CN109400682B - cyclic polypeptide simulating aflatoxin B1 epitope and detection kit thereof - Google Patents
cyclic polypeptide simulating aflatoxin B1 epitope and detection kit thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
Abstract
The invention discloses a cyclic polypeptide simulating aflatoxin B1 epitope and a detection kit thereof, wherein the amino acid sequence of the cyclic polypeptide is CVPSKPGLC, and is shown as SEQ ID No. 1. The kit comprises AFB1 competitive antigen, an enzyme label plate embedded with an anti-AFB 1 monoclonal antibody, standard solution of aflatoxin B1, horseradish peroxidase labeled streptavidin and the like; the polypeptide sequence can simulate the epitope of AFB1, can be specifically combined with a screened antibody, has high sensitivity and strong specificity, reduces the exposure of AFB1 to operators, and is a safer and more efficient detection method. The method has important practical significance for monitoring pollution of AFB1 in agricultural products.
Description
Technical Field
The invention relates to the technical field of immunoassay chemistry, in particular to a cyclic polypeptide simulating aflatoxin B1 epitope and a detection kit thereof.
background
Aflatoxins (AFTs) are a class of highly toxic secondary metabolites produced primarily by aspergillus flavus and aspergillus parasiticus. AFTs are largely classified into groups B, G and M. The group B AFTs can emit blue fluorescence under 365nm ultraviolet excitation, and can be further divided into Aflatoxin B1(AFB1) and AFB 2. Among them, AFB1 is the most toxic and is defined by the world health organization cancer research institute as a class I carcinogen, i.e., a substance that has confirmed carcinogenicity in humans. The hot and humid environment, improper harvesting and storage can result in the production of AFB1, which mainly contaminates rice, peanuts, corn, grain and oil products, etc.
most of the conventional immunoassay methods for detecting aflatoxin B1 are competitive assays. First, preparation of competitive antigen: carboxyl is introduced to AFB1 molecules through a succinimide ester method to generate aflatoxin B1 oxime, the aflatoxin B1 oxime is coupled with carrier protein (bovine serum albumin or ovalbumin) through an active ester method, the epitope exposed by the conjugate can compete with AFB1 for anti-AFB 1 antibody, and the conjugate is often coated on an ELISA plate to be used as a coating antigen. However, in the process of preparation and application of the coating antigen, operators can be exposed in the AFB1 environment, professional equipment such as high temperature and high pressure is needed when aflatoxin B1 oxime is prepared, certain potential hazard is caused, and the competitive antigen is high in preparation cost and large in batch influence.
Therefore, there is an urgent need to prepare and develop a safer and more efficient rapid immunoassay kit and assay method.
Disclosure of Invention
the invention aims to overcome the defects of the prior art and provides a cyclic polypeptide simulating the aflatoxin B1(AFB1) epitope and a detection kit thereof, wherein the kit has the characteristics of high sensitivity, high specificity, simple operation method, safety and high efficiency; it can be used for the rapid screening and monitoring of AFB1 in agricultural products.
in order to achieve the purpose, the invention designs a cyclic polypeptide simulating aflatoxin B1 epitope, which is characterized in that: the amino acid sequence is CVPSKPGLC, as shown in SEQ ID No. 1.
One of the preparation methods of the cyclic polypeptide simulating the aflatoxin B1 epitope is as follows:
1) The method comprises the steps of purifying an anti-AFB 1 monoclonal antibody, coating the monoclonal antibody on an enzyme label plate, adding a commercial phage display cyclic heptapeptide library Ph.D. -C7C (the peptide library is purchased from New England Biolabs, and the peptide library is displayed on PIII protein of filamentous phage M13), then competitively eluting with AFB1 to obtain phage display polypeptide with specificity simulating AFB1 epitope, determining the DNA sequence of the phage display polypeptide, translating the polypeptide into amino acid sequence, and chemically synthesizing the polypeptide sequence, wherein the sequence is shown as SEQ ID No. 1.
2) The amino acid sequence is CVPSKPGLC which is obtained by artificial synthesis according to the sequence shown in SEQ ID No. 1.
The invention discloses a kit for detecting aflatoxin B1, which comprises the following components:
(1) AFB1 competes for an antigen, and the AFB1 competes for the antigen as a biotin-labeled cyclic polypeptide of claim 1;
(2) ELISA plate embedded with anti-AFB 1 monoclonal antibody
(3) standard solutions of aflatoxin B1;
(4) horse radish peroxidase is marked with streptavidin, namely an enzyme-labeled secondary antibody;
(5) Washing the buffer solution;
(6) A sample diluent;
(7) Developing solution A;
(8) Developing B liquid;
(9)2M sulfuric acid solution.
further, the AFB1 competitive antigen is formed by coupling a cyclic polypeptide for detecting aflatoxin AFB1 and biotin. Still further, the formula of the washing buffer PBST is as follows: 40g of sodium chloride, 8g of monopotassium phosphate, 29g of disodium phosphate, 8g of potassium chloride, 202.5 mL of Tween-and 20mL of distilled water.
the standard solution concentration of the aflatoxin B1 comprises 30, 10, 2, 1, 0.2 and 0.02 ng/mL;
The sample diluent is: the phosphate buffer solution containing 10% methanol by volume comprises 8g of sodium chloride, 0.2g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate and 0.2g of potassium chloride.
The color developing solution A comprises 1g of carbamide peroxide, 10.3g of citric acid, Na 2 HPO 4.12H 2 O35.8g, Tween-20100 mu L, 1000mL of distilled water and pH 5;
the color developing liquid B: tetramethylbenzidine (TMB)300mg (dissolved in 50mL DMSO), 10.3g citric acid, 1000mL distilled water, pH 2.4-2.6.
The invention also discloses a competitive immunoassay method for detecting aflatoxin B1 by using the kit, which is characterized by comprising the following steps: the method comprises the following steps:
1) After mixing the AFB1 competitive antigen with a sample to be detected in an equal volume, adding the mixture into an ELISA plate embedded with an anti-AFB 1 monoclonal antibody;
2) Washing the ELISA plate by using a washing buffer solution, adding horseradish peroxidase labeled streptavidin (enzyme-labeled secondary antibody), washing the ELISA plate by using the washing buffer solution, adding a mixed solution of a developing solution A and a developing solution B for developing, and then adding 2M sulfuric acid solution for stopping;
3) And (3) measuring the OD value of the hole to be measured by using an enzyme-labeling instrument at the wavelength of 450nm, establishing a standard curve by using a standard solution of the aflatoxin B1, and correspondingly converting according to the established standard curve and the OD value of the hole to be measured to obtain the content of the aflatoxin B1 in the sample to be measured.
The invention also provides application of the kit for detecting aflatoxin B1 in checking the content of AFB1 in rice.
The principle of the invention is as follows:
The principle of the competitive immunoassay method for detecting AFB1 based on the biotin-labeled cyclic polypeptide is as follows: each hole on the enzyme label plate is coated with anti-AFB 1 antibody with the same amount, a mixture of the cyclic polypeptide labeled by equal volume of biotin and AFB1 standard substance or sample to be detected is added, the cyclic polypeptide labeled by biotin and AFB1 to be detected compete with each other to react with the antibody, because the content of the coating antibody in each hole is consistent with that of the cyclic polypeptide labeled by added biotin, when the concentration of AFB1 to be detected is high, the cyclic polypeptide labeled by biotin bound on the solid-phase coating antibody is less, the binding amount of the added enzyme-labeled secondary antibody and the cyclic polypeptide labeled by fixed biotin is less, a substrate color developing solution is added after washing by using a washing solution, the color developing reaction is light, the value detected by an enzyme label instrument is low, and the OD (OD) value is high, so that the inhibition rate is high; on the contrary, when the concentration of the AFB1 to be detected is low, the detected OD value is high, and the inhibition rate is low. According to a standard curve made by using the known AFB1 standard substance concentration detection, the concentration of AFB1 in a sample to be detected can be calculated.
the polypeptide is used as an element competing with AFB1, so that the epitope bound by AFB1 and an antibody can be simulated, the detection signal can be amplified by means of the high affinity of biotin and streptavidin, and a safe and non-toxic competitive immunoassay method of AFB1 is established. Therefore, the biotin-labeled cyclic polypeptide is a very safe and efficient AFB1 competitive antigen.
The sequence is CVPSKPGLC, the sequence can effectively simulate the binding epitope of AFB1 and an antibody, can be combined with an anti-AFB 1 monoclonal antibody, and the rice sample is added with quantitative AFB1, is measured by an established immunoassay method after simple pretreatment, and is verified by the method.
The invention has the beneficial effects that:
1. the general immunoassay method for detecting AFB1 in agricultural products is an indirect immunoassay method, generally needs to connect AFB1 after being processed with carrier protein to be used as competitive antigen, and operators in the process can be exposed in the environment of AFB1 and have potential hazard.
The invention obtains a polypeptide with specificity simulating AFB1 epitope and sequence CVPSKPGLC by screening, the polypeptide can be combined with an anti-AFB 1 monoclonal antibody, the polypeptide is marked by biotin, the traditional competitive antigen is successfully replaced, and the established method is a safe and nontoxic immunoassay method for detecting AFB 1.
2. the biotin-labeled cyclic polypeptide is used as a competitive antigen, a conjugate of streptavidin and horseradish peroxidase is used as an enzyme-labeled secondary antibody, the sensitivity of the detection method can be obviously improved, the IC 50 of the method established by using the biotin-labeled cyclic polypeptide as the competitive antigen is 0.92ng/mL, and the content of AFB1 in the rice sample can be accurately measured.
3. the kit to be protected in the invention comprises reagents such as biotin-labeled cyclic polypeptide, anti-AFB 1 monoclonal antibody, enzyme-labeled secondary antibody and the like, can meet the detection requirements of agricultural product samples, and is a safe, simple and portable detection method of AFB 1.
in summary, the following steps: the invention relates to screening of phage polypeptide of specificity competition AFB1, amino acid sequence of the polypeptide, coupling of the polypeptide and biotin, and establishment of an immunoassay method,
The invention uses the phage-derived cyclic polypeptide labeled by biotin as a competitive antigen to detect the content of AFB1 in agricultural products for the first time. The method replaces the preparation and the use of the traditional AFB1 coated antigen, and greatly reduces the exposure of AFB1 to operators, so the method is an efficient, safe and sensitive determination method.
drawings
FIG. 1 is a schematic representation of a cyclic polypeptide derived from a phage display system;
FIG. 2 is a schematic diagram showing the identification of positive plaques by the Phage ELISA method,
FIG. 3 is a standard curve established for a standard solution of aflatoxin B1 in the kit.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
The following experimental samples were purchased from the market, and some commercial descriptions were purchased from:
Example 1 specific panning and amplification of phage display Polypeptides
Panning of phage display specific polypeptides
Coating 5 holes on a 96-hole enzyme label plate, wherein the 1 st hole is 100 mu g/mL antibody, the rest 4 holes are coated with 3% BSA, the mixture is coated overnight at 4 ℃, and 200 mu L of 0.05% PBST is added for washing the plate for 3 times the next day; blocking the ELISA plate by 1% BSA, incubating for 2h at 25 ℃, and washing the plate for 3 times; adding 1 mu L of phage display cyclic heptapeptide library into a hole 1, incubating for 1h, washing the plate, adding 500ppb AFB1 for competitive elution, incubating for 1h at 25 ℃, transferring the eluate into holes 2, 3, 4 and 5, incubating for 1h, collecting supernatant, and determining the titer of phage in the eluate by using a top-layer plate method. The antibody concentrations in the second and third rounds of panning were reduced by half (50. mu.g/mL, 25. mu.g/mL) in sequence, using 0.1% and 0.15% Tween-20 in the PBST wash solution, respectively.
amplification of eluted phage
Inoculating escherichia coli E.coli ER2738 bacterial liquid into 20mL LB liquid culture medium, adding the eluted phage when OD 600nm is 0.6-1.0, shaking and culturing at 37 ℃ for 4.5h, transferring the bacterial liquid into a 50mL centrifuge tube, centrifuging at 4 ℃ and 8000rpm for 10min, adding 1/6 volume of 20% PEG/NaCl into the supernatant to precipitate phage, standing overnight at 4 ℃, centrifuging at 4 ℃ and 10000rpm for 15min, dissolving the precipitate with 1mL of PBS solution, centrifuging at 4 ℃ and 10000rpm for 5min, taking the supernatant, adding 1/6 volume of 20% PEG/2.5M NaCl into the supernatant to precipitate again, incubating on ice for 15-60 min, centrifuging at 4 ℃ and 10000rpm for 10min, dissolving the precipitate with 200 mu L of sterile PBS buffer solution, mixing uniformly to obtain the amplified phage solution, and measuring the titer of the amplified phage peptide library, and performing the next round of selection.
example 2 identification and sequencing of specific Polypeptides
2 4Picking single clone from the plate for determining eluted Phage in the third round, determining the capability of Phage display polypeptide clone simulating AFB1 epitope and binding antibody by adopting the method of Phage ELISA, specifically, inoculating the picked Phage display polypeptide clone in 2mL LB culture medium (containing 1:100 log prophase E.coli ER2738), shaking and culturing for 6h at 37 ℃, centrifuging for 10min at 10000rpm, collecting supernatant, determining each clone by using 3 holes, coating 100 muL (10 muG/mL) of antibody used for screening by using hole 1 and hole 2, coating 1% BSA in hole 3 as blank control, washing by PBST, adding 3% skim milk to seal for 2h (300 muL/hole), adding 50 muL 500ppb AFB1 and 50 muL of mixed solution of Phage culture supernatant into hole 1, adding 50 muL of 10% methanol PBS and 50 muL of Phage culture supernatant into hole 2 and hole 3, incubating for 2 min at 37 ℃ for 30min, adding 3 times of washed plate for 3 times, adding 100 muL of washed hole 100 muL of AFB and 100 muL of Phage culture supernatant, adding 100 muL of anti-80 mu L of Phage display polypeptide to react with light-10 μ L of HRP substrate, adding 30min after adding 50 muL of HRP 3-54 to obtain light absorption reaction, adding light-7 nm, determining the light-absorbance value after adding the light-absorbance value of Phage amplification reaction for 3-30 min, adding the Phage amplification reaction after adding 50 muL of positive amplification reaction for 3-30 min, adding 365.
the competitive binding capacity of the plaques is determined by Phage ELISA, as shown in figure 2, wherein the clone 3-C-43 has the best competitive inhibition effect, so that the clone is amplified and sequenced, the clone DNA sequence is GTGCCTAGTAAGCCGGGGCTG, and the translated amino acid sequence is CVPSKPGLC, namely the cyclic polypeptide for detecting aflatoxin AFB 1.
example 3 Synthesis of Biotin-labeled Cyclic Polypeptides
Biotin is reacted with-NH 2 in a cyclic polypeptide sequence having a total of two free-NH 2, one at the N-terminus and the other at the R group of lysine, thus linking the cyclic polypeptide to biotin in a 1:2 ratio. Weighing 2mg of cyclic polypeptide, adding 50 mu L of DMF and 2mL of PBS buffer solution to dissolve the polypeptide;
Weighing 4.6mg of biotin, adding 50 mu of LDMF (low density polyethylene) for dissolving, dropwise adding 22 mu of dissolved biotin into the dissolved polypeptide, carrying out ice bath for 2 hours, and dialyzing for 24 hours to obtain the cyclic polypeptide marked by the biotin.
Example 4 kit for the detection of AFB1 based on the biotin-labelled cyclic polypeptide immunoassay method, comprising the following parts:
(1) The biotin-labeled cyclic polypeptide;
(2) A 96-well enzyme label plate coated with an AFB1 monoclonal antibody;
(3) Standard solutions of aflatoxin B1;
(4) Horse radish peroxidase labeled streptavidin (enzyme-labeled secondary antibody);
(5) the formulation of the concentrated wash buffer (PBST) was: 40g of sodium chloride, 8g of monopotassium phosphate, 14.5g of disodium hydrogen phosphate, 8g of potassium chloride, 202.5 ml of Tween-and 20ml of distilled water;
(6) Sample diluent: phosphate buffer solution containing 10% methanol, wherein the formula of the phosphate buffer solution comprises 8g of sodium chloride, 0.2g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate and 0.2g of potassium chloride;
(7) The color developing solution A comprises 1g of carbamide peroxide, 10.3g of citric acid, 35.8g of Na 2 HPO 4.12H 2 O, Tween-20100 mu l of distilled water, 1000mL of pH5, and the color developing solution B comprises 300mg of tetramethyl benzidine (TMB) (dissolved in 50mL of DMSO), 10.3g of citric acid, 1000mL of distilled water, and pH 2.4-2.6.
(8)2M sulfuric acid solution.
After the kit is assembled, the antibody coated plate and the biotin-labeled cyclic polypeptide can be stored for at least 1 year at-20 ℃, and other reagents can be stored at normal temperature.
example 5 immunoassay method for biotin-labeled cyclic polypeptide-based determination of AFB1
the anti-AFB 1 monoclonal antibody is used as a coating antibody, biotin-labeled cyclic polypeptide and an AFB1 sample are mixed in equal volume, added into an enzyme-labeled hole coated with the anti-AFB 1 monoclonal antibody, compete for the antibody together, then an enzyme-labeled secondary antibody and a substrate are added, and the content of AFB1 in the sample is judged according to the degree of substrate color development. AFB1 was determined by a competitive immunoassay method, which was performed as follows:
(1) Balancing: taking the ELISA plate treated with the anti-AFB 1 monoclonal antibody out of the package, and balancing for 15min at room temperature;
(2) Washing the plate: adding 200 μ L of PBST (PBS (pH 7.4) containing 0.05% (v/v) Tween 20) into each well, standing for 1min, removing the washing solution, repeating for 3 times, and drying the PBST remained in the plate on absorbent paper;
(3) and (3) competitive reaction: mixing the biotin-labeled cyclic polypeptide with an AFB1 standard or a sample extracting solution to be detected in the same volume, wherein the standard solution and the sample diluent are PBST containing 10% methanol; adding 100 mu L of the enzyme-labeled reagent into the closed enzyme-labeled hole, reacting for 1h at room temperature, and washing the plate;
(4) adding an enzyme-labeled secondary antibody: diluting enzyme-labeled secondary antibody (conjugate of streptavidin and horseradish peroxidase) with PBST at a ratio of 1:1,000, adding 100 μ L of the diluted secondary antibody into each well, reacting at room temperature for 1h, and washing the plate;
(5) developing, wherein the solution A is prepared from 1g of carbamide peroxide, 10.3g of citric acid, 35.8g of Na 2 HPO 4.12H 2 O, Tween-20100 mu L of distilled water, 1000mL of distilled water and pH5, the solution B is prepared from 700mg of tetramethyl benzidine (TMB) (dissolved in 40mL of DMSO), 10.3g of citric acid and 1000mL of distilled water and pH2.4-2.6, the solution A and the solution B are mixed according to the ratio of 1 to 1, then 100 mu L of solution A is added into each hole, and the solution A and the solution B are developed for 10-15min in a dark place at room temperature;
(6) Termination and measurement: the reaction was stopped by adding 50. mu.L of 2M sulfuric acid solution to each well, and the OD value of each well was immediately measured at a wavelength of 450nm using a microplate reader.
The standard inhibition curve of AFB1 is detected by an immunoassay method established based on biotin-labeled cyclic polypeptide by using an anti-AFB 1 monoclonal antibody used for screening as a coating antigen, the series standard concentration of AFB1 is set to be in the range of 0-30 ng/mL, a four-parameter equation is fitted to the standard curve (see figure 3), the sensitivity is expressed by the concentration of AFB1 required when half of the highest OD value is inhibited, namely the IC 50 value of the medium inhibition concentration, and the IC 50 value of the standard curve is 0.92ng/mL, and the linear range of the IC 20 -IC 80 value of the curve is 0.23-3.36 ng/mL.
Example 6 specificity test
The method comprises the steps of selecting aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, ochratoxin A and citrinin as substances to be tested, measuring IC 50 values of the substances, and calculating the cross reactivity of the antibody to the substances by using the following equation, wherein the cross reactivity is smaller, the specificity of the polypeptide to AFB1 is stronger, and the specificity is worse.
Cross-reaction (CR%) -IC 50 (AFB1)/IC 50 (test substance). times.100%
the experimental determination results are shown in table 1, and by adopting the method of embodiment 3, the biotin-labeled cyclic polypeptide can not identify the substance to be determined (the cross-reaction rate is less than 1%) and can only identify AFB1, so that the method has strong specificity and can ensure the reliability of the determination result of the content of AFB1 in the sample.
TABLE 1 Cross-reactivity of polypeptide-based immunoassays with different mycotoxins
example 7 sample testing
A rice sample is purchased from a supermarket and is identified to be free of AFB1 by HPLC-FLD. 5.0g rice was weighed into a 50mL beaker and 1.5mL LAFB1 standard (dissolved in methanol) was added to the sample to give final concentrations of AFB1 in the rice sample of 10ng/g,20ng/g, and 40 ng/g. Sample pretreatment: adding 15mL of methanol into a 5g rice sample, mixing uniformly, performing ultrasonic extraction for 5min, centrifuging at 5000rpm for 10min, diluting the supernatant by 10 times with PBS, and detecting the content of AFB1 by using the method described in example 4. The detection results of the addition recovery are shown in table 2, the average recovery rate of the in-batch analysis is 84.1-96.7%, and the variation coefficient is 2.1-6.3%; the average recovery rate of the inter-batch analysis is 80.5-89.1%, and the coefficient of variation is 5.4-9.2%. The accuracy and precision of the method meet the actual detection requirements, and the method can be used for rapidly screening AFB1 in rice samples.
TABLE 2 measurement of AFB1 addition recovery in rice samples
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> aflatoxin B1 epitope-mimicking cyclic polypeptide and detection kit thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Synthetic sequence (Synthetic sequence)
<400> 1
Cys Val Pro Ser Lys Pro Gly Leu Cys
1 5
Claims (5)
1. a cyclic polypeptide simulating aflatoxin B1 epitope, which is characterized in that: the amino acid sequence is CVPSKPGLC, as shown in SEQ ID No.1, and cysteine on both sides of the amino acid is oxidized to form a ring.
2. A kit for detecting aflatoxin B1 comprising the polypeptide of claim 1, wherein: the kit comprises the following parts:
(1) AFB1 competes for an antigen, and the AFB1 competes for the antigen as a biotin-labeled cyclic polypeptide of claim 1; wherein the AFB1 competitive antigen is formed by coupling a cyclic polypeptide for detecting aflatoxin AFB1 and biotin;
(2) An ELISA plate embedded with an anti-AFB 1 monoclonal antibody;
(3) Standard solutions of aflatoxin B1;
(4) Horse radish peroxidase is marked with streptavidin, namely an enzyme-labeled secondary antibody;
(5) washing the buffer solution;
(6) A sample diluent;
(7) Developing solution A;
(8) developing B liquid;
(9)2M sulfuric acid solution.
3. the kit for detecting aflatoxin B1 of claim 2, which is characterized in that: the formula of the washing buffer PBST is as follows: 40g of sodium chloride, 8g of monopotassium phosphate, 29g of disodium hydrogen phosphate, 8g of potassium chloride, 202.5 mL of Tween-and 20mL of distilled water;
The standard solution concentration of the aflatoxin B1 comprises 30, 10, 2, 1, 0.2 and 0.02 ng/mL;
The sample diluent is: the phosphate buffer solution containing 10% methanol by volume comprises 8g of sodium chloride, 0.2g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate and 0.2g of potassium chloride;
The color developing solution A comprises 1g of carbamide peroxide, 10.3g of citric acid, 35.8g of Na 2 HPO 4.12H 2 O, 1000mL of Tween-20100 mu L of distilled water and the pH value of 5;
The color developing liquid B: 300mg of tetramethylbenzidine, 10.3g of citric acid and 1000mL of distilled water, and the pH value is 2.4-2.6.
4. a competitive immunoassay method for detecting aflatoxin B1 using the kit of claim 2, characterized in that: the method comprises the following steps:
1) After mixing the AFB1 competitive antigen with a sample to be detected in an equal volume, adding the mixture into an ELISA plate embedded with an anti-AFB 1 monoclonal antibody;
2) Washing the ELISA plate by using a washing buffer solution, adding horseradish peroxidase labeled streptavidin, washing the ELISA plate by using the washing buffer solution, adding a mixed solution of a developing solution A and a developing solution B for developing, and then adding 2M sulfuric acid solution for stopping;
3) And (3) measuring the OD value of the hole to be measured by using an enzyme-labeling instrument at the wavelength of 450nm, establishing a standard curve by using a standard solution of the aflatoxin B1, and correspondingly converting according to the established standard curve and the OD value of the hole to be measured to obtain the content of the aflatoxin B1 in the sample to be measured.
5. The application of the aflatoxin B1 detection kit in claim 2 in checking the AFB1 content of rice.
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