titer.A week after the 4th immunity; Tail vein blood; Separation of serum adopts indirect elisa method monitoring mice serum antibody titer, and measures mice serum sensitivity with the indirect competitive ELISA method; The mouse that the serum that selection is tired, sensitivity is all higher relatively is corresponding carries out last booster immunization, and immunizing dose is before 2 times.AFB1 purchases the company in Sigma-Aldrich.
2. cytogamy
Last booster immunization is after 3 days, and adopting the polyoxyethylene glycol of 50% (weight percentage) is that PEG (molecular weight is 1450) makes fusogen, carries out cytogamy by ordinary method, and concrete steps are following:
The cervical vertebra dislocation method is put to death immune mouse, under aseptic condition, gets spleen, separating Morr. cell; With mouse source myeloma cell SP2/0 with 5: 1 number than mixing, wash cell mixing with the RPMI-1640 basic culture solution, merge with 50%PEG then; Time of fusion 1 minute is filled it up with the RPMI-1640 basic culture solution then, and is centrifugal; Remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with 72mL RPMI-1640 basic culture solution, and resuspended cell is added drop-wise in the 96 porocyte culture plates; 2/hole; Put 37 ℃ of CO2gas incubators and cultivate, described RPMI-1640 basic culture solution is for containing 20% (percent by volume) foetal calf serum, 2% (weight percentage) growth factor and 1% (weight percentage) xanthoglobulin-aminopterin-thymidine.Above-mentioned SP2/0 purchases in last ingression Ke bio tech ltd; The RPMI-1640 basic culture solution is purchased the company in Hyclone; 1% xanthoglobulin-aminopterin-thymidine (HAT) is purchased the company in Sigma-Aldrich.
3. the screening of cell strain and clone
Treated after the cytogamy about the 12nd day, cell colony long at the bottom of account for the hole 1/2 area big or small, the nutrient solution flavescence can be carried out antibody test.Adopt the ELISA method that the culture hole that the hybridoma growth is arranged is screened, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out aspergillus flavus resisting toxin B1 and the positive hole of not anti-carrier proteins BSA; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected; Former with AFB1 as competition; (it was that 0 hole is that the final measured value of negative control hole is higher originally that the higher finger of light absorption value is competed, and the higher finger inhibiting rate of sensitivity is 50% o'clock competition original content that is IC to select all higher hole of light absorption value and sensitivity 50Be worth less), adopt limiting dilution assay to clone, clone and adopted same two-step approach to detect in back about 10 days, so behind repeated cloning 2-3 time, acquisition hybridoma cell strain 3G1.
(2) strain of aspergillus flavus resisting toxin B1 monoclonal antibody hybridoma cell is a 3G1 antibody variable region sequencing
1. extract total RNA: adopt day total RNA extraction reagent box of root company and extract to specifications to produce total RNA that hybridoma cell strain is 3G1;
2. synthetic cDNA: the total RNA that obtains with step 1 is a template, oligo (dT) 15Be primer, according to SuperScript TM-2 II ThermoScript II specification sheetss carry out reverse transcription, synthetic cDNA first chain; Primer oligo (dT) 15Buy by Invitrogen;
3. the PCR method is cloned variable region gene: according to the conservative site design primer of the medium and small mouse antibody genes sequence of GENEBANK, be light, the heavy chain variable region gene of masterplate amplification antibody with cDNA.The PCR program is: 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.After the agarose gel electrophoresis separation of PCR product through 1% (weight percentage); With test kit purifying and recovering dna fragmentation, be connected among the carrier pMD18-T transformed into escherichia coli DH5 α competent cell; The picking positive colony is delivered to Shanghai Sani's bio tech ltd and is checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is 5 ,-AGG TSM ARC TGC AGS AGT CWG G-3 ,(22mer) with 5 ,-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3 ,(32mer) wherein S, M, R and W are the merger base, M=A/C, and R=A/G, S=C/G, W=A/T, variable region of light chain primer are 5 ,-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3 ,(24mer) with 5 ,-CCG TTT CAG CTC CAG CTT GGT CCC-3 ,(24mer).
The gene order result who obtains: the long 347bp of variable region of heavy chain coding gene sequence; Sequence is shown in SEQ ID NO:1; Derive the coded variable region of heavy chain of this gene order according to the gene order that is obtained and be made up of 115 amino acid, sequence is shown in SEQ ID NO:3.The long 338bp of variable region of light chain coding gene sequence, sequence is derived the coded variable region of light chain of this gene order according to the gene order that is obtained and is made up of 110 amino acid shown in SEQ ID NO:2, and sequence is shown in SEQ ID NO:4.
(3) aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR, hypotype and CHARACTERISTICS IDENTIFICATION:
With the strain of aspergillus flavus resisting toxin B1 monoclonal antibody hybridoma cell is the BALB/c mouse that the 3G1 injection was handled with freund 's incomplete adjuvant in advance, collects the ascites of this mouse, adopts sad-ammonium sulfate method antibody purification, and concrete operations are: with double-deck filter paper filtering mouse ascites; 4 ℃, the centrifugal 15min of 12000r/min draws supernatant, and gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes; Stir the slow down n-caprylic acid that adds, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, and 4 ℃ leave standstill 2h; 4 ℃ then, the centrifugal 30min of 12000r/min abandons deposition, with the supernatant that obtains with double-deck filter paper filtering after; The volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffered saline buffer, with the pH value to 7.4 that the sodium hydroxide solution of 2 mol/L is regulated this mixed solution, and 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL; 4 ℃ leave standstill 2h, and 4 ℃ then, the centrifugal 30min of 12000r/min; Abandon supernatant, the gained deposition is resuspended with the 0.01mol/L phosphate buffered saline buffer of former ascites volume 1/10, the dialysis tubing of packing into; To the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, uses the freeze drier freeze-drying afterwards; Collect lyophilized powder, promptly get the good aspergillus flavus resisting toxin B1 monoclonal antibody of purifying, antibody is put in-20 ℃ of refrigerators subsequent use;
Described acetate buffer is the 0.29g sodium-acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is a 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g adds water and is settled to the 100mL gained.
Use commercially available hypotype identification kit to identify that the hypotype of hybridoma cell strain 3G1 excretory aspergillus flavus resisting toxin B1 monoclonal antibody is IgG 2a
The tiring of mouse ascites fluid antibody that records 3G1 with conventional non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method can reach 6.4 * 10 6, promptly the mouse ascites fluid antibody dilution 6.4 * 10 6Times the time measured in solution result positive.Use conventional indirect competitive ELISA method to identify that its sensitivity to AFB1 is 1.6ng/mL, with AFB 2 cross reacting rates be 6.4%, the cross reacting rate of aflatoxin G 1 and G2 is all less than 1%.
2. hybridoma cell strain 1C8, this cell strain has been preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, and the preservation address is, China, Wuhan, Wuhan University, deposit number is CCTCC NO. C201017.Its screening method, aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR and hypotype characteristic thereof are following:
(1) preparation of hybridoma cell strain 1C8
1. antigen synthesizes and animal immune
Buy commercially available aflatoxin G 1 standard substance, carry out the synthetic of AFG1-BSA complete antigen according to the compound method of disclosed AFG1-BSA among the embodiment 1 of the embodiment among the CN101270146.X;
6 of purchase female BALB/c mouses in 6 ages in week, immunity is synthetic aflatoxin G 1 complete A antigen FG1-BSA voluntarily.Immunity is with complete A antigen FG1-BSA and equivalent Fu Shi Freund's complete adjuvant mixing and emulsifying, the subcutaneous multi-point injection in mouse carotid back then for the first time.For the second time be immune to head and exempt to carry out after 4 weeks, adopt freund 's incomplete adjuvant and equal-volume complete A antigen FG1-BSA emulsification, the mouse peritoneal injection.Immunity for the third time and immunity 4 weeks of pitch time for the second time, immunization ways and dosage be with for the second time identical, carries out after being immune to immune for the third time 3 weeks the 4th time, and immunization ways and immunizing dose are immune identical with for the second time, and immunizing dose is every mouse 50 μ g at every turn.3 times each immunity one week of back, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum antibody titer.A week after the 4th immunity; Tail vein blood; Separation of serum adopts indirect elisa method monitoring mice serum antibody titer, and measures mice serum sensitivity with the indirect competitive ELISA method; The mouse that the serum that selection is tired, sensitivity is all higher relatively is corresponding carries out last booster immunization, and immunizing dose is before 2 times.
2. cytogamy
After 3 days, adopting the polyoxyethylene glycol of 50% (weight percentage) is that PEG (molecular weight is 1450) makes fusogen, carries out cytogamy by ordinary method in last booster immunization; Concrete steps: kill immune mouse under the aseptic condition, separating Morr. cell, with mouse source myeloma cell SP2/0 with 5: 1 number than mixing; Wash cell mixing with the RPMI-1640 basic culture solution, merge, merged 1 minute with 50%PEG; Fill it up with the RPMI-1640 basic culture solution then, centrifugal, remove supernatant; The fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with the 72mLRPMI-1640 basic culture solution; Resuspended cell is added drop-wise in the 96 porocyte culture plates, 2/hole, puts 37 ℃ of CO2gas incubators and cultivate; Described RPMI-1640 basic culture solution is for containing 20% (percent by volume) foetal calf serum, 2% (weight percentage) growth factor and 1% (weight percentage) xanthoglobulin-aminopterin-thymidine is HAT.Above-mentioned SP2/0 purchases in last ingression Ke bio tech ltd; The RPMI-1640 basic culture solution is purchased the company in Hyclone; 1% xanthoglobulin-aminopterin-thymidine is that HAT purchases the company in Sigma-Aldrich.
3.The screening of cell strain and clone treated after the cytogamy about the 12nd day, and cell colony is long to accounting for 1/2 size at the bottom of the hole, and the nutrient solution flavescence can be carried out antibody test.Adopt the ELISA method that the culture hole that the hybridoma growth is arranged is screened, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out aspergillus flavus resisting toxin G1 and the positive hole of not anti-carrier proteins BSA; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected; Former with aflatoxin G 1 as competition; (it was that 0 hole is that the final measured value in positive control hole is higher originally that the higher finger of light absorption value is competed, and the higher finger inhibiting rate of sensitivity is 50% o'clock competition original content that is IC to select all higher hole of light absorption value and sensitivity 50Be worth less), adopt limiting dilution assay to clone, clone and adopted same two-step approach to detect in back about 10 days, so behind repeated cloning 2-3 time, acquisition hybridoma cell strain 1C8.
(2) strain of aspergillus flavus resisting toxin G1 monoclonal antibody hybridoma cell is a 1C8 antibody variable region sequencing
1. extract total RNA: adopt day total RNA extraction reagent box of root company and extract to specifications to produce total RNA that hybridoma cell strain is 1C8;
2. synthetic cDNA: the total RNA that obtains with step 1 is a template, oligo (dT) 15Be primer, according to SuperScript TM-2 II ThermoScript II specification sheetss carry out reverse transcription, synthetic cDNA first chain; Primer oligo (dT) 15Buy by Invitrogen;
3.PCR method clone variable region gene:, be light, the heavy chain variable region gene of masterplate amplification antibody with cDNA according to the conservative site design primer of the medium and small mouse antibody genes sequence of GENEBANK.The PCR program is: 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.After the agarose gel electrophoresis separation of PCR product through 1% (weight percentage); With test kit purifying and recovering dna fragmentation, be connected among the carrier pMD18-T transformed into escherichia coli DH5 α competent cell; The picking positive colony is delivered to Shanghai Sani's bio tech ltd and is checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is 5 ,-AGG TSM ARC TGC AGS AGT CWG G-3 ,(22mer) with 5 ,-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3 ,(32mer) wherein S, M, R and W are the merger base, M=A/C, and R=A/G, S=C/G, W=A/T, variable region of light chain primer are 5 ,-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3 ,(24mer) with 5 ,-CCG TTT CAG CTC CAG CTT GGT CCC-3 ,(24mer).
The gene order result who obtains: the long 332bp of variable region of light chain coding gene sequence; Sequence is shown in SEQ ID NO:1; Derive the coded variable region of light chain of this gene order according to the gene order that is obtained and be made up of 110 amino acid, sequence is shown in SEQ ID NO:3.The long 362bp of variable region of heavy chain coding gene sequence, sequence is derived the coded variable region of heavy chain of this gene order according to the gene order that is obtained and is made up of 120 amino acid shown in SEQ ID NO:2, and sequence is shown in SEQ ID NO:4.
(3) aspergillus flavus resisting toxin G1 MONOCLONAL ANTIBODIES SPECIFIC FOR purifying, hypotype and CHARACTERISTICS IDENTIFICATION
The aspergillus flavus resisting toxin G1 monoclonal antibody hybridoma cell strain that embodiment 2 is obtained is the BALB/c mouse that the 1C8 injection was handled with freund 's incomplete adjuvant in advance, collects the ascites of this mouse, adopts sad-ammonium sulfate method antibody purification, and concrete operations are: with double-deck filter paper filtering mouse ascites; 4 ℃, the centrifugal 15min of 12000r/min draws supernatant, and gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes; Stir the slow down n-caprylic acid that adds, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min; 4 ℃ leave standstill 2h, and 4 ℃ then, the centrifugal 30min of 12000r/min; Abandon deposition, with the supernatant that obtains with double-deck filter paper filtering after, the volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffered saline buffer; With the pH value to 7.4 that the sodium hydroxide solution of 2mol/L is regulated this mixed solution, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL; 4 ℃ leave standstill 2h, and 4 ℃ then, the centrifugal 30min of 12000r/min; Abandon supernatant, the gained deposition is resuspended with the 0.01mol/L phosphate buffered saline buffer of former ascites volume 1/10, the dialysis tubing of packing into; To the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, uses the freeze drier freeze-drying afterwards; Collect lyophilized powder, promptly get the good aspergillus flavus resisting toxin G1 monoclonal antibody of purifying, antibody is put in-20 ℃ of refrigerators subsequent use;
Described acetate buffer is the 0.29g sodium-acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is a 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g adds water and is settled to the 100mL gained.
Use commercially available hypotype identification kit to identify that the hypotype of hybridoma cell strain 1C8 excretory aspergillus flavus resisting toxin G1 monoclonal antibody is IgG 2a
The tiring of mouse ascites fluid antibody that records 1C8 with conventional non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method can reach 8.19 * 10 5, promptly the mouse ascites fluid antibody dilution 8.19 * 10 5Times the time measured in solution result positive.Identify its sensitivity (IC with conventional indirect competitive ELISA method to aflatoxin G 1 50) be 13.92ng/mL, with AFB1, B2 and M1 do not have cross reaction.
3. the preparation of the general surrogate of standard substance:
Mouse source aspergillus flavus resisting toxin B1 monoclonal antibody 3G1, aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 and aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 equimolar amount are mixed; And as hybrid antigen; Adopt ordinary method immunity nz screech owl White Rabbit; Carotid artery is got blood, and serum affinity purification, purified serum are the general surrogate of standard substance;
Described aspergillus flavus resisting toxin B1 monoclonal antibody 3G1 is the hybridoma cell strain 3G1 secretion generation of CCTCC NO. C201014 by deposit number; Described aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 is the hybridoma cell strain 2C9 secretion generation of CCTCC NO. C201018 by deposit number, and described aspergillus flavus resisting toxin G1 monoclonal antibody is to be the hybridoma cell strain 1C8 secretion generation of CCTCC NO. C201017 by deposit number.
Among the following embodiment 2-4:
The said damping fluid that encapsulates is: Na 2CO 31.59 g, NaHCO 32.93 g adds pure water and is settled to 1 L.
Said OVA is an oralbumin.
Said PBST solution is: KH 2PO 40.2 g, KCl 0.2 g, Na 2HPO 412H 2O 2.9 g, NaCl 8.0 g, 0.5mL Tween-20 adds pure water and is settled to 1 L.
Said colour developing liquid is that 9.5 mL substrate buffer solutions+0.5 mL substrate use liquid+32 μ L concentration are 3% ydrogen peroxide 50
Form.
Substrate buffer solution is: Na 2HPO 412H 2O 1.841 g, C 6H 7O 8H 2O 0.933 g is dissolved in the 100 mL ultrapure waters.
Substrate uses liquid to be: 2 mg TMB are dissolved in the 100 mL absolute ethyl alcohols.
The mensuration of AFB1 in embodiment 2 peanut samples
(1) foundation of the corresponding relation conversion equation of general surrogate concentration of standard substance and AFB1 standard substance concentration
1. AFB1 standard substance ELISA standard analysis curvilinear equation
At first with encapsulating damping fluid dilution AFB1 complete antigen (AFB 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, and 37 ° of C react 1 h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin B1 monoclonal anti liquid solutions, adds AFB1 standard substance (2.4 to 600 pg/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no AFB1 standard substance hole is B 0, inhibiting rate is B/B 0AFB1 standard substance elisa assay curve is seen Fig. 1 " ■-" part, gets AFB1 standard substance ELISA standard analysis curvilinear equation through non-linear logistic regression fit to be:
y 2=75.78/ [1+(x 2/25.25) 2.37]+15.89 equations (1)
Y wherein 2The expression inhibiting rate, x 2Expression AFB1 concentration.
2. the general surrogate ELISA of standard substance standard analysis curvilinear equation
With encapsulating damping fluid dilution AFB1 complete antigen (AFB 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, 37 ° of C reaction 1h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin B1 monoclonal anti liquid solutions, adds the general surrogate of 50 μ L standard substance (0.4 to 100 μ g/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in the general surrogate of no standard substance hole is B 0, inhibiting rate is B/B 0Substitute the AFB1 standard substance with the general surrogate of standard substance; The general surrogate ELISA of the standard substance that obtain standard analysis curve is seen " ■-" part among Fig. 2, gets the general surrogate ELISA of standard substance standard analysis curvilinear equation through non-linear logistic regression fit to be:
y 1=86.22/[1+(x 1/ 5.26) 1.23]+12.92 equations (2)
Y wherein 1The expression inhibiting rate, x 1The general surrogate concentration of expression standard substance.
3. the corresponding relation conversion equation of general surrogate concentration of standard substance and AFB1 standard substance concentration
According to equation (1) and equation (2), calculate same inhibiting rate pairing AFB1 standard substance concentration and the general surrogate concentration of standard substance in 20%～80% inhibiting rate scope, be spaced apart 5% inhibiting rate.Be X-coordinate with AFB1 concentration then; The general surrogate concentration of standard substance is the ordinate zou mapping; Obtain the corresponding relation curve of AFB1 concentration and the general surrogate concentration of standard substance under the same inhibiting rate; See Fig. 3, and obtain the corresponding relation equation of general surrogate concentration of standard substance and AFB1 standard substance concentration through non-linear logistic regression analysis match:
x 1=98.26/[1+(x 2/ 115.97) 1.89]+0.54 equation (3)
X wherein 1The general surrogate concentration of expression standard substance, x 2Expression AFB1 concentration.
(2) AFB in the peanut sample 1The mensuration of concentration
1. do under the typical curve inhibiting rate and AFB1 concentration curve equation at the general surrogate of standard substance
Obtain at the general surrogate of standard substance according to equation (2) and (3) and to do under the typical curve, the relation curve equation of inhibiting rate and AFB1 concentration is:
y 1=86.22/(1+((98.26/(1+(x 2/ 115.97) 1.89)+0.54)/5.26) 1.23)+12.92 equations (4)
Y wherein 1The expression inhibiting rate, x 2Expression AFB1 concentration.
2. AFB in the peanut sample 1The mensuration of concentration
Take by weighing 5 g peanut samples (being designated as 1#) and be dissolved in 15 mL, 80% methanol aqueous solution (containing 4% NaCl), whirlpool 30 s mixings are put in the ultrasonic oscillator, 50 ° of C water-baths, 15 min that vibrate.Get supernatant and cross the organic filter membrane of 0.45 μ m.Filtrating is with 10 times of PBS dilutions, gets 50 μ L on and kind to make test sample liquid.
With encapsulating damping fluid dilution AFB1 complete antigen (AFB 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, 37 ° of C reaction 1h.PBST washes plate three times, and every hole adds the anti-AFB of 50 μ L 1The monoclonal anti liquid solution adds 50 μ L peanut sample liquid again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no testing sample hole is B 0, calculate the inhibiting rate of sample.
In the inhibiting rate value substitution equation (4) with peanut sample 1#, try to achieve that AFB1 concentration is 15.6 μ g/kg among the peanut sample 1#; AFB1 concentration is 14.3 μ g/kg among the standard method NY/T 1286-2007 detection gained peanut sample 1# that the employing Ministry of Agriculture announces, the limit of error of two results' relative deviation conformance with standard regulation.
Take by weighing 5 g peanut samples 2 (being designated as 2#) and be dissolved in 15 mL, 80% methanol aqueous solution (containing 4% NaCl), whirlpool 30 s mixings are put in the ultrasonic oscillator, 50 ° of C water-baths, 15 min that vibrate.Get supernatant and cross the organic filter membrane of 0.45 μ m.Filtrating is with 10 times of PBS dilutions, gets 50 μ L on and kind to make test sample liquid.
Recording the concentration of trying to achieve AFB1 among this peanut sample 2# in inhibiting rate and the substitution equation (4) of peanut sample 2# according to the as above detection method of peanut sample 1# is 9.6 μ g/kg; AFB1 concentration is 8.7 μ g/kg among the standard method NY/T 1286-2007 detection gained peanut sample 2# that the employing Ministry of Agriculture announces, the limit of error of two results' relative deviation conformance with standard regulation.
The mensuration of aflatoxin G 1 concentration in embodiment 3 peanut samples
(1) foundation of the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin G 1 standard substance concentration
1. aflatoxin G 1 standard substance elisa assay curvilinear equation
At first with encapsulating damping fluid dilution aflatoxin G 1 complete antigen (AFG 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, and 37 ° of C react 1 h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin G1 monoclonal anti liquid solutions, adds aflatoxin G 1 standard substance (82.3 to 20000pg/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no aflatoxin G 1 standard substance hole is B 0, inhibiting rate is B/B 0Aflatoxin G 1 standard substance ELISA standard analysis curve is seen Fig. 1 " ▼-" part, gets aflatoxin G 1 standard substance ELISA standard analysis curvilinear equation through non-linear logistic regression fit to be:
y 2=79.52/ [1+(x 2/ 3150.46) 1.03]+18.59 equations (5)
Y wherein 2The expression inhibiting rate, x 2The expression aflatoxin G 1.
2. the general surrogate ELISA of standard substance standard analysis curvilinear equation
With encapsulating damping fluid dilution aflatoxin G 1 complete antigen (AFG 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, 37 ° of C reaction 1h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin G1 monoclonal anti liquid solutions, adds the general surrogate of 50 μ L standard substance ((0.4 to 100 μ g/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 M sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in the general surrogate of no standard substance hole is B 0, inhibiting rate is B/B 0The general surrogate ELISA of standard substance standard analysis curve with the alternative aflatoxin G 1 standard substance of the general surrogate of standard substance obtain is seen Fig. 2 " ▼-" part, gets the general surrogate ELISA of standard substance standard analysis curvilinear equation through non-linear logistic regression fit and is:
y 1=261.72/[1+(x 1/ 471.09) 0.37] 149.45 equations (6)
Y wherein 1The expression inhibiting rate, x 1The general surrogate concentration of expression standard substance.
3. the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin G 1 standard substance concentration
According to equation (5) and equation (6), pairing aflatoxin G 1 concentration of same inhibiting rate in 20%～80% inhibiting rate scope and the general surrogate concentration of standard substance are calculated again, be spaced apart 5% inhibiting rate.Be X-coordinate with aflatoxin G 1 concentration then; The general surrogate concentration of standard substance is the ordinate zou mapping; Obtain the corresponding relation curve of pairing aflatoxin G 1 concentration and the general surrogate concentration of standard substance under the same inhibiting rate; See Fig. 4, and obtain the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin G 1 standard substance concentration through non-linear logistic regression analysis match:
x 1=86.11/[1+(x 2/ 11200.22) 1.39] 0.43 equation (7)
X wherein 1The general surrogate concentration of expression standard substance, x 2Expression aflatoxin G 1 concentration.
(2) mensuration of AFG1 concentration in the peanut sample
1. do under the typical curve inhibiting rate and aflatoxin G 1 concentration curve equation at the general surrogate of standard substance
Obtain at the general surrogate of standard substance according to equation (6) and (7) and to do under the typical curve, the relation curve equation of inhibiting rate and aflatoxin G 1 concentration is:
y 1=261.72/ (1+((86.11/ (1+(x 2/ 11200.22) 1.39) 0.43)/471.09) 0.37) 149.45 equations (8)
Y wherein 1The expression inhibiting rate, x 2Expression aflatoxin G 1 concentration.
2. AFG in the peanut sample 1The mensuration of concentration
Take by weighing 5 g peanut samples (note is made 1#) and be dissolved in 15 mL, 80% methanol aqueous solution (containing 4% NaCl), whirlpool 30 s mixings are put in the ultrasonic oscillator, 50 ° of C water-baths, 15 min that vibrate.Get supernatant and cross the organic filter membrane of 0.45 μ m.Filtrating is got appearance do detection liquid on the 50 μ L with 10 times of PBS dilutions.
With encapsulating damping fluid dilution aflatoxin G 1 complete antigen (AFG 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, 37 ° of C reaction 1h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin G1 monoclonal anti liquid solutions, adds 50 μ L peanut sample liquid again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no testing sample hole is B 0, inhibiting rate is B/B 0, calculate the inhibiting rate of sample.
In the inhibiting rate value substitution equation (8) with peanut sample 1#, the concentration of obtaining aflatoxin G 1 among the peanut sample 1# is 4.2 μ g/kg; The concentration that the standard method SN 0637-1997 that adopts State General Administration for Quality Supervision to announce measures aflatoxin G 1 among the peanut sample 1# is 3.9 μ g/kg, the limit of error of two results' relative deviation conformance with standard regulation.
Take by weighing 5 g peanut samples (note is made 2#) and be dissolved in 15 mL, 80% methanol aqueous solution (containing 4% NaCl), whirlpool 30 s mixings are put in the ultrasonic oscillator, 50 ° of C water-baths, 15 min that vibrate.Get supernatant and cross the organic filter membrane of 0.45 μ m.Filtrating is got appearance do detection liquid on the 50 μ L with 10 times of PBS dilutions.
Record according to the as above detection method of peanut sample 1# in inhibiting rate and the substitution equation (8) of peanut sample 2# and calculate; The aflatoxin G 1 concentration of finally trying to achieve among this peanut sample 2# is 1.2 μ g/kg; The concentration that the standard method SN 0637-1997 that adopts State General Administration for Quality Supervision to announce measures aflatoxin G 1 among the peanut sample 2# is 1.1 μ g/kg, the limit of error of two results' relative deviation conformance with standard regulation.
The mensuration of aflatoxin M 1 concentration in embodiment 4 milk samples
(1) foundation of the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin M 1 standard substance concentration
1. aflatoxin M 1 standard substance ELISA standard analysis curvilinear equation
At first with encapsulating damping fluid dilution aflatoxin M 1 complete antigen (AFM 1-OVA, 0.06 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, and 37 ° of C react 1 h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin M1 monoclonal anti liquid solutions, adds aflatoxin M 1 standard substance (4.1 to 1000 pg/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no aflatoxin M 1 standard substance hole is B 0, inhibiting rate is B/B 0Aflatoxin M 1 standard substance ELISA standard analysis curve is seen Fig. 1 " ●-" part, gets aflatoxin M 1 standard substance ELISA standard analysis curvilinear equation through non-linear logistic regression fit and is:
y 2=78.84/ [1+(x 2/ 121.97) 1.19]+12.27 equations (9)
Y wherein 2The expression inhibiting rate, x 2Expression aflatoxin M 1 concentration.
2. the general surrogate ELISA of standard substance standard analysis curvilinear equation
With encapsulating damping fluid dilution aflatoxin M 1 complete antigen (AFM 1-OVA, 0.06 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, and 37 ° of C react 1 h.PBST washes plate three times, and every hole adds 50 μ l aspergillus flavus resisting toxin M1 monoclonal anti liquid solutions, adds the general surrogate of 50 μ L standard substance ((0.4 to 100 μ g/mL) again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in the general surrogate of no standard substance hole is B 0, inhibiting rate is B/B 0The elisa assay curve that substitutes aflatoxin M 1 standard substance with the general surrogate of standard substance is seen Fig. 2 " ●-" part, gets the general surrogate ELISA of standard substance standard analysis curvilinear equation through non-linear logistic regression fit and is:
y 1=133.42/[1+(x 1/ 61.81) 0.80] 30.98 equations (10)
Y wherein 1The expression inhibiting rate, x 1The general surrogate concentration of expression standard substance.
3. the foundation of the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin M 1 standard substance concentration
According to equation (9) and equation (10), pairing aflatoxin M 1 concentration of same inhibiting rate in 20%～80% inhibiting rate scope and the general surrogate densitometer of standard substance are calculated, be spaced apart 5% inhibiting rate.With aflatoxin M 1 concentration is X-coordinate; The general surrogate concentration of standard substance is the ordinate zou mapping; Obtain the corresponding relation curve of pairing aflatoxin M 1 concentration and the general surrogate concentration of standard substance under the same inhibiting rate; See Fig. 4, and obtain the corresponding relation conversion equation of general surrogate concentration of standard substance and aflatoxin M 1 standard substance concentration through non-linear logistic regression fit:
x 1=146.41/[1+(x 2/ 340.89) 1.30]+3.22 equations (11)
X wherein 1The general surrogate concentration of expression standard substance, x 2Expression AFM 1Concentration.
(2) AFM in the milk sample 1The mensuration of concentration
1. do under the typical curve inhibiting rate and aflatoxin M 1 concentration curve equation at the general surrogate of standard substance
Obtain at the general surrogate of standard substance according to equation (10) and (11) and to do under the typical curve, the relation curve equation of inhibiting rate and aflatoxin M 1 concentration is:
y 1=133.42/ (1+((146.41/ (1+(x 2/ 340.89) 1.30)+3.22)/61.81) 0.80) 30.98 equations (12)
Y wherein 1The expression inhibiting rate, x 2Expression AFM 1Concentration.
2. AFM in the milk sample 1The mensuration of concentration
Get 20 mL milk samples (note is made 1#) and add in the 50 mL centrifuge tubes, centrifugal 15 min remove the upper strata oil layer under 3500 g, cast out the lower sediment thing, get the middle layer, get appearance detection on the 50 μ L.
With encapsulating damping fluid dilution aflatoxin M 1Complete antigen (AFM 1-OVA, 2 μ g/mL), add in the enzyme plate, every hole 100 μ L, 4 ° of C spend the night.After PBST solution was washed plate three times, every hole added 200 μ L, the 1.5% OVA aqueous solution as confining liquid, and 37 ° of C react 1 h.PBST washes plate three times, and every hole adds 50 μ L aspergillus flavus resisting toxin M 1The monoclonal anti liquid solution adds 50 μ L milk sample liquid again.After 37 ° of C reacted 1 h, PBST washed plate three times, and every hole adds 100 μ L commercialization sheep anti mouse ELIAS secondary antibodies, and 37 ° of C react 1 h.PBST washes plate six times, adds colour developing liquid and reacts 15 min.Add 50 μ L, 2 mol/L sulfuric acid termination reactions.Under 450 nm, measure absorbance value, treat that the gaging hole absorbance value is B, the absorbance value in no testing sample hole is B 0, inhibiting rate is B/B 0, calculate the inhibiting rate of sample.
In the inhibiting rate value substitution equation (12) with milk sample 1#, the concentration of obtaining aflatoxin M 1 among the milk sample 1# is 0.92 μ g/kg The concentration that adopts standard GB/T 23212-2008 to record aflatoxin M 1 among the milk sample 1# is 0.95 μ g/kg, the limit of error of two results' relative deviation conformance with standard regulation.
Get 20 mL milk samples (note is made 2#) and add in the 50 mL centrifuge tubes, centrifugal 15 min remove the upper strata oil layer under 3500 g, cast out the lower sediment thing, get the middle layer, get appearance detection on the 50 μ L.
Record according to the as above detection method of milk sample 1# in inhibiting rate and the substitution equation (12) of milk sample 2# and calculate, aflatoxin M 1 concentration of finally trying to achieve in this milk sample is 0.37 μ g/kg; The concentration that adopts standard GB/T 23212-2008 to record aflatoxin M 1 among the milk sample 2# is 0.34 μ g/kg, the limit of error of two results' relative deviation conformance with standard regulation.
The foregoing description 2-4 all is the experiment first of AFB1, aflatoxin G 1, aflatoxin M 1 in the corresponding mensuration testing sample.After comprising that in subsequent detection placement is long-time, invariable because of the corresponding relation conversion equation of the general surrogate concentration of the corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each, need not to rebulid.So concrete steps can directly be reduced to: set up the general surrogate ELISA of each corresponding Toxins, afla standard substance standard analysis curvilinear equation; The corresponding relation conversion equation of the general surrogate concentration of the corresponding then combination corresponding Toxins, afla standard substance with each of each corresponding Toxins, afla standard substance concentration is (as being measured as example with AFB1 in the sample; The square journey of corresponding relation conversion equation (3) of this AFB1 standard substance concentration and the general surrogate concentration of standard substance then); Obtain: do under the ELISA standard analysis curve curvilinear equation e (y of the corresponding Toxins, afla concentration of inhibiting rate at the general surrogate of standard substance with each 1, x 2), measure the inhibiting rate value of testing sample then, the above-mentioned Equation for Calculating of corresponding substitution gets final product.
Sequence table
＜110＞Inst. of Oil Crops, Chinese Academy of Agriculture
＜120＞be used for the general surrogate of standard substance, its preparation method and Toxins, afla ELISA detection method that ELISA detects Toxins, afla
＜160＞ 8
＜210＞ 1
＜211＞ 347bp
＜212＞ DNA
＜213＞mouse
＜400＞ 1
tgaggagacg gtgaccgtgg tcccttggcc ccagtagtcc atagcccagt 50
aggccgatct tgcacagtaa tatgtggctg tgtcctcagt agtcacagaa 100
tttaactgca ggtagtactg gttcttggat gtgtctcgag tgatggagat 150
tcgactcttg agagatggat tgtagtaagg gttaccactg tagctcatga 200
accccatgta ctcaagttta ttcccaggga atttccggat ccagtgccag 250
taatcactgg tgatggagtc gccagtgaca gaacaggtga gggacagagt 300
ctgagaaggt ttcacgaggc taggtcctga ctcctgcagc tgcacct 347
＜210＞ 2
＜211＞ 338bp
＜212＞ DNA
＜213＞mouse
＜400＞ 2
gacattgagc tcacccagtc tccaaaattc atgtccacat cagtaggaga 50
cagggtcagc atctcctgca aggccagtca ggatgtgggt actgatgtag 100
cctggtacgg agtccctgat cgcctcacag gcagtggatc tgggacagat 150
ttcactctca ccattagcaa tgtgcagtct gaagacttgg cagattattt 200
ctgtcaacaa tatagcagct atattcacgt tcggctcggg gacaaagttg 250
gaaataaaac gtttttgtgg agaggggcat gtcatagtcc tcactgtgtc 300
tcacgttcgg tgctgggacc aagctggagc tgaaacgg 338
＜210＞ 3
＜211＞ 115
＜212＞ PRT
＜213＞mouse
＜400＞ 3
Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
1 5 10 15 20
Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asp Tyr Trp His Trp Ile Arg Lys Phe Pro
25 30 35 40
Gly Asn Lys Leu Glu Tyr Met Gly Phe Met Ser Tyr Ser Gly Asn Pro Tyr Tyr Asn Pro
45 50 55 60
Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu Gln
65 70 75 80
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Ala Tyr Trp
85 90 95 100
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
105 110 115
＜210＞ 4
＜211＞ 110
＜212＞ PRT
＜213＞mouse
＜400＞ 4
Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser
1 5 10 15 20
Ile Ser Cys Lys Ala Ser Gln Asp Val Gly Thr Asp Val Ala Trp Tyr Gly Val Pro Asp
25 30 35 40
Arg Leu Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
45 50 55 60
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Ile His Val Arg Leu Gly
65 70 75 80
Asp Lys Val Gly Asn Lys Thr Phe Leu Trp Arg Gly Ala Cys His Ser Pro His Cys Val
85 90 95 100
Ser Arg Ser Val Leu Gly Pro Ser Trp Ser
105 110
＜210＞ 5
＜211＞ 332bp
＜212＞ DNA
＜213＞mouse
＜400＞ 5
ccgttttatt tccagcttgg tcccccctcc gaacgtgtaa gctccctaat 50
gtgctgacag taataggttg cagcatcctc ctcctccaca ggatggatgt 100
tgagggtgaa gtctgtccca gacccactgc cactgaacct ggcagggacc 150
ccagattcta ggttggatac aagatagatg aggagtctgg gtggctgtcc 200
tggtttctgt tggttccagt gcatataact atagccagat gtactgacac 250
ttttgctggc cctgtatgag atggcggccc tctgccccag agatacagct 300
aaggaagctg gagactgggt gagctcaatg tc 332
＜210＞ 6
＜211＞ 362bp
＜212＞ DNA
＜213＞mouse
＜400＞ 6
aggtgcagct gcagcagtca ggacctgagc tggtgaggcc tgggacttca 50
gtgaagatat cctacaaggc ttctggctgt accttcctca cctactggat 100
gagctgggtg aagttgaggc ctgcacaggg ccttgagtgg attggacaga 150
tttttcctgc aggtggtagt gctaacttca atgagatttt cagggtcaag 200
gccacattga ctgtaggcac atcctccagt tcagcctaca tgcagctaat 250
cagcctgaca tctgaggact ctgcggtcaa tttctgtgca agagaggagg 300
gcctattact atcttatggt agggacttct ggggccaagg gaccacggtc 350
accgtctcct ca 362
＜210＞ 7
＜211＞ 110
＜212＞ PRT
＜213＞mouse
＜400＞ 7
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Ala
1 5 10 15 20
Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Thr Ser Tyr Met His Trp Asn
25 30 35 40
Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
45 50 55 60
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ale Ale Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg
85 90 95 100
Ser Glu Gly Gly Pro Ser Trp Lys Leu Asn
105 110
＜210＞ 8
＜211＞ 120
＜212＞ PRT
＜213＞mouse
＜400＞ 8
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser
1 5 10 15 20
Tyr Lys Ala Ser Gly Cys Thr Phe Leu Thr Tyr Trp Met Ser Trp Val Lys Leu Arg Pro
25 30 35 40
Ala Gln Gly Leu Glu Trp Ile Gly Gln Ile Phe Pro Ala Gly Gly Ser Ala Asn Phe Asn
45 50 55 60
Glu Ile Phe Arg Val Lys Ala Thr Leu Thr Val Gly Thr Ser Ser Ser Ser Ala Tyr Met
65 70 75 80
Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Asn Phe Cys Ala Arg Glu Glu Gly
85 90 95 100
Leu Leu Leu Ser Tyr Gly Arg Asp Phe Asn Trp Gly Gln Gly Thr Thr Val Thr Val Ser
105 110 115 120

Claims (11)

1. be used for the general surrogate of standard substance that ELISA detects Toxins, afla, it is characterized in that: it is for discerning the rabbit anti-mouse antibody of each mouse source aspergillus flavus resisting toxin monoclone antibody.
2. the general surrogate of standard substance that is used for ELISA detection Toxins, afla according to claim 1, it is characterized in that: said aspergillus flavus resisting toxin monoclone antibody is aspergillus flavus resisting toxin B1 monoclonal antibody, aspergillus flavus resisting toxin M1 monoclonal antibody and aspergillus flavus resisting toxin G1 monoclonal antibody.
3. the preparation method who is used for the general surrogate of standard substance of ELISA detection Toxins, afla according to claim 1 and 2; It is characterized in that: it is as hybrid antigen with mouse source aspergillus flavus resisting toxin B1 monoclonal antibody, aspergillus flavus resisting toxin M1 monoclonal antibody and aspergillus flavus resisting toxin G1 monoclonal antibody; Through immune nz screech owl White Rabbit; Carotid artery is got blood, obtains behind the serum affinity purification.
4. the preparation method who is used for the general surrogate of standard substance of ELISA detection Toxins, afla according to claim 3 is characterized in that: described aspergillus flavus resisting toxin B1 monoclonal antibody is for by deposit number being the aspergillus flavus resisting toxin B1 monoclonal antibody 3G1 of the hybridoma cell strain 3G1 secretion generation of CCTCC NO. C201014; Described aspergillus flavus resisting toxin M1 monoclonal antibody is to be the aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 of the hybridoma cell strain 2C9 secretion generation of CCTCC NO. C201018 by deposit number, and described aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 is for by deposit number being the aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 of the hybridoma cell strain 1C8 secretion generation of CCTCC NO. C201017; Said aspergillus flavus resisting toxin B1 monoclonal antibody 3G1, aspergillus flavus resisting toxin M1 monoclonal antibody 2C9 and aspergillus flavus resisting toxin G1 monoclonal antibody 1C8 make hybrid antigen for the equimolar amount proportioning.
5. be used for ELISA and detect of the application of the general surrogate of standard substance of Toxins, afla in Toxins, afla ELISA detection.
6. Toxins, afla ELISA detection method, it is characterized in that: it may further comprise the steps:
(1) adopts conventional indirect competitive ELISA method, the general surrogate of standard substance is replaced each Toxins, afla standard substance, set up the general surrogate ELISA of each corresponding Toxins, afla standard substance standard analysis curvilinear equation a (x respectively 1, y 1),
---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate;
(2) adopt conventional indirect competitive ELISA method, measure the inhibiting rate of testing sample;
In the curvilinear equation of the corresponding substitution step of testing sample inhibiting rate (1) that (3) step (2) is obtained, the corresponding relation conversion equation b (x of the general surrogate concentration of the concentration results substitution of the general surrogate of the corresponding Toxins, afla standard substance of each that will calculate the then corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, calculate the concentration of each corresponding Toxins, afla in the testing sample.
7. Toxins, afla ELISA detection method according to claim 6 is characterized in that: said step (3) is: the general surrogate elisa assay of each corresponding Toxins, afla standard substance of corresponding combination curvilinear equation a (x 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of corresponding Toxins, afla standard substance concentration with each with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, obtain: the curvilinear equation e (y that does the corresponding Toxins, afla concentration of inhibiting rate under the ELISA standard analysis curve at the general surrogate of standard substance with each 1, x 2),---y 1Be inhibiting rate, x 2Be each corresponding Toxins, afla concentration;
The above-mentioned curvilinear equation e of the testing sample inhibiting rate substitution (y that step (2) is obtained 1, x 2), calculate the concentration of each corresponding Toxins, afla in the testing sample.
8. according to claim 6 or 7 described Toxins, afla ELISA detection methods, it is characterized in that: the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of the corresponding Toxins, afla standard substance concentration of each in the said step (3) with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance---, adopt following method to obtain:
1. adopt conventional indirect competitive ELISA method to set up the ELISA standard analysis curvilinear equation c (x of each corresponding Toxins, afla standard substance respectively 2, y 2),---x 2Be the concentration of each corresponding Toxins, afla standard substance, y 2Be inhibiting rate;
2. adopt conventional indirect competitive ELISA method, the general surrogate of standard substance is substituted each corresponding Toxins, afla standard substance, set up the general surrogate ELISA of each corresponding Toxins, afla standard substance standard analysis curvilinear equation d (x respectively 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate;
3. each corresponding Toxins, afla standard substance analytic curve equation c (x of corresponding combination 2, y 2) the general surrogate elisa assay of corresponding Toxins, afla standard substance with each curvilinear equation d (x 1, y 1), obtain the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance.
9. Toxins, afla ELISA detection method according to claim 8, it is characterized in that: 3. said step is: each corresponding Toxins, afla standard substance analytic curve equation c (x of corresponding combination 2, y 2),---x 2Be the concentration of each corresponding Toxins, afla standard substance, y 2Be inhibiting rate---the general surrogate elisa assay of corresponding Toxins, afla standard substance with each curvilinear equation d (x 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---, the general surrogate concentration of the pairing corresponding Toxins, afla standard substance with each of each corresponding Toxins, afla standard substance concentration of same inhibiting rate in the inhibiting rate scope of calculating 20%～80% is then with each corresponding Toxins, afla standard substance concentration x 2Be X-coordinate, the general surrogate concentration of each corresponding Toxins, afla standard substance x 1Be ordinate zou, map match respectively and obtain the corresponding relation conversion equation b (x of the general surrogate concentration of the corresponding Toxins, afla standard substance of each corresponding Toxins, afla standard substance concentration with each 1, x 2),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, x 2Concentration for each corresponding Toxins, afla standard substance.
10. according to claim 6 or 7 described Toxins, afla ELISA detection methods, it is characterized in that: the measuring method of inhibiting rate is following in the described conventional indirect competitive ELISA method: get the micropore that encapsulates the corresponding Toxins, afla complete antigen of each Toxins, afla and encapsulate plate, add the corresponding aspergillus flavus resisting toxin monoclone antibody of each Toxins, afla; Add testing sample or each corresponding Toxins, afla standard substance or the general surrogate of standard substance then, after the reaction, the washings washing; It is anti-to add enzyme labelling sheep anti mouse two; Carry out the labelled immune reaction, after the washings washing, add colour developing liquid; After leaving standstill, the dark place adds stop buffer; Under 450 nm, measure absorbance value, according to treating that the gaging hole absorbance value is B, the absorbance value of control wells is B 0, the calculating inhibiting rate is B/B 0
11., it is characterized in that: the general surrogate ELISA of said each corresponding Toxins, afla standard substance standard analysis curvilinear equation d (x according to claim 6 or 7 described Toxins, afla ELISA detection methods 1, y 1),---x 1Be the concentration of the general surrogate of each corresponding Toxins, afla standard substance, y 1Be inhibiting rate---foundation in, the concentration laying scope of the general surrogate of each corresponding Toxins, afla standard substance is 0.4-100 μ g/mL.
CN2012101176172A 2012-04-20 2012-04-20 Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin Pending CN102746403A (en)

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CN113831396A (en) * 2021-09-07 2021-12-24 中国农业科学院油料作物研究所 Molecule for indicating aflatoxin toxigenicity of toxigenic bacteria and application thereof

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WO2013155883A1 (en) * 2012-04-20 2013-10-24 中国农业科学院油料作物研究所 Hybridoma cell line 3g1 and monoclonal antibody produced thereby against aflatoxin b1
CN103792359A (en) * 2012-11-05 2014-05-14 江苏维赛科技生物发展有限公司 Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit
CN103091494A (en) * 2013-01-14 2013-05-08 华南农业大学 Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method
CN103091494B (en) * 2013-01-14 2015-07-29 华南农业大学 Aflatoxin M 1chemical luminescence ELISA detection kit and using method
CN104569380A (en) * 2015-01-23 2015-04-29 天津伯克生物科技有限公司 Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit
CN105624119B (en) * 2016-02-04 2019-10-15 中国农业科学院油料作物研究所 The general monoclonal antibody of anti-capsaicine, Dihydrocapsaicin, the synthetic capsaicin of hybridoma cell strain YQQD8 and its generation
CN105624119A (en) * 2016-02-04 2016-06-01 中国农业科学院油料作物研究所 Hybridoma cell line YQQD8 and general monoclonal antibodies produced by same to capsaicine, dihydrocapsaicin and nonivamide
CN113061188A (en) * 2021-03-26 2021-07-02 中国农业科学院油料作物研究所 AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M1ELISA immunoassay method for standard substance
CN113061188B (en) * 2021-03-26 2022-06-17 中国农业科学院油料作物研究所 AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M1ELISA immunoassay method for standard substance
CN113607949A (en) * 2021-05-28 2021-11-05 中国农业科学院油料作物研究所 Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland
CN113607949B (en) * 2021-05-28 2023-06-27 中国农业科学院油料作物研究所 Method for rapidly identifying and comparing relative abundance of toxigenic fungi of farmland aflatoxins
CN113831396A (en) * 2021-09-07 2021-12-24 中国农业科学院油料作物研究所 Molecule for indicating aflatoxin toxigenicity of toxigenic bacteria and application thereof
CN113831396B (en) * 2021-09-07 2023-12-19 中国农业科学院油料作物研究所 Molecule for indicating toxicity of aflatoxin-producing bacteria and application thereof

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