WO2023155876A1 - 基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用 - Google Patents

基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用 Download PDF

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WO2023155876A1
WO2023155876A1 PCT/CN2023/076761 CN2023076761W WO2023155876A1 WO 2023155876 A1 WO2023155876 A1 WO 2023155876A1 CN 2023076761 W CN2023076761 W CN 2023076761W WO 2023155876 A1 WO2023155876 A1 WO 2023155876A1
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mycotoxin
nanobody
fusion protein
bifunctional fusion
alkaline phosphatase
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PCT/CN2023/076761
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English (en)
French (fr)
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王松雪
刘洪美
叶金
倪保霞
陈金男
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国家粮食和物资储备局科学研究院
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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention belongs to the technical field of food safety detection, and relates to a mycotoxin magnetochemiluminescence immunoassay kit based on a bifunctional fusion protein and an application thereof.
  • Mycotoxins are secondary metabolites produced by toxin-producing fungi under certain environmental conditions, which widely contaminate plant-derived products such as agricultural products, food, Chinese medicinal materials, and feed.
  • the more common mycotoxins in agricultural products, food and feed include: deoxynivalenol (also known as vomitoxin, DON), aflatoxin B1 (aflatoxin B1, AFB1), aflatoxin B2 (AFB2) , Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2), Ochratoxin A (ochratoxin A, OTA), Fumonisin B1 (fumonisin B1, FB1), Fumonisin B2 (FB1), Gibberella Zea Enone (zearalenone, ZEN), T-2 toxin (T-2toxin), HT-2 toxin (HT-2toxin) or aflatoxin M1 (aflatoxin M1, AFM1), patulin (pat ⁇ Lin), etc.
  • Mycotoxins can cause acute or chronic poisoning of humans and animals, damage the liver, kidneys, nerve tissue, hematopoietic tissue, and skin tissue of the body, and have serious impacts on the health of humans and animals.
  • countries around the world have strictly limited their content. Carrying out research on mycotoxin residue pollution, and developing highly sensitive, low-cost, and reliable automated detection technology will ensure the quality and safety of agricultural products, food, traditional Chinese medicine, and feed, break foreign technical barriers, and protect my country's economic interests in international trade. It is of great significance to increase foreign exchange earnings through exports.
  • the detection methods of mycotoxins include thin-layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, capillary electrophoresis, and liquid chromatography-mass chromatography.
  • thin-layer chromatography is the earliest method used to detect mycotoxins. Its advantages are that it is suitable for operation by personnel without special training, and it is low in cost and does not require expensive instruments.
  • the sample processing of thin-layer chromatography is cumbersome, the experimental process is complicated, the required detection cycle is long, and it is easy to be interfered by impurities.
  • Semi-quantitative visual inspection has disadvantages such as large subjective influence and low sensitivity, which are far from meeting the requirements of modern detection.
  • ELISA has the advantages of good detection specificity, high sensitivity, and low detection cost. It is suitable for the screening and general survey of a large number of samples in grassroots institutions, which can greatly save time and cost.
  • the main problem with ELISA is that it is prone to false positives. Therefore, it is mainly used for screening and detection at the grassroots level.
  • Instrumental analysis methods such as high performance liquid chromatography, capillary electrophoresis, and liquid chromatography-mass spectrometry have the advantages of high accuracy, strong sensitivity, and micro-determination, and are currently commonly used methods for the detection of toxins in food.
  • some pre-treatment processes are required, resulting in high detection costs and long cycles, which cannot meet the requirements of rapid screening of large-scale samples.
  • Magnetic particles have superparamagnetic particle properties, and have magnetic particle field responsiveness under the magnetic particle field. Using magnetic particles as a solid-phase carrier for immunoassays will greatly increase the surface area of the reaction and make it easier to separate solid and liquid phases. The detection sensitivity can be improved. The use of tiny magnetic particles as the solid phase can increase the surface area of the coating, thereby increasing the amount of antigen or antibody adsorption, which not only speeds up the reaction speed, but also makes cleaning and separation easier.
  • the preparation method of immunoenzyme labeling reagents is to use chemical labeling methods, such as using bifunctional reagents, glutaraldehyde, periodate, SMCC reagent ⁇ 4-(N-maleimidomethyl)cyclohexane-1 -Carboxylic acid succinimide ester, etc.) ⁇ , 2-IT reagent (iminothiophene hydrochloride), etc., the antibody is coupled to the enzyme, but the chemical coupling method is complicated to operate and the coupling efficiency is low. Due to the coupling site The spot is not fixed and the conditions are severe, which will easily lead to the decrease of antibody or enzyme activity, and the binding of antibody and enzyme is not uniform.
  • chemical labeling methods such as using bifunctional reagents, glutaraldehyde, periodate, SMCC reagent ⁇ 4-(N-maleimidomethyl)cyclohexane-1 -Carboxylic acid succinimide ester, etc.) ⁇ , 2-IT reagent (
  • the first object of the present invention is to provide a mycotoxin magnetochemiluminescence immunoassay kit, which integrates chemiluminescence, magnetic particle separation and genetic engineering technologies, and has good accuracy, sensitivity and repeatability.
  • the second object of the present invention is to provide the application of the above kit in the detection of mycotoxins.
  • the present invention adopts the following technical solutions:
  • the present invention provides a mycotoxin magnetochemiluminescence immunoassay kit, which kit includes streptavidin magnetic particles, biotin-labeled mycotoxin antigen, mycotoxin standard solution, mycotoxin nanobody- Alkaline phosphatase (ScFV-AP) Bifunctional fusion protein, sample diluent, washing solution, substrate solution.
  • kit includes streptavidin magnetic particles, biotin-labeled mycotoxin antigen, mycotoxin standard solution, mycotoxin nanobody- Alkaline phosphatase (ScFV-AP) Bifunctional fusion protein, sample diluent, washing solution, substrate solution.
  • the invention uses micron-sized magnetic particles as the carrier, uses the carboxyl active group provided by the surface organic matter to covalently bond with the amino group of streptavidin, and uses biotin-streptavidin to "bridge” to form magnetic particles—streptavidin Avidin-biotin-mycotoxin-ScFV-AP bifunctional fusion protein complex.
  • novelties of this technology are: (1) using magnetic particles as a solid phase carrier to increase the contact area of antigens and antibodies and the luminescent area of the substrate to improve the sensitivity of the reaction, and to use the rotating magnetic particle field to make the magnetic particles stir and Separation of the magnetic particle-streptavidin-biotin-mycotoxin-ScFV-AP bifunctional fusion protein complex and the effect of the mycotoxin-scFV-AP bifunctional fusion protein complex. (2) The scFV-AP bifunctional fusion protein improves the specificity and stability of the reaction. (3) Edible oil can be directly sampled and tested without extraction, centrifugation and other operations, which greatly improves the detection efficiency.
  • scFv-AP bifunctional fusion protein is obtained by fused expression of mycotoxin nanobody (scFv) and alkaline phosphatase (AP) using alkaline phosphatase (AP) as a catalyst for bioluminescence.
  • the mycotoxin nanobody is selected from heavy chain antibodies of camelids or sharks.
  • amino acid sequence of the mycotoxin nanobody-alkaline phosphatase bifunctional fusion protein of the present invention may be any one or more of the following:
  • the amino acid sequence of the aflatoxin B1 nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:1;
  • amino acid sequence of the deoxynivalenol nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:2;
  • amino acid sequence of the zearalenone nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:3;
  • T-2 toxin nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:4;
  • amino acid sequence of the fumonisin B1 nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:5;
  • the amino acid sequence of the ochratoxin A nanobody-alkaline phosphatase bifunctional fusion protein is shown in SEQ ID NO:6.
  • the corresponding biotin-labeled mycotoxin antigen, mycotoxin standard solution, and mycotoxin nanobody-alkaline phosphatase bifunctional fusion protein are selected.
  • the kit of the present application should include biotin-labeled deoxynivalenol antigen, deoxynivalenol standard solution, and deoxynivalenol Fusarium enol nanobody-alkaline phosphatase bifunctional fusion protein.
  • scFv-AP bifunctional fusion protein can be synthesized according to the amino acid sequence provided above, or can be prepared by the following method:
  • the invention fuses and expresses the mycotoxin nanobody and alkaline phosphatase, which can not only overcome various defects of the chemical labeling method, but also obtain high specific activity enzyme-labeled antibody.
  • the prepared fusion protein can be applied to the field of immunodiagnosis, such as chemiluminescent immunoassay, enzyme-linked immunosorbent assay or enzyme-catalyzed fluorescent immunoassay, etc. as detection reagents.
  • the ScFV-AP bifunctional fusion protein has the following advantages: (1) The traditional chemical coupling method has a long process flow, complex coupling process and severe conditions, resulting in unstable process , the coupling product yield is low and the stability between batches is not good.
  • the fusion expression of mycotoxin nanobody and alkaline phosphatase is simple and the process is stable, avoiding the cumbersome and inefficient chemical cross-linking of enzymes and proteins.
  • the fusion expression method of mycotoxin nanobody and alkaline phosphatase overcomes this shortcoming.
  • the enzyme and antibody complexes after coupling are not uniform, and the mycotoxin nanobody in the fusion expression product of mycotoxin nanobody and alkaline phosphatase
  • the molecular ratio of body to monomer alkaline phosphatase is 1:1 or 1:2, and it can be separated during nickel column purification to ensure the homogeneity of the enzyme and antibody complex.
  • the binding position of the coupling chemically active reagent since the binding position of the coupling chemically active reagent is not fixed, it may bind to the variable region of the antibody, thereby affecting the binding of the antibody to the target antigen; or the coupling of the chemically active reagent to the alkaline phosphatase activity
  • the vicinity of the site affects the binding of alkaline phosphatase to the substrate; both of these situations can lead to a decrease in the activity of the coupling complex, and it is easy to get false negative results during detection.
  • the fusion expression of mycotoxin nanobody and alkaline phosphatase does not affect the activity of mycotoxin nanobody and alkaline phosphatase.
  • the scFv-AP fusion protein provided by the present invention has better specificity and signal amplification.
  • the scFv-AP fusion protein is used to establish a supersensitive bioluminescence immunoassay kit for detecting grain, oil, food,
  • the content of mycotoxins in substrates such as feed and Chinese herbal medicine has good application value and prospects.
  • the magnetic particle is made of ferric iron tetroxide or ferric oxide superparamagnetic material as the inner core, and the periphery is coated with polystyrene or dextran, and activated by physical or chemical methods to generate amino groups on the surface of the magnetic particle ( NH2-), tosyl (Tosyl), carboxyl (COOH-) or epoxy (—CH(O)) groups, particle size 1-2 ⁇ m.
  • streptavidin magnetic particles are coupled with streptavidin and magnetic particles.
  • biotin-labeled mycotoxin antigen is firstly coupled with the mycotoxin and bovine serum albumin, and then the obtained bovine serum albumin-mycotoxin complex is coupled with biotin.
  • the mycotoxin standard solution is prepared by dissolving the mycotoxin standard in a methanol-water mixed solution.
  • the mycotoxin standard solution in the kit can be a mother solution of a certain concentration, which can be diluted into a standard working solution of different concentrations according to needs during use for drawing a standard curve; it can also be used in the kit A series of concentrations of mycotoxin standard solution were directly dispensed into the medium for the drawing of the standard curve.
  • the volume ratio of methanol to water is 50:50.
  • the substrate solution is (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridiniumethylene)phosphate disodium salt solution with a concentration of 0.5-2mmol/L.
  • the sample diluent consists of 0.01M phosphate buffer, 0.1% Tween 20 and 0.5% bovine serum albumin.
  • the washing solution is composed of 0.01M Tris-HCl buffer and 0.1% Tween 20.
  • the kit also includes a reaction tube; preferably, the material of the reaction tube is transparent polystyrene, polyethylene, polypropylene or glass.
  • the present invention also claims the application of the above kit in the detection of mycotoxins, especially in the detection of mycotoxins in grain, oil, food, feed or Chinese herbal medicine.
  • the edible oil includes one of peanut oil, corn oil, soybean oil, rapeseed oil, sunflower oil, rapeseed oil, sesame oil, and olive oil.
  • the mycotoxins include one or more of aflatoxin, zearalenone, deoxynivalenol, fumonisins, ochratoxin A, and T-2 toxin.
  • the invention adopts the principle of indirect competition to measure mycotoxins in food, agricultural products and feedstuffs, and adds biotin-labeled mycotoxin antigen working solution, scFV-AP bifunctional fusion protein and sample.
  • the mycotoxin in the sample competes with the biotin-labeled mycotoxin antigen for a limited number of ScFV-AP bifunctional fusion proteins, and forms magnetic particles-streptavidin through the affinity reaction of streptavidin and biotin and the antigen-antibody reaction, respectively.
  • Avidin-biotin-mycotoxin-scFV-AP bifunctional fusion protein complex and mycotoxin-scFV-AP bifunctional fusion protein complex magnetic particles are directly precipitated in an external magnetic particle field, and the composite The substance is adsorbed at the bottom of the reagent tube, the free components are washed away, and the substrate solution is added. After the alkaline phosphatase catalyzes the hydrolysis of the phosphate radical of the substrate, a decomposition reaction occurs immediately and a 475nm photon is released, and the concentration of each sample well is measured within 5 minutes. Luminescence value (RLU). The RLU of a sample was inversely correlated with the concentration of mycotoxins in the sample. The mycotoxin concentration in the sample is quantified according to the four-parameter mathematical model established by the mycotoxin standard concentration and the corresponding RLU, so as to detect the mycotoxin content in the sample.
  • the above-mentioned detection can use a fully automatic chemiluminescent immunoassay analyzer, so as to realize the fully automatic detection of mycotoxins.
  • Each kit does not interfere with each other, and can detect multiple samples at the same time in real time, which is highly in line with the development needs of on-site rapid detection of mycotoxins.
  • kits of the present invention to detect mycotoxins also has the following advantages: (1) The detection speed is fast, stable, and free of radioactive pollution, and it can be detected without magnetic micro In the presence of a particle field, the magnetic particles are suspended in the liquid, making the antigen-antibody reaction similar to a homogeneous reaction; the magnetic particles can be easily separated and washed quickly under the action of an external magnetic particle field, and the detection can be judged within 24 minutes result. (2) High sensitivity. (3) Strong specificity. (4) The precision is good.
  • Figure 1 is a schematic diagram of the magnetochemiluminescent immunoassay of mycotoxins based on bifunctional fusion proteins.
  • test materials used in the following examples were purchased from conventional biochemical reagent suppliers unless otherwise specified.
  • the AFB1-scFv-AP bifunctional fusion protein is obtained by fused expression of aflatoxin B1 nanobody (AFB1-scFv) and alkaline phosphatase (AP) using alkaline phosphatase as a catalyst for bioluminescence.
  • the AFB1-scFV-AP bifunctional fusion protein is prepared by the following method:
  • AFB1 nanobody gene according to the amino acid sequence of AFB1 nanobody SEQ ID NO: 7, the high-efficiency expression of the gene in Escherichia coli was realized by optimizing the structure of the gene, including the use of E. coli preferred codons to eliminate the possibility Existing secondary structure makes GC/AT balance etc., design and synthesize the nucleotide fragment of AFB1 Nanobody gene (nucleotide sequence is shown in SEQ ID NO:8).
  • AFB1-scFV-AP expression vector Cloning the aflatoxin B1 nanobody gene synthesized in step (1) into the prokaryotic expression vector pET25b(+) to obtain pET25b(+)-scFV; Endonucleases XhoI and NotI double-digest alkaline phosphatase expression vector pET25b(+)-AP (provided by Suzhou Synbio Biotechnology Co., Ltd.) to obtain alkaline phosphatase gene fragments; after recovery from agarose gel, use T4 DNA ligase was used to connect the alkaline phosphatase gene fragment to the pET25b(+)-scFV expression vector cut with restriction endonucleases XhoI and NotI at a molar ratio of 1:3, and heat shock (42°C, 45s) ) Transform the ligation product into Escherichia coli DH5 ⁇ competent cells, inoculate the transformed bacteria on an
  • Alkaline phosphatase activity assay of a bifunctional fusion protein the p-nitrophenyl phosphate disodium method.
  • a bifunctional fusion protein and AP diluted 50, 100, 200, 400, 800, 1000 times respectively, and then add 100 ⁇ L of PNPP reagent (2mg/mL, dissolved in 2mM MgCl 2 0.1M diethanolamine buffer, pH 9.8), mixed well, incubated at 37°C for 10min, and finally added 50 ⁇ L of 4M NaOH solution to terminate the reaction, measured the absorbance at 405nm with a microplate microplate reader, and calculated the Concentration of phenol (mM).
  • the alkaline phosphatase activity of the bifunctional fusion protein was evaluated by comparing the specific activity of the bifunctional fusion protein and AP.
  • the Kon value, Koff value and KD value (Kon value/Koff value) of the toxin are used to characterize the antibody affinity of the bifunctional fusion protein by comparing the KD value.
  • Kon represents the formation rate of the antigen-antibody complex, the larger the Kon value, the faster the antigen-antibody binding;
  • Koff reflects the stability of the formed complex, the larger the Koff value, the faster the complex dissociation; KD can reflect the antigen and antibody
  • Example 2 Using the method of Example 1, a DON-scFV-AP bifunctional fusion protein was prepared, the amino acid sequence of which is shown in SED ID NO:2.
  • Example 2 Using the method of Example 1, a ZEN-scFv-AP bifunctional fusion protein was prepared, the amino acid sequence of which is shown in SED ID NO:3.
  • T-2-scFV-AP bifunctional fusion protein was prepared, the amino acid sequence of which is shown in SED ID NO:4.
  • the FB1-scFV-AP bifunctional fusion protein was prepared, and its amino acid sequence is shown in SED ID NO:5.
  • the OTA-scFv-AP bifunctional fusion protein was prepared, and its amino acid sequence is shown in SED ID NO:6.
  • Dispersion of magnetic particles disperse 10 mg of magnetic particles with 10 mL of 0.1M 2-morpholineethanesulfonic acid solution (MES buffer), so that the final concentration of magnetic particles is 1 mg/mL;
  • MES buffer 2-morpholineethanesulfonic acid solution
  • step (4) the magnetic particles in step (4) are separated from the reaction system, and the magnetic particle beads are washed twice with a phosphate buffer solution of pH 7.4 to obtain streptavidin magnetic particles;
  • the streptavidin magnetic particles are configured into the streptavidin magnetic particle working solution: the streptavidin in step (5) Sumagnetic particles are dispersed in phosphate buffer solution of 0.5g/mL casein, 1g/mL PEG200, 0.2% Tween 20, 0.5% bovine serum albumin, the pH value is 7-8, and the final concentration of magnetic particles is 1mg/mL mL, ready to use.
  • AFB1 and carboxymethylhydroxylamine hemihydrochloride were placed in pyridine, stirred and reacted at room temperature in the dark for 24 hours to obtain the first reaction solution;
  • the AFB1-scFV-AP bifunctional fusion protein prepared in Example 1 was diluted with 0.01M Tris salt buffer solution and 0.5% bovine serum albumin solution at a ratio of 1:20000 to obtain the AFB1-scFV-AP bifunctional fusion protein working solution.
  • Sample diluent consisted of 0.01M phosphate buffer, 0.1% Tween 20 and 0.5% bovine serum albumin.
  • the washing solution consisted of 0.01M Tris-HCl buffer and 0.1% Tween 20.
  • the AFB1 magnetochemiluminescence immunoassay kit prepared in Example 7 was used to conduct a methodological investigation test for the detection of aflatoxin B1.
  • Stability test Treat with advanced stabilizer (0.01M phosphate buffer, 0.1% bovine serum albumin solution, 0.1% proclin 300 and 1% trehalose), store at 37°C after treatment for accelerated stability test Test, the measured stability results are shown in Table 1. According to experience, storing at 37°C for 1 day is equivalent to storing at 4°C for 2 months, that is, storing this kit at 4°C for 12 months will not affect the use.
  • the detection limit LOD of this method is calculated by the mean value of 20 negative samples plus 3 times the standard deviation, and the result is 0.2 ⁇ g/kg, and the quantification limit LOQ is determined by 20 negative samples The mean value of the result plus 10 times the standard deviation was calculated, and the result was 1.0 ⁇ g/kg.
  • the luminescence value is inversely proportional to the concentration of aflatoxin B1 antigen, and the aflatoxin B1 content of the sample to be tested is calculated according to the fitted standard curve of aflatoxin B1 concentration-luminescence value.
  • Aflatoxin B1 magnetochemiluminescence immunoassay reagents were used to detect structural analogs (AFB1, AFB2, AFG1 and AFG2) and other common mycotoxins (deoxynivalenol, DON; fumonisin B1, FB1; Fumonisins B1, FB2; Fumonisins B1, FB3; Zearalenone, ZEN; T-2 Toxin, T-2; Ochratoxin A, OTA; 3-Acetyldeoxynivalenol, 3 -ADON; 15-acetyl deoxynivalenol, 15-ADON; deoxynivalenol-3-glucoside, DON-3G; nivalenol, NIV), the results are shown in Table 1 It was shown that only AFB2 and AFG1 had obvious cross-reactions, and the cross-reaction rates were 26.3% and 23.8%, respectively. sex is good.
  • Pretreatment of the sample to be tested Weigh 5.0 g of the pulverized sample to be tested in a 50 mL centrifuge tube, add 25.0 mL of methanol/water (70:30) solution, and place it on a multi-tube vortex mixer at 2500 rpm. Spin for 5 minutes (or use a high-speed homogenizer at 11,000 rpm for 3 minutes, or shake at 200 rpm for 40 minutes), add 20 ⁇ L of the sample extract to the test tube, and then centrifuge at 7,000 rpm for 5 minutes. min, to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or Aflatoxin B1 standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L aflatoxin B1 nanobody-alkaline phosphatase bifunctional fusion protein and 400 ⁇ L diluent, mix well, and incubate at 37°C for 5 minutes; After washing 3 times, measure the luminescence value, and analyze the results according to the built-in four-parameter fitting curve. The determination of 8 samples can be completed within 20 minutes, and a test report will be issued.
  • Reference Example 7 prepared a deoxynivalenol magnetochemiluminescence immunoassay kit and conducted a methodological investigation on the detection of deoxynivalenol.
  • Stability test Treat with advanced stabilizer (0.01M phosphate buffer, 0.1% bovine serum albumin solution, 0.1% proclin300 and 1% trehalose), store at 37°C after treatment for accelerated stability test , the measured stability results are shown in Table 1. According to experience, storage at 37°C for 1 day is equivalent to storage at 4°C for 2 months, that is, the kit can be stored at 4°C for 12 months, which will not affect the use.
  • the detection limit LOD of this method is calculated by the mean value of 20 negative samples plus 3 times the standard deviation, and the result is 77.0 ⁇ g/kg, and the quantification limit LOQ is determined by 20 negative samples The mean value of the result plus 10 times the standard deviation was calculated, and the result was 222.8 ⁇ g/kg.
  • the luminescence value is inversely proportional to the concentration of deoxynivalenol antigen, and the deoxynivalenol concentration-luminescence value standard curve fitted is used to calculate the deoxynivalenol concentration of the sample to be tested. Fusarium enol content.
  • the deoxynivalenol magnetochemiluminescence immunoassay reagent was used to detect structural analogues (3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15- ADON) and nivalenol, NIV) and other common mycotoxins (aflatoxin B1, DON zearalenone, ZEN; T-2 toxins, T-2 and ochratoxin A, OTA), results As shown in Table 8, only 3-ADON has a relatively obvious cross-reactivity, and other toxins have no cross-reactivity, indicating that the deoxynivalenol magnetochemiluminescence immunoassay kit has good specificity.
  • Pretreatment of the sample to be tested Weigh 5.0 g of the pulverized sample to be tested in a 50 mL centrifuge tube, add 25.0 mL of water, and place it on a multi-tube vortex mixer at 2500 rpm for 5 minutes (or use a high-speed homogenizer Homogenize at 11000rpm for 3min on a shaker, or shake at 200rpm for 40min on a shaker) Add 20 ⁇ L of the sample extract to be tested into the test tube, and then centrifuge at 7000rpm for 5min to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or Deoxynivalenol standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L LDON-scFV-AP bifunctional fusion protein and 400 ⁇ L diluent, mix well, incubate at 37°C for 5 min; wash 3 times in sequence , Measure the luminescence value, and analyze the results according to the built-in four-parameter fitting curve, complete the determination of 8 samples within 20 minutes, and issue a test report.
  • Example 7 a zearalenone magnetochemiluminescence immunoassay kit was prepared.
  • Pretreatment of the sample to be tested Weigh 5.0 g of the pulverized sample to be tested in a 50 mL centrifuge tube, add 25.0 mL of 80% acetonitrile aqueous solution, and place it on a multi-tube vortex mixer at 2500 rpm for 5 min (or use a high-speed Homogenize on a homogenizer at 11000rpm for 3min, or shake at 200rpm on a shaker for 40min) Add 20 ⁇ L of the sample extract to be tested into the test tube, and then centrifuge at 7000rpm for 5min to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or Zearalenone standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L ZEN-scFV-AP bifunctional fusion protein and 400 ⁇ L diluent, mix well, incubate at 37°C for 5 min; after washing 3 times in sequence, measure The luminescence value is analyzed according to the built-in four-parameter fitting curve, and the determination of 8 samples can be completed within 20 minutes, and a test report is issued.
  • the detection limit LOD of this method is calculated by the mean value of 20 negative samples plus 3 times the standard deviation, and the result is 3.9939 ⁇ g/kg, and the quantification limit LOQ is determined by 20 negative samples The mean value of the result plus 10 times the standard deviation was calculated, and the result was 9.4483 ⁇ g/kg.
  • T-2 toxin magnetochemiluminescence immunoassay kit was prepared.
  • Pretreatment of samples to be tested Weigh 5.0 g of crushed corn negative samples into 50 mL centrifuge tubes, add different amounts of T-2 toxin standard substances, and the final spiked levels are 47.8 ⁇ g/Kg and 108 ⁇ g/Kg, respectively. Kg, 7 in parallel, add 25.0mL80% acetonitrile aqueous solution, place on a multi-tube vortex mixer and vortex at 2500rpm for 5min (or use a high-speed homogenizer to homogenize at 11000rpm for 3min, or shake at 200rpm for 40min) in the detection tube Add 20 ⁇ L of the sample extract to be tested, and then centrifuge at 7000rpm for 5min to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or T-2 toxin standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L LT-2-scFV-AP bifunctional fusion protein and 400 ⁇ L diluent, mix well, and incubate at 37°C for 5 min; after washing 3 times in sequence, Measure the luminescence value and analyze the results according to the built-in four-parameter fitting curve. The measurement of 8 samples can be completed within 20 minutes and a test report will be issued.
  • Example 7 a fumonisin B1 magnetochemiluminescence immunoassay kit was prepared.
  • Pretreatment of samples to be tested Weigh 5.0 g of crushed corn negative samples into 50 mL centrifuge tubes, add different amounts of FB1 standard substances, and the final spiked levels are 47.8 ⁇ g/Kg and 108 ⁇ g/Kg, respectively, 7 In parallel, add 25.0 mL of 80% acetonitrile aqueous solution, place on a multi-tube vortex mixer and vortex at 2500 rpm for 5 min (or use a high-speed homogenizer to homogenize at 11000 rpm for 3 min, or shake at 200 rpm for 40 min on a shaker) and add 20 ⁇ L of The sample extract to be tested is then centrifuged at 7000rpm for 5min to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or Fumonisin B1 toxin standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L FB1-scFV-AP bifunctional fusion protein and 400 ⁇ L diluent, mix well, incubate at 37°C for 5 min; after washing 3 times in sequence, measure The luminescence value is analyzed according to the built-in four-parameter fitting curve, and the determination of 8 samples can be completed within 20 minutes, and a test report is issued.
  • the extract of the sample to be tested obtained in step (1) was measured with the FB1 toxin magnetochemiluminescence immunoassay kit, and the recoveries, mean values and RSDs of 7 parallels were shown in Table 17, and the recoveries were between 89.2% and 111.3%. Between, the RSD is less than 6%.
  • Pretreatment of the sample to be tested Weigh 5.0 g of the pulverized sample into a 50 mL centrifuge tube, add 25.0 mL of 80% acetonitrile aqueous solution, and place it on a multi-tube vortex mixer at 2500 rpm for 5 minutes (or use a high-speed homogenizer Homogenize at 11000rpm for 3min on a shaker, or shake at 200rpm for 40min on a shaker) Add 20 ⁇ L of the sample extract to be tested into the test tube, and then centrifuge at 7000rpm for 5min to obtain the sample extract to be tested;
  • sample determination Add 40-200 ⁇ L of the sample extract to be tested into the sample well, place it in the automatic chemiluminescence immunoassay analyzer, click Run, and the instrument will add 50 ⁇ L of the sample to be tested extract or Ochratoxin A standard solution, 50 ⁇ L streptavidin magnetic particle working solution, 50 ⁇ L LOTA-scFV-AP bifunctional fusion protein and 400 ⁇ L diluent, mix well, incubate at 37°C for 5 min; after washing 3 times in sequence, measure the luminescence Value, and analyze the results according to the built-in four-parameter fitting curve, the determination of 8 samples can be completed within 20 minutes, and a test report will be issued.
  • the detection limit LOD of this method is calculated by the mean value of 20 negative samples plus 3 times the standard deviation, and the result is 2.0610 ⁇ g/kg, and the quantification limit LOQ is determined by 20 negative samples The mean value of the result plus 10 times the standard deviation was calculated, and the result was 4.5105 ⁇ g/kg.
  • ochratoxin A magnetic chemiluminescence immunoassay kit was tested with the sample extract after adding the standard, and the recoveries, mean values and RSDs of 7 parallels were shown in Table 18, Table 19 and Table 20, and the recoveries were between 80% and 120% %, the RSD is less than 8%.

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Abstract

一种基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用。该试剂盒包括链霉亲和素磁微粒、生物素标记的真菌毒素抗原、真菌毒素标准品溶液、真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白、样本稀释液、洗涤液、底物液。该试剂盒将化学发光、磁微粒分离和基因工程技术汇聚在一起，可实现真菌毒素的快速、准确、全自动化检测，食用油可直接加样检测，无需提取、离心等操作，减少检测人员的操作误差，降低对检测人员的能力要求，显著提高基层大批量样品的检测效率，为真菌毒素检测、监测提供有力的手段。

Description

基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用 技术领域
本发明属于食品安全检测技术领域，涉及一种基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用。
背景技术
真菌毒素(Mycotoxins)是产毒真菌在一定环境条件下产生的次级代谢产物，广泛污染农产品、食品、中药材及饲料等植物源性产品。在农产品、食品和饲料中比较常见的真菌毒素有：脱氧雪腐镰刀菌烯醇(deoxynivalenol，又称呕吐毒素，DON)、黄曲霉毒素B1(aflatoxin B1，AFB1)、黄曲霉毒素B2(AFB2)、黄曲霉毒素G1(AFG1)、黄曲霉毒素G2(AFG2)、赭曲霉毒素A(ochratoxin A，OTA)、伏马毒素B1(fumonisin B1，FB1)、伏马毒素B2(FB1)、玉米赤霉烯酮(zearalenone，ZEN)、T-2毒素(T-2toxin)、HT-2毒素(HT-2toxin)或黄曲霉毒素M1(aflatoxin M1，AFM1)、展青霉素(patμLin)等。真菌毒素可以引起人类和动物的急性或慢性中毒，可损害机体的肝脏、肾脏、神经组织、造血组织及皮肤组织等，对人类和动物健康可产生严重的影响。鉴于真菌毒素的危害，世界各国对其含量进行了严格的限定。开展真菌毒素残留污染研究，开发高灵敏、低成本、可靠的自动化检测技术对保证农产品、食品、中药和饲料等的质量和安全，打破国外的技术壁垒，保护我国在国际贸易中的经济利益，增加出口创汇具有重要意义。
目前真菌毒素的检测方法有薄层色谱法，高效液相色谱法、酶联免疫吸附法，毛细管电泳法、液质联用法等。其中薄层色谱法是最早使用的检测真菌毒素的方法，其优点是适合于没有经过专门培训的人员操作，且成本低、无需价格昂贵的仪器。但是，薄层色谱法的样品处理繁琐，实验过程复杂，所需检测周期较长，容易受到杂质的干扰。测定时用目测半定量，存在主观影响较大，灵敏度不高等缺点，已远远不能满足现代检测要求。酶联免疫吸附法具有检测特异性好、灵敏度高、并且检测成本较低的优点，适用于基层机构大量样品的筛选和普查，可以大大节省时间和费用。酶联免疫吸附法的主要问题是容易造成假阳性。因此，主要用于基层的筛查检测。高效液相色谱法、毛细管电泳法和液质联用法等仪器分析方法具有准确度高、灵敏性强、可微量测定等优点，是目前常用的食品中毒素检测的方法。但因其对样品纯度要求较高，需要经过一些前处理过程，导致检测成本高、周期长，无法满足大批量样品快速筛选的要求。磁微粒具有超顺磁微粒性，在磁微粒场下具有磁微粒场响应性，将磁微粒应作免疫检测的固相载体，将大大提高反应的表面积，更容易进行固相、液相分离，可提高检测的灵敏度。采用微小的磁微粒作为固相可增加包被表面积，从而增加抗原或抗体的吸附量，既加快了反应速度，也使清洗和分离更简便。目前免疫酶标试剂制备方式是采用化学标记方法，如使用双功能试剂，戊二醛，过碘酸盐，SMCC试剂{4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯等)}，2-IT试剂(亚氨基噻吩盐酸盐)等，将抗体与酶偶联，但是化学偶联方法操作复杂，偶联效率低，由于偶联位点不固定且条件剧烈，易导致抗体或酶活性降低，抗体与酶的结合物不均一，需要分离去除未结合的酶和抗体，且去除未结合的抗体至关重要，因为游离的抗体会与酶标记抗体竞争相应的抗原，减低结合到固相上的酶标抗体量，因而降低了检测的灵敏度。
由于真菌毒素危害较大，因而寻找一种简单、快速、准确、自动化的检测方法，对于研究真菌毒素的检测和污染控制具有重要意义。
发明内容
本发明的第一个目的在于提供一种真菌毒素磁化学发光免疫分析试剂盒，该试剂盒将化学发光、磁微粒分离和基因工程技术汇聚在一起，具有良好的准确性、灵敏度和重复性。
本发明的第二个目的在于提供上述试剂盒在检测真菌毒素中的应用。
为达到上述目的，本发明采用下述技术方案：
第一方面，本发明提供一种真菌毒素磁化学发光免疫分析试剂盒，该试剂盒包括链霉亲和素磁微粒、生物素标记的真菌毒素抗原、真菌毒素标准品溶液、真菌毒素纳米抗体-碱性磷酸酶(ScFV-AP) 双功能融合蛋白、样本稀释液、洗涤液、底物液。
本发明以微米级磁微粒为载体，利用表面有机物提供的羧基活性基团与链霉亲和素氨基共价结合，采用生物素-链霉亲和素进行“搭桥”形成磁微粒——链霉亲和素——生物素——真菌毒素——ScFV-AP双功能融合蛋白复合物。该技术的新颖之处在于：(1)利用磁微粒为固相载体，增加抗原、抗体的接触面积及底物发光面积，提高反应的灵敏度，并采用旋转磁微粒场使磁微粒起搅拌作用及分离磁微粒——链霉亲和素——生物素——真菌毒素——ScFV-AP双功能融合蛋白复合物和真菌毒素——scFV-AP双功能融合蛋白复合物的作用。(2)scFV-AP双功能融合蛋白提高了反应的特异性及稳定性。(3)食用油可直接加样检测，无需提取、离心等操作，大幅提高检测效率。
进一步的，所述scFV-AP双功能融合蛋白是以碱性磷酸酶(AP)作为生物发光的催化剂，将真菌毒素纳米抗体(scFv)与碱性磷酸酶(AP)融合表达获得。
优选的，真菌毒素纳米抗体(scFV)选自骆驼科或鲨鱼科动物的重链抗体。
进一步的，本发明所述真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列可以是如以下中的任一种或多种：
黄曲霉毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:1所示；
脱氧雪腐镰刀菌烯醇纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:2所示；
玉米赤霉烯酮纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:3所示；
T-2毒素纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:4所示；
伏马毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:5所示；
赭曲霉毒素A纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:6所示。
根据本申请的具体实施方式，依据要检测的毒素种类，选择相应的生物素标记的真菌毒素抗原、真菌毒素标准品溶液、以及真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白。例如，当检测脱氧雪腐镰刀菌烯醇时，本申请的试剂盒中，应包括生物素标记的脱氧雪腐镰刀菌烯醇抗原，脱氧雪腐镰刀菌烯醇标准品溶液，以及脱氧雪腐镰刀菌烯醇纳米抗体-碱性磷酸酶双功能融合蛋白。
进一步的，所述的scFv-AP双功能融合蛋白可以根据上述提供的氨基酸序列进行合成，也可以通过下述方法制备获得：
(1)真菌毒素纳米抗体基因的设计与合成：根据真菌毒素纳米抗体的氨基酸序列，通过优化基因的结构实现基因在大肠杆菌中的高效表达，包括使用大肠杆菌偏爱密码子，消除可能存在的二级结构，使GC/AT均衡，设计并合成真菌毒素纳米抗体基因的核苷酸片段；
(2)真菌毒素纳米抗体-碱性磷酸酶双功能融合表达载体的构建：将步骤(1)合成的真菌毒素纳米抗体基因与碱性磷酸酶基因克隆到原核表达载体中，构建真菌毒素纳米抗体-碱性磷酸酶双功能融合表达载体；
(3)真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白的表达：将双功能融合表达载体转化至表达菌株E.coli BL21或Rosetta DE3的感受态细胞中，诱导表达，收集菌体细胞，加入菌体蛋白提取试剂，离心收集含有可溶性蛋白的上清液；
(4)双功能融合蛋白的纯化：采用Ni2+-NTA亲和层析柱进行融合蛋白的纯化。
本发明将真菌毒素纳米抗体与碱性磷酸酶融合表达，既能克服化学标记方法的各种缺陷，又可获取高比活酶标抗体。所制备的融合蛋白可应用于免疫诊断领域，如化学发光免疫法，酶联免疫吸附法或酶促荧光免疫法等作检测试剂用。相比较传统的化学偶联碱性磷酸酶方式，ScFV-AP双功能融合蛋白，具有如下优势：(1)传统化学偶联方式，工艺流程长，偶联工艺复杂，条件剧烈，导致工艺不稳定，偶联产物得率低且批次之间稳定性不佳。真菌毒素纳米抗体与碱性磷酸酶融合表达简单且工艺稳定，避免了繁琐且低效率的酶与蛋白质的化学交联。(2)传统化学偶联方式，为了获取较佳的偶联产物，通常需要对目标偶联产物进行纯化，从而去除未连接的酶与抗体分子，纯化不完全，在检测时会产生假阳性结果。真菌毒素纳米抗体与碱性磷酸酶融合表达方法则克服了这一缺点。(3)传统化学偶联方式，偶联后的酶与抗体复合物不均一，而真菌毒素纳米抗体与碱性磷酸酶融合表达产物中真菌毒素纳米抗 体与单体碱性磷酸酶分子比例为1:1或1:2，且在镍柱纯化时可分离开，保证酶与抗体复合物均一。(4)传统化学偶联方式，由于偶联化学活性试剂结合位置不固定，可能会结合到抗体可变区，从而影响抗体与目标抗原结合；或者偶联化学活性试剂结合到碱性磷酸酶活性位点附近，影响碱性磷酸酶与底物结合；这两种情况均可导致偶联复合物活性下降，在检测时易得到假阴性的结果。而真菌毒素纳米抗体与碱性磷酸酶融合表达方式，不影响真菌毒素纳米抗体与碱性磷酸酶活性。(5)本发明提供的scFv-AP融合蛋白，该融合蛋白具有较好特异性和信号放大作用，采用scFv-AP融合蛋白建立超敏的生物发光免疫分析试剂盒，用于检测粮油、食品、饲料和中草药等基质中真菌毒素的含量，具有良好的应用价值及前景。(6)纳米抗体相较于单链抗体，在抗体稳定性，批间重复性、效价等方面有较大优势，除本发明所示意的，其他所有抗体分子均可通过这种方式生产碱性磷酸酶偶联分子。
进一步的，所述磁微粒是以四氧化三铁或三氧化二铁超顺磁性材料为内核，***包覆聚苯乙烯或葡聚糖，通过物理或化学的方法活化使磁微粒表面产生氨基(NH2-)、甲苯磺酰基(Tosyl)、羧基(COOH-)或环氧基(—CH(O))基团，粒径为1-2μm。
进一步的，所述链霉亲和素磁微粒是将链霉亲和素与磁微粒偶联。
进一步的，所述生物素标记的真菌毒素抗原是先将真菌毒素和牛血清白蛋白偶联，再将得到的牛血清白蛋白-真菌毒素复合物和生物素偶联。
进一步的，所述真菌毒素标准品溶液是将真菌毒素标准品溶于甲醇-水混合溶液中配制而成。根据本发明的具体实施方式，试剂盒中的真菌毒素标准品溶液可以是一定浓度的母液，使用时根据需要稀释成不同浓度的标准品工作液，用于标准曲线的绘制；也可以在试剂盒中直接分装放置一系列浓度的真菌毒素标准品溶液，用于标准曲线的绘制。
优选的，所述甲醇-水混合溶液中，甲醇与水的体积比为50:50。
进一步的，所述底物液是浓度为0.5-2mmol/L的(4-氯苯巯基)(10-甲基-9,10-二氢化吖啶亚甲基)磷酸二钠盐溶液。
优选的，所述样本稀释液由0.01M磷酸盐缓冲液、0.1％吐温20和0.5％牛血清白蛋白组成。
优选的，所述洗涤液由0.01MTris-HCl缓冲液和0.1％吐温20组成。
进一步的，所述试剂盒还包括反应管；优选的，所述反应管的材料为透明聚苯乙烯、聚乙烯、聚丙烯或玻璃。
第二方面，本发明还要求保护上述试剂盒在真菌毒素检测中的应用，特别是在检测粮油、食品、饲料或中草药中真菌毒素的应用。
进一步的，所述食用油包括花生油、玉米油、大豆油、菜籽油、葵花籽油、油菜籽油、芝麻油、橄榄油中的一种。
进一步的，所述真菌毒素包括黄曲霉毒素、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、伏马毒素、赭曲霉毒素A、T-2毒素中的一种或多种。
本发明采用间接竞争的原理测定食品、农产品及饲料中的真菌毒素，在链霉亲和素磁微粒悬浮液中加入生物素标记的真菌毒素抗原工作液、scFV-AP双功能融合蛋白和待测样本。样本中的真菌毒素与生物素标记的真菌毒素抗原竞争数量有限的ScFV-AP双功能融合蛋白，通过链霉亲和素和生物素的亲和反应以及抗原抗体反应，分别形成磁微粒-链霉亲和素-生物素-真菌毒素-scFV-AP双功能融合蛋白复合物和真菌毒素-scFV-AP双功能融合蛋白复合物，磁微粒在外加磁微粒场中直接沉淀，用磁微粒场将复合物吸附在试剂管底部，清洗掉游离的成分，加入底物液，碱性磷酸酶催化底物的磷酸根水解后，立即发生分解反应释放出475nm的光子，于5min内测定各加样孔的发光值(RLU)。样本的RLU与样本中真菌毒素的浓度呈负相关。样本中的真菌毒素浓度依据由真菌毒素标准品浓度和对应的RLU建立的四参数数学模型进行定量，从而检测样本中的真菌毒素含量。
进一步，上述检测可以使用全自动化学发光免疫分析仪，从而实现真菌毒素全自动化检测。每个试剂盒互不干扰，可即时多样本同时检测，高度切合了真菌毒素现场快速检测的发展需求。此外，使用本发明试剂盒检测真菌毒素还具有以下优点：(1)检测速度快、稳定、无放射性污染，在没有磁微 粒场存在的情况下，磁微粒悬浮在液体中，使得抗原-抗体反应类似于均相反应；磁微粒在外加磁微粒场的作用下可方便地分离、洗涤快速，可在24分钟内判断检测结果。(2)灵敏度高。(3)特异性强。(4)精密度良好。(5)链霉亲和素与生物素级联放大体系，链霉亲和素磁微粒，生物素标记的真菌毒素抗原，较磁微粒直接联真菌毒素抗原，大大提高了反应效率，操作简单，同时增加了试剂盒的有效期，经37℃加速稳定性及2-8℃真实稳定性试验证实，本产品在37℃可存放7天以上，在2-8℃可存放1年。(6)成本低，与市场上同类产品比较，本试剂盒性能良好，成本低，具有实际应用价值。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1为基于双功能融合蛋白的真菌毒素磁化学发光免疫分析原理图。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
下述实施例中所用的试验材料，如无特殊说明，均为常规生化试剂供应商购买得到。
实施例1制备黄曲霉毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白
AFB1-scFV-AP双功能融合蛋白是以碱性磷酸酶作为生物发光的催化剂，将黄曲霉毒素B1纳米抗体(AFB1-scFv)与碱性磷酸酶(AP)融合表达获得的。
AFB1-scFV-AP双功能融合蛋白是通过下述方法制备的：
(1)AFB1纳米抗体基因的设计与合成：根据AFB1纳米抗体的氨基酸序列SEQ ID NO:7，通过优化基因的结构实现基因在大肠杆菌中的高效表达，包括使用大肠杆菌偏爱密码子，消除可能存在的二级结构，使GC/AT均衡等，设计并合成AFB1纳米抗体基因的核苷酸片段(核苷酸序列如SEQ ID NO:8所示)。
(2)AFB1-scFV-AP表达载体的构建：将步骤(1)合成的黄曲霉毒素B1纳米抗体基因克隆到原核表达载体pET25b(+)中，得到pET25b(+)-scFV；然后用限制性内切酶XhoI和NotI双酶切碱性磷酸酶表达载体pET25b(+)-AP(由苏州泓迅生物科技股份有限公司提供)，得到碱性磷酸酶基因片段；琼脂糖凝胶回收后，利用T4DNA连接酶按摩尔比1：3将碱性磷酸酶基因片段连接到经限制性内切酶XhoI和NotI双酶切的pET25b(+)-scFV表达载体上，通过热击法(42℃，45s)将连接产物转化到大肠杆菌DH5α感受态细胞中，将转化的细菌接种到含有100μg/mL氨苄青霉素(Amp)的LB平板上，37℃过夜培养。采用菌落聚合酶链式反应(PCR)和双酶切验证进行克隆初步筛选，将筛选的阳性克隆进行测序，最终得到双功能融合表达载体AFB1-scFV-AP。
(3)AFB1-scFv-AP双功能融合蛋白的表达：将双功能融合表达载体热击转化至表达菌株E.coli BL21(Rosetta，DE3)的感受态细胞中，均匀涂布于含有100μg/mL Amp和34μg/mL氯霉素(CAP)的LB平板上，37℃倒置培养12h。挑取单菌落接入5mL LB/Amp/CAP液体培养基中，37℃220rpm摇振培养12h；按1％接种量将上述培养物转接至200mL LB/Amp/CAP液体培养基中，37℃，220rpm摇振培养至OD600为0.4～0.6；加入IPTG进行诱导培养，移取1mL诱导后培养物离心收集菌体细胞并于-20℃冻存备用，5000g离心10min收集剩余菌体；在上述每克剩余菌体中加入4mL菌体蛋白提取试剂(Bacterical Protein Extraction Reagent,B-PER)，且每毫升B-PER中含有2μL溶菌酶和2μL核酸内切酶DNase I，充分重悬菌体后室温静置10-15min；15000g离心5min，收集含有可溶性蛋白的上清液，采用SDS-PAGE和Western blot分析目的蛋白的表达情况。通过改变IPTG的终浓度(0mmol/L～1mmol/L，诱导温度为16℃)和诱导温度(16℃，20℃，30℃，37℃，IPTG终浓度为0.5mmol/L)对原核表达条件进行优化，探究融合蛋白的最佳表达条件。
(4)双功能融合蛋白的纯化与表征：将最佳表达条件下的上清液经0.45μm无菌过滤器除菌后上样到Ni2+-NTA亲和层析柱中，用十倍柱床体积的结合缓冲液(TBS缓冲液，pH 8.0)洗涤后，用含不同浓度(20mmol/L、50mmol/L、100mmol/L、200mmol/L)咪唑的结合缓冲液洗脱，收集各洗 脱组分。利用12％SDS-PAGE表征表达产物及双功能融合蛋白的纯化情况，使用Image J软件调节凝胶图片的灰度，对洗脱后的蛋白质条带进行灰度分析，确定纯化后融合蛋白的纯度。
通过上述方法，最终得到纯化后的AFB1-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO.1所示。
(5)双功能融合蛋白的活性分析：
双功能融合蛋白的碱性磷酸酶活性测定：对硝基苯磷酸二钠法。在酶标板中，分别加入20μL稀释50，100，200，400，800，1000倍的双功能融合蛋白和AP，再向孔中添加100μL PNPP试剂(2mg/mL，溶于含有2mM MgCl2的0.1M二乙醇胺缓冲液，pH9.8)，混匀后，置于37℃下孵育10min，最后加入50μL 4M NaOH溶液终止反应，利用微孔板酶标仪测定405nm处的吸光度，分别计算对硝基苯酚的浓度(mM)。酶活力(U/mL)＝对硝基苯酚浓度/反应时间*样品稀释倍数，酶的比活力(U/mg)＝酶活力(U/mL)/蛋白浓度(mg/mL)。通过比较双功能融合蛋白和AP的比活力大小，评估双功能融合蛋白的碱性磷酸酶活性。
双功能融合蛋白的抗体亲和力测定：生物膜干涉技术。用PBS缓冲液平衡传感器60s后，加入生物素标记的目标真菌毒素进行固定，用PBST缓冲液(含0.02％Tween-20的PBS缓冲液，pH 7.4)封闭180s。在30℃的恒温条件下观察不同梯度下双功能融合蛋白及特异性纳米抗体与目标真菌毒素的结合和解离情况，并通过Octet RED96e数据处理程序计算双功能融合蛋白和特异性纳米抗体与目标真菌毒素的Kon值、Koff值和KD值(Kon值/Koff值)，通过比较KD值的大小表征双功能融合蛋白的抗体亲和力情况。Kon表示抗原抗体复合物的形成速率，Kon值越大，抗原抗体结合越快；Koff反映了形成的复合物的稳定性，Koff值越大，复合物解离越快；KD则可以反映抗原与抗体相互作用的结合能力的大小，当KD值达到10-8mol/L时，表明抗原抗体之间具有很高的亲和性。
实施例2制备脱氧雪腐镰刀菌烯醇纳米抗体-碱性磷酸酶双功能融合蛋白
使用实施例1的方法，制备DON-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO:2所示。
实施例3制备玉米赤霉烯酮纳米抗体-碱性磷酸酶双功能融合蛋白
使用实施例1的方法，制备ZEN-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO:3所示。
实施例4制备T-2毒素纳米抗体-碱性磷酸酶双功能融合蛋白
使用实施例1的方法，制备T-2-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO:4所示。
实施例5制备伏马毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白
使用实施例1的方法，制备FB1-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO:5所示。
实施例6制备赭曲霉毒素A纳米抗体-碱性磷酸酶双功能融合蛋白
使用实施例1的方法，制备OTA-scFV-AP双功能融合蛋白，其氨基酸序列如SED ID NO:6所示。
实施例7制备AFB1磁化学发光免疫分析试剂盒
1.链霉亲和素磁微粒制备：
(1)磁微粒的分散：将10mg磁微粒用10mL 0.1M 2-吗啉乙磺酸溶液(MES缓冲液)分散，使磁微粒的终浓度为1mg/mL；
(2)磁微粒的清洗：用3倍磁微粒溶液体积的MES缓冲液清洗磁微粒后，再用MES缓冲液重新分散，使磁微粒的终浓度为1mg/mL；
(3)磁微粒的活化与链接：将3mg链霉亲和素和3.5μL 10mg/mL碳二亚胺盐酸盐溶液依次加入步骤(2)所得的磁微粒分散液中，室温下搅拌反应2小时；
(4)磁微粒的封闭：步骤(3)中将磁微粒与反应体系分离后，用MES缓冲液洗涤磁微粒2次，再用MES缓冲液重新分散磁微粒，磁微粒的终浓度为2-30mg/mL；然后加入2％甘氨酸封闭液，室温下搅拌反应2小时；
(5)磁微粒的清洗：将步骤(4)的磁微粒与反应体系分离，用pH7.4的磷酸盐缓冲液清洗磁微粒珠两次，获得链霉亲和素磁微粒；
(6)使用时，将链霉亲和素磁微粒配置成链霉亲和素磁微粒工作液：将步骤(5)中的链霉亲和 素磁微粒分散于0.5g/mL酪蛋白、1g/mL PEG200、0.2％吐温20、0.5％牛血清白蛋白的磷酸盐缓冲液，pH值为7-8，磁微粒的终浓度为1mg/mL，即得。
2.生物素标记的AFB1抗原制备：
(1)将AFB1与羧甲基羟氨半盐酸盐置于吡啶中，室温避光搅拌反应24小时，得到第一反应液；
(2)将所述第一反应液冷冻干燥，得到固体干燥物；
(3)将固体干燥物用纯水溶解后，并用盐酸调节pH为3.0，用乙酸乙酯抽提沉淀物，真空干燥后得到AFB1肟；
(4)将AFB1肟溶解于KOH的吡啶溶液中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，在70℃的温度下反应12小时，得到第二反应液；
(5)向第二反应液中加入N-羟基琥珀酰亚胺，室温搅拌反应2小时，得到第三反应液；将所述第三反应液；
(6)将阳离子牛血清白蛋白用蒸馏水溶解后，加入至第三反应液中，避光室温搅拌下反应24小时，得到第四反应液；
(7)将第四反应液在0.01摩尔/升的磷酸盐缓冲液中透析3天，将透析液冷冻干燥得到AFB1-牛血清白蛋白复合物；
(8)将AFB1-牛血清白蛋白复合物与PBS缓冲液混合，加入活化生物素标记2h，而后透析得到所述生物素标记的AFB1抗原；
(9)使用时，将生物素标记的AFB1抗原用0.01M磷酸盐缓冲液和0.5％牛血清白蛋白溶液按1：15000比例稀释得到生物素标记的AFB1抗原工作液。
3.AFB1-scFV-AP双功能融合蛋白工作液的制备
将实施例1制备的AFB1-scFV-AP双功能融合蛋白用0.01M Tris盐缓冲液和0.5％牛血清白蛋白溶液按1：20000比例稀释得到AFB1-scFV-AP双功能融合蛋白工作液。
4.样品稀释液的配制：
样品稀释液由0.01M磷酸盐缓冲液、0.1％吐温20和0.5％牛血清白蛋白组成。
5.洗涤液的配制
洗涤液由0.01M Tris-HCl缓冲液和0.1％吐温20组成。
6.AFB1标准品溶液的配制
将AFB1标准品用甲醇-水(50：50)溶液稀释至工作浓度，分别为0.25ng/mL，0.5ng/mL，1ng/mL，2.5ng/mL，5ng/mL，10ng/mL，20ng/mL，即为各标准品点浓度。
7.底物液的制备：
将(4-氯苯巯基)(10-甲基-9,10-二氢化吖啶亚甲基)磷酸二钠盐用0.01M PBS稀释成1mmol/L得到底物液。
8.组装：将上述试剂组装成盒，2-8℃储存。
基于双功能融合蛋白的AFB1磁化学发光免疫分析原理图如图1所示。
实施例8黄曲霉毒素B1磁化学发光免疫分析试剂盒检测黄曲霉毒素B1的方法学考察
对实施例7制备的AFB1磁化学发光免疫分析试剂盒进行检测黄曲霉毒素B1的方法学考察试验。
(1)稳定性实验：采用先进的稳定剂(0.01M磷酸盐缓冲液、0.1％牛血清白蛋白溶液、0.1％proclin 300和1％海藻糖)处理，处理后37℃保存进行加速稳定性试验测试，测得稳定性结果见表1，根据经验在37℃下保存1天相当于4℃保存2个月，即本试剂盒在4℃保存12个月，不影响使用。
(2)灵敏度分析
通过对20个独立的玉米阴性样品进行测定，该方法检出限LOD以阴性样品20次测定结果的均值加3倍标准偏差计算，结果为0.2μg/kg，定量限LOQ以阴性样品20次测定结果的均值加10倍标准偏差计算，结果为1.0μg/kg。
(3)线性关系考察
①标准曲线的绘制：根据黄曲霉毒素B1标准品溶液的浓度-发光值系列数值进行四参数拟合，获得基于的浓度-发光值标准曲线。
②样本浓度的计算：在一定范围内发光值与黄曲霉毒素B1抗原浓度呈反比，根据拟合的黄曲霉毒素B1的浓度-发光值标准曲线计算待测样本的黄曲霉毒素B1含量。
(4)黄曲霉毒素B1磁化学发光免疫分析试剂盒特异性考察
将黄曲霉毒素B1磁化学发光免疫分析试剂用于检测结构类似物(AFB1，AFB2，AFG1和AFG2)和其他常见真菌毒素(脱氧雪腐镰刀菌烯醇，DON；伏马毒素B1，FB1；；伏马毒素B1，FB2；伏马毒素B1，FB3；玉米赤霉烯酮，ZEN；T-2毒素，T-2；赭曲霉毒素A，OTA；3-乙酰脱氧雪腐镰刀菌烯醇，3-ADON；15-乙酰脱氧雪腐镰刀菌烯醇，15-ADON；脱氧雪腐镰刀菌烯醇-3-葡萄糖苷，DON-3G；雪腐镰刀菌烯醇，NIV)，结果如表1所示，仅AFB2和AFG1存在较为明显的交叉反应，交叉反应率分别为26.3％和23.8％，其他毒素无交叉(交叉反应率小于1％)，表明黄曲霉毒素B1磁化学发光免疫分析试剂盒特异性良好。
表1.黄曲霉毒素B1磁化学发光免疫分析试剂盒特异性考察
实施例9黄曲霉毒素B1磁化学发光免疫分析试剂盒的使用
(1)待测样品前处理：称取5.0g粉碎后的待测样品于50mL离心管中，加入25.0mL甲醇/水(70：30)溶液，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5 min，得到待测样品提取液；
(2)黄曲霉毒素B1磁化学发光免疫分析试剂盒(实施例7制备)的使用步骤：
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或黄曲霉毒素B1标准品溶液，50μL链霉亲和素磁微粒工作液，50μL黄曲霉毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)黄曲霉毒素B1毒素磁化学发光免疫分析试剂盒的准确性和重复性
分别称取5g阴性糙米、大米、玉米、花生酱、小麦、和花生油样品，并加入不同量的黄曲霉毒素B1标准品，7个平行，用黄曲霉毒素B1磁化学发光免疫分析试剂盒加标后的样品提取液进行测定，7个平行的回收率、均值及RSD如表2、表3、表4、表5、表6和表7所示，回收率在79.1％-120％之间，RSD小于10％。
表2.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定糙米中AFB1的回收率和重复性
表3.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定大米的回收率和重复性
表4.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定玉米的回收率和重复性

表5.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定花生酱的回收率和重复性
表6.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定小麦的回收率和重复性
表7.黄曲霉毒素B1磁化学发光免疫分析试剂盒测定花生油的回收率和重复性

实施例10脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒检测脱氧雪腐镰刀菌烯醇的方法学考察
参考实施例7制备脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒并进行检测脱氧雪腐镰刀菌烯醇的方法学考察。
(1)稳定性实验：采用先进的稳定剂(0.01M磷酸盐缓冲液、0.1％牛血清白蛋白溶液、0.1％proclin300和1％海藻糖)处理，处理后37℃保存进行加速稳定性试验测试，测得稳定性结果见表1，根据经验在37℃下保存1天相当于4℃保存2个月，即本试剂盒在4℃保存12个月，不影响使用。
(2)灵敏度分析
通过对20个独立的玉米阴性样品进行测定，该方法检出限LOD以阴性样品20次测定结果的均值加3倍标准偏差计算，结果为77.0μg/kg，定量限LOQ以阴性样品20次测定结果的均值加10倍标准偏差计算，结果为222.8μg/kg。
(3)线性关系考察
①标准曲线的绘制：根据脱氧雪腐镰刀菌烯醇标准品溶液的浓度-发光值系列数值进行四参数拟合，获得基于的浓度-发光值标准曲线。
②样本浓度的计算：在一定范围内发光值与脱氧雪腐镰刀菌烯醇抗原浓度呈反比，根据拟合的脱氧雪腐镰刀菌烯醇的浓度-发光值标准曲线计算待测样本的脱氧雪腐镰刀菌烯醇含量。
(4)脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒特异性考察
将脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂用于检测结构类似物(3-乙酰脱氧雪腐镰刀菌烯醇(3-ADON)，15-乙酰脱氧雪腐镰刀菌烯醇(15-ADON)和雪腐镰刀菌烯醇，NIV)和其他常见真菌毒素(黄曲霉毒素B1，DON玉米赤霉烯酮，ZEN；T-2毒素，T-2和赭曲霉毒素A，OTA)，结果如表8所示，仅3-ADON存在较为明显的交叉反应，其他毒素无交叉，表明脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒特异性良好。
表8.脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒的特异性

实施例11脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒的使用
(1)待测样品前处理：称取5.0g粉碎后的待测样品于50mL离心管中，加入25.0mL水，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5min，得到待测样品提取液；
(2)脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒的使用步骤
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将待测试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或脱氧雪腐镰刀菌烯醇标准品溶液，50μL链霉亲和素磁微粒工作液，50μLDON-scFV-AP双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒的准确性和重复性
分别称取5g阴性玉米、小麦和面粉样品，并加入不同量的脱氧雪腐镰刀菌烯醇标准品，7个平行，用脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒加标后的样品提取液进行测定，7个平行的回收率、均值及RSD如表9、表10和表11所示，回收率在80％-120％之间，RSD小于9％。。
表9.脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒测定玉米中DON的准确性和重复性
表10.脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒测定小麦中DON的准确性和重复性

表11.脱氧雪腐镰刀菌烯醇磁化学发光免疫分析试剂盒测定面粉中DON的准确性和重复性
实施例12玉米赤霉烯酮磁化学发光免疫分析试剂盒的使用
参考实施例7制备玉米赤霉烯酮磁化学发光免疫分析试剂盒。
(1)待测样品前处理：称取5.0g粉碎后的待测样品于50mL离心管中，加入25.0mL80％乙腈水溶液，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5min，得到待测样品提取液；
(2)玉米赤霉烯酮磁化学发光免疫分析试剂盒的使用步骤
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将待测试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或玉米赤霉烯酮标准品溶液，50μL链霉亲和素磁微粒工作液，50μLZEN-scFV-AP双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)玉米赤霉烯酮磁化学发光免疫分析试剂盒的灵敏度分析
通过对20个独立的玉米阴性样品进行测定，该方法检出限LOD以阴性样品20次测定结果的均值加3倍标准偏差计算，结果为3.9939μg/kg，定量限LOQ以阴性样品20次测定结果的均值加10倍标准偏差计算，结果为9.4483μg/kg。
(4)玉米赤霉烯酮磁化学发光免疫分析试剂盒的准确性和重复性
分别称取5g阴性玉米、小麦、糙米和面粉样品，并加入不同量的玉米赤霉烯酮标准品，7个平行，用玉米赤霉烯酮磁化学发光免疫分析试剂盒加标后的样品提取液进行测定，7个平行的回收率、均值及RSD如表12、表13、表14和表15所示，回收率在80％-120％之间，RSD小于10％。
表12.玉米赤霉烯酮磁化学发光免疫分析试剂盒测定玉米中ZEN的准确性和重复性

表13.玉米赤霉烯酮磁化学发光免疫分析试剂盒测定小麦中ZEN的准确性和重复性
表14.玉米赤霉烯酮磁化学发光免疫分析试剂盒测定糙米中ZEN的准确性和重复性
表15.玉米赤霉烯酮磁化学发光免疫分析试剂盒测定面粉中ZEN的准确性和重复性

实施例13 T-2毒素磁化学发光免疫分析试剂盒的使用
参考实施例7制备T-2毒素磁化学发光免疫分析试剂盒。
(1)待测样品前处理：称取5.0g粉碎后的玉米阴性样品于50mL离心管中，分别加入不同量的T-2毒素标准品，最终加标水平分别为47.8μg/Kg和108μg/Kg，7个平行，加入25.0mL80％乙腈水溶液，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5min，得到待测样品提取液；
(2)T-2毒素磁化学发光免疫分析试剂盒的使用步骤
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将待测试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或T-2毒素标准品溶液，50μL链霉亲和素磁微粒工作液，50μLT-2-scFV-AP双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)T-2毒素磁化学发光免疫分析试剂盒的准确性和重复性
用T-2毒素磁化学发光免疫分析试剂盒对步骤(1)得到的待测样品提取液进行测定，7个平行的回收率、均值及RSD如表16所示，回收率在89.0％-107.5％之间，RSD小于3％。
表16.T-2毒素磁化学发光免疫分析试剂盒的准确性和重复性
实施例14伏马毒素B1磁化学发光免疫分析试剂盒的使用
参考实施例7制备伏马毒素B1磁化学发光免疫分析试剂盒。
(1)待测样品前处理：称取5.0g粉碎后的玉米阴性样品于50mL离心管中，分别加入不同量的FB1标准品，最终加标水平分别为47.8μg/Kg和108μg/Kg，7个平行，加入25.0mL80％乙腈水溶液，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5min，得到待测样品提取液；
(2)伏马毒素B1毒素磁化学发光免疫分析试剂盒的使用步骤
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将待测试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或伏马毒素B1毒素标准品溶液，50μL链霉亲和素磁微粒工作液，50μLFB1-scFV-AP双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)FB1毒素磁化学发光免疫分析试剂盒的准确性和重复性
用FB1毒素磁化学发光免疫分析试剂盒对步骤(1)得到的待测样品提取液进行测定，7个平行的回收率、均值及RSD如表17所示，回收率在89.2％-111.3％之间，RSD小于6％。
表17.FB1毒素磁化学发光免疫分析试剂盒的准确性和重复性
实施例15赭曲霉毒素A磁化学发光免疫分析试剂盒的使用
参考实施例7制备赭曲霉毒素A磁化学发光免疫分析试剂盒。
(1)待测样品前处理：称取5.0g粉碎后的样品于50mL离心管中，加入25.0mL80％乙腈水溶液，置于多管涡旋混匀仪上2500rpm涡旋5min(或使用高速均质器上11000rpm均质3min，或摇床上200rpm振荡40min)在检测管中加入20μL待测样品提取液，然后7000rpm离心5min，得到待测样品提取液；
(2)赭曲霉毒素A磁化学发光免疫分析试剂盒的使用步骤
①物理检查：液体组分应澄清，无沉淀或絮状物；其他组分应无包装破损。
②将待测试剂盒在室温(2-8℃)下平衡30min；
③样本测定：将40-200μL待测样品提取液加入样本孔中，放置于全自动化学发光免疫分析仪中，点击运行后，仪器依据预先设置好的运行程序依次加入50μL待测样品提取液或赭曲霉毒素A标准品溶液，50μL链霉亲和素磁微粒工作液，50μLOTA-scFV-AP双功能融合蛋白以及400μL稀释液，混匀，37℃温育5min；依次洗涤3次后，测度发光值，并根据内置的四参数拟合曲线进行结果分析，可20min内完成8个样本的测定，并出具检测报告。
(3)赭曲霉毒素A磁化学发光免疫分析试剂盒的灵敏度
过对20个独立的小麦阴性样品进行测定，该方法检出限LOD以阴性样品20次测定结果的均值加3倍标准偏差计算，结果为2.0610μg/kg，定量限LOQ以阴性样品20次测定结果的均值加10倍标准偏差计算，结果为4.5105μg/kg。
(4)赭曲霉毒素A磁化学发光免疫分析试剂盒的准确性和重复性
分别称取5g阴性玉米、小麦和糙米样品，并加入不同量的赭曲霉毒素A标准品，7个平行，用 赭曲霉毒素A磁化学发光免疫分析试剂盒加标后的样品提取液进行测定，7个平行的回收率、均值及RSD如表18、表19和表20所示，回收率在80％-120％之间，RSD小于8％。
表18.赭曲霉毒素A磁化学发光免疫分析试剂盒测定玉米中OTA的准确性和重复性
表19.赭曲霉毒素A磁化学发光免疫分析试剂盒测定小麦中OTA的准确性和重复性
表20.赭曲霉毒素A磁化学发光免疫分析试剂盒测定糙米中OTA的准确性和重复性

显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (10)

  1. 一种真菌毒素磁化学发光免疫分析试剂盒，其特征在于，该试剂盒包括链霉亲和素磁微粒、生物素标记的真菌毒素抗原、真菌毒素标准品溶液、真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白、样本稀释液、洗涤液、底物液。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白是以碱性磷酸酶作为生物发光的催化剂，将真菌毒素纳米抗体与碱性磷酸酶融合表达获得；
    优选的，真菌毒素纳米抗体选自骆驼科或鲨鱼科动物的重链抗体。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如以下中的任一种或多种：
    黄曲霉毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:1所示；
    脱氧雪腐镰刀菌烯醇纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:2所示；
    玉米赤霉烯酮纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:3所示；
    T-2毒素纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:4所示；
    伏马毒素B1纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:5所示；
    赭曲霉毒素A纳米抗体-碱性磷酸酶双功能融合蛋白的氨基酸序列如SEQ ID NO:6所示。
  4. 根据权利要求2或3所述的试剂盒，其特征在于，所述的真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白是通过下述方法制备的：
    (1)真菌毒素纳米抗体基因的设计与合成：根据真菌毒素纳米抗体的氨基酸序列，通过优化基因的结构实现基因在大肠杆菌中的高效表达，包括使用大肠杆菌偏爱密码子，消除可能存在的二级结构，使GC/AT均衡，设计并合成真菌毒素纳米抗体基因的核苷酸片段；
    (2)真菌毒素纳米抗体-碱性磷酸酶双功能融合表达载体的构建：将步骤(1)合成的真菌毒素纳米抗体基因与碱性磷酸酶基因克隆到原核表达载体中，构建真菌毒素纳米抗体-碱性磷酸酶双功能融合表达载体；
    (3)真菌毒素纳米抗体-碱性磷酸酶双功能融合蛋白的表达：将双功能融合表达载体转化至表达菌株E.coli BL21或Rosetta DE3的感受态细胞中，诱导表达，收集菌体细胞，加入菌体蛋白提取试剂，离心收集含有可溶性蛋白的上清液；
    (4)双功能融合蛋白的纯化：采用Ni2+-NTA亲和层析柱进行融合蛋白的纯化。
  5. 根据权利要求1所述的试剂盒，其特征在于，所述磁微粒是以四氧化三铁或三氧化二铁超顺磁性材料为内核，***包覆聚苯乙烯或葡聚糖，通过物理或化学的方法活化使磁微粒表面产生氨基、甲苯磺酰基、羧基或环氧基基团，粒径为1-2μm。
  6. 根据权利要求1所述的试剂盒，其特征在于，所述链霉亲和素磁微粒是将链霉亲和素与磁微粒偶联。
  7. 根据权利要求1所述的试剂盒，其特征在于，所述生物素标记的真菌毒素抗原是先将真菌毒素和牛血清白蛋白偶联，再将得到的牛血清白蛋白-真菌毒素复合物和生物素偶联。
  8. 根据权利要求1所述的试剂盒，其特征在于，所述真菌毒素标准品溶液是将真菌毒素标准品溶于甲醇-水混合溶液中配制而成；
    优选的，所述甲醇-水混合溶液中，甲醇与水的体积比为20:80-80:20。
  9. 根据权利要求1所述的试剂盒，其特征在于，所述底物液是浓度为0.5-2mmol/L的(4-氯苯巯基)(10-甲基-9,10-二氢化吖啶亚甲基)磷酸二钠盐溶液；
    优选的，所述样本稀释液由0.01M磷酸盐缓冲液、0.1％吐温20和0.5％牛血清白蛋白组成；
    优选的，所述洗涤液由0.01 MTris-HCl缓冲液和0.1％吐温20组成；
    优选的，所述试剂盒还包括反应管；
    优选的，所述反应管的材料为透明聚苯乙烯、聚乙烯、聚丙烯或玻璃。
  10. 权利要求1-9任一所述的试剂盒在检测真菌毒素中的应用；
    优选的，所述试剂盒在检测食用油、食品、粮食、饲料或中草药中真菌毒素的应用；
    优选地，所述食用油包括花生油、玉米油、大豆油、菜籽油、葵花籽油、油菜籽油、芝麻油、橄榄油中的一种；
    优选的，使用真菌毒素全自动分析仪进行真菌毒素检测。
    优选的，所述真菌毒素包括黄曲霉毒素、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、伏马毒素、赭曲霉毒素A、T-2毒素中的一种或多种。
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