WO2018161905A1 - 同步检测黄曲霉毒素b1等五种真菌毒素混合污染的免疫层析时间分辨荧光试剂盒及应用 - Google Patents

同步检测黄曲霉毒素b1等五种真菌毒素混合污染的免疫层析时间分辨荧光试剂盒及应用 Download PDF

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WO2018161905A1
WO2018161905A1 PCT/CN2018/078198 CN2018078198W WO2018161905A1 WO 2018161905 A1 WO2018161905 A1 WO 2018161905A1 CN 2018078198 W CN2018078198 W CN 2018078198W WO 2018161905 A1 WO2018161905 A1 WO 2018161905A1
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monoclonal antibody
time
ochratoxin
aflatoxin
fumonisin
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PCT/CN2018/078198
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English (en)
French (fr)
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张兆威
李培武
张奇
王督
张文
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中国农业科学院油料作物研究所
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Priority to US16/492,303 priority Critical patent/US20210132066A1/en
Publication of WO2018161905A1 publication Critical patent/WO2018161905A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7759—Dipstick; Test strip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/8483—Investigating reagent band
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/40—Rare earth chelates

Definitions

  • the invention relates to a mycotoxin immunochromatography time-resolved fluorescent kit, in particular to an immunochromatography time-resolved fluorescent kit for synchronously detecting mixed contamination of five mycotoxins such as aflatoxin B1 and application thereof.
  • Mycotoxins are a series of toxic and harmful substances produced by the growth and metabolism of fungi in grain oil or feed. At present, there are more than 400 kinds of mycotoxins found in nature. According to the main toxigenic strains, mycotoxins can be classified into aspergillus toxins (such as aflatoxins, variegated toxins, etc.), penicillin toxins, and fusarium toxins (such as zearalenone). Mycotoxins have carcinogenic, teratogenic and mutagenic effects, which can cause acute or chronic poisoning in humans and seriously endanger human health. Mycotoxins contaminate crops, food and feed, posing a huge threat to the health of humans and livestock.
  • aspergillus toxins such as aflatoxins, variegated toxins, etc.
  • penicillin toxins such as zearalenone
  • fusarium toxins such as zearalenone
  • the main fungal toxins currently contaminating agricultural products such as food and feed include aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone, and variegyr toxin.
  • Aflatoxins are mainly found in cereals and legumes. Aflatoxins are also detected in milk, dairy products, eggs and meat if the birds are exposed to aflatoxin-contaminated feed. Aflatoxins are mainly infected by food digestion. Long-term skin contact can also cause chronic infection, causing acute or chronic liver damage, acute hepatitis, hepatic steatosis, and eventually liver cancer.
  • Fumonisin is a water-soluble metabolite produced by Fusarium oxysporum. It consists mainly of diesters composed of polyhydric alcohol and triglyceride. 11 fumonisins have been discovered, of which fumonisin B1 is the most serious and mainly infects corn. , sorghum, rice and other crops. Fumonisin is a carcinogen that causes brain edema, brain necrosis and liver toxicity, and severely damages the immune system. Ochratoxin is mainly infected with cereal crops and legumes such as corn, barley, wheat, coffee beans, peas, cocoa beans, and the like.
  • Ochratoxin is absorbed in the gastrointestinal tract of the body, such as the duodenum and jejunum, and has strong nephrotoxicity.
  • Zearalenone is widely polluted and can be detected in cereals and agricultural by-products on all continents, contaminating corn, oats, wheat, barley, millet and other agricultural products, as well as dairy products, beef and other foods.
  • the variegated toxin is mainly a secondary metabolite produced by fungi such as Aspergillus flavus, Aspergillus variabilis, Aspergillus nidulans, Aspergillus fumigatus, etc. Its toxicity is second only to aflatoxin, which can induce liver cancer, lung cancer and other tumors.
  • the detection methods of mycotoxins mainly include thin layer chromatography, enzyme-linked immunosorbent assay, liquid chromatography, and liquid chromatography-mass spectrometry.
  • Thin-layer chromatography is simple and convenient, but has poor reproducibility and low precision.
  • Enzyme-linked immunosorbent assay has strong specificity and simple pretreatment, but the false positive rate is high, which cannot be used as a confirmatory method.
  • Liquid chromatography, liquid chromatography-mass spectrometry The stability is good and the sensitivity is high, but the sample preparation process is complicated, the detection time is long, the instruments used are expensive, and the requirements for the experimental environment and the inspection personnel are high, and it is difficult to achieve rapid detection.
  • Immunochromatographic fluorescence detection technology is an immunoassay developed in the early 1980s. It is a rapid detection technology developed on the basis of monoclonal antibody technology, fluorescent labeling immunotechnology and new material technology.
  • a microporous membrane is used as a solid phase carrier, and a known specific antigen is immobilized on a nitrocellulose membrane as a detection line.
  • the diffusion of the sample through the capillary is first combined with the immunoprobe and the chromatography is continued to the detection line, and the complex of the label and the analyte is intercepted by the antigen of the detection line, thereby presenting a fluorescent band.
  • the method has the advantages of rapidity, high sensitivity, strong specificity, good stability and simple operation. The result is intuitive and reliable, and it is easy to be mastered by grassroots units. It is suitable for on-site rapid screening and large-scale promotion of grassroots, especially suitable for agricultural products and feed. Synchronous and rapid monitoring of the mixed contamination of mycotoxins in agricultural products and feeds is urgently needed in such fields as the urgent need for rapid and simultaneous detection of mixed contamination of mycotoxins.
  • the problem to be solved by the present invention is to provide an immunochromatographic time-resolved fluorescent kit capable of simultaneously detecting mixed contamination of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone and variegyr toxin. Preparation method and its application.
  • the immunochromatographic time-resolved fluorescent kit can be used for synchronous detection of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone and variegated toxin, and has simple, rapid and high sensitivity.
  • the technical solution adopted by the present invention is:
  • An immunochromatographic time-resolved fluorescent kit for simultaneous detection of mixed contamination of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone, variegyroxone characterized in that it comprises immunochromatographic time-resolved Fluorescent test strips and aflatoxin B1 monoclonal antibodies containing tritiated markers, tritiated fumonisin B1 monoclonal antibodies, tritiated ochratoxin A monoclonal antibodies, tritiated zearalenone monoclonal antibodies , a sample reaction bottle of sputum-labeled Aspergillus fumigatus monoclonal antibody lyophilized product.
  • the immunochromatographic time-resolved fluorescent test strip comprises a PVC substrate, and one side of the substrate is pasted with an absorbent pad, a test pad and a sample pad from top to bottom, and adjacent pads are overlapped at the joint.
  • the test pad is based on a nitrocellulose membrane, and a transverse quality control line and five detection lines are arranged from top to bottom on the nitrocellulose membrane, and the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the 5 Aflatoxin B1-bovine serum albumin conjugate, fumonisin B1-bovine serum albumin conjugate, ochratoxin A-bovine serum albumin conjugate, zearalenone a bovine serum albumin conjugate, a ochratoxin-bovine serum albumin conjugate, the monoclonal antibody of fumonisin B1 being secreted by the hybridoma cell line Fm7A11 deposited under accession number CCTCC No. C201636 Cloning
  • the tritiated monoclonal antibody is prepared according to the following method:
  • the ⁇ -labeled aflatoxin B1 monoclonal antibody mixes the aflatoxin B1 monoclonal antibody and the activated guanidine labeling reagent in a boric acid buffer, oscillates the reaction, and then obtains the target product by the centrifugation, reconstitution, and blocking steps.
  • Aflatoxin B1 monoclonal antibody; 1 mL of activated guanidine labeling reagent can be coupled to aflatoxin B1 monoclonal antibody: 30 ⁇ g-80 ⁇ g.
  • the ⁇ -labeled zearalenone monoclonal antibody dissolves and oscillates the zearalenone monoclonal antibody and the activated guanidine labeling reagent in boric acid buffer, and then obtains the target product by centrifugation, reconstitution and blocking steps.
  • Labeled zearalenone monoclonal antibody; 1 mL of activated sputum labeling reagent can be coupled to zearalenone monoclonal antibody: 30 ⁇ g - 90 ⁇ g.
  • the ⁇ -labeled Aspergillus fumigatus monoclonal antibody dissolves and oscillates the Aspergillus fumigatus monoclonal antibody and the activated ruthenium labeling reagent in a boric acid buffer solution, and then obtains the target product ⁇ mark variegated by centrifugation, reconstitution, and blocking steps.
  • Aspergillus monoclonal antibody; 1 mL of activated sputum labeling reagent can be coupled to Aspergillus variabilis monoclonal antibody: 30 ⁇ g-80 ⁇ g.
  • the activation method is a labeling reagent, ultrasonically dispersed in a boric acid buffer solution of pH 8.2 0.2 mol/L, and then slowly added to the carbodiimide solution, activated by shaking at room temperature, and centrifuged to remove the supernatant.
  • the boric acid buffer solution of pH 8.2 0.2 mol/L was reconstituted, and the activation time was 15-30 min.
  • the cloned antibody and tritiated Aspergillus fumigatus monoclonal antibody were reconstituted in 0.01 mol/L pH 8.2 phosphate buffer containing 1.5% (m/v) trehalose and 2% (m/v) bovine serum albumin. When in use, it is placed in a sample reaction bottle, and placed in a freeze dryer to be lyophilized to obtain a lyophilized product of each sputum-labeled monoclonal antibody, which is ready for use.
  • the absorbent pad in the immunochromatographic time-resolved fluorescent test strip has a length of 10-16 mm and a width of 3-5 mm; the test pad has a length of 25-33 mm and a width of 3-5 mm; and the sample pad has a length of 12-18 mm and a width of 3 -5mm, the overlapping length of adjacent pads is 1-2mm; the distance between the detection line on the detection pad near the quality control line and the upper edge of the nitrocellulose membrane in the immunochromatography time-resolved fluorescent test strip is 5-10mm The spacing between each adjacent two detection lines is 1.5-4.5 mm, and the detection line close to the quality control line and the quality control line is 3-6 mm; the sample reaction bottle is a 1-5 mL bayonet bottle.
  • the amount of the aflatoxin B1-bovine serum albumin conjugate required for detecting the detection line per centimeter on the detection pad in the immunochromatographic time-resolved fluorescent test strip is 100-400 ng, and the detection line per centimeter
  • the required amount of fumonisin B1-bovine serum albumin conjugate is 100-400 ng
  • the amount of ochratoxin A-bovine serum albumin conjugate required per cm of detection line is 100- 400ng
  • zearalenone-bovine serum albumin conjugate required per cm of detection line is 100-400ng
  • variegated toxin-bovine serum albumin conjugate required per cm of detection line The coating amount is 100-400 ng; the amount of rabbit anti-mouse polyclonal antibody required per centimeter of the control line is 80-200 ng.
  • the content of the sputum-labeled aflatoxin B1 monoclonal antibody lyophilized product in the sample reaction bottle is 0.1-0.3 ⁇ g, and the content of the sputum-labeled fumonisin B1 monoclonal antibody lyophilized product in the sample reaction bottle is 0.1.
  • the content of the sputum-labeled ochratoxin A monoclonal antibody lyophilizate in the sample reaction flask is 0.1-0.3 ⁇ g
  • the lysin-labeled zearalenone monoclonal antibody is lyophilized in the sample reaction bottle
  • the content of the product is 0.1-0.3 ⁇ g
  • the content of the sputum-labeled variegated toxin monoclonal antibody lyophilized product in the sample reaction bottle is 0.1-0.3 ⁇ g.
  • the glass fiber membrane is placed in a blocking solution to be wetted, taken out, and dried at 37-40 ° C for 4-6 hours to obtain a sample pad, which is then stored in a desiccator at room temperature;
  • the preparation of the immunochromatographic time-resolved fluorescent test strip prepares aflatoxin B1-bovine serum albumin conjugate coating solution, fumonisin B1-bovine serum albumin conjugate, Aspergillus oryzae Toxin A-bovine serum albumin conjugate coating solution, zearalenone-bovine serum albumin conjugate coating solution, variegated toxin-bovine serum albumin conjugate coating solution
  • the coating buffer is: 0.1g of bovine serum albumin per 10mL, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride, potassium dihydrogen phosphate 0.002g;
  • the coating buffer used in preparing the rabbit anti-mouse polyclonal antibody coating solution comprises: 0.002 g of sodium azide per 10 mL, 0.08 g of sodium chloride, 0.029 g of disodium hydrogen phosphate dodecahydrate, potassium chloride. 0.002g, potassium dihydrogen phosphate 0.002g;
  • the ratio of the fluorescence intensity of the upper detection line (T) to the fluorescence intensity of the quality control line (C); the ratio of the fluorescence intensity of the detection line to the fluorescence intensity of the quality control line based on the previously obtained immunochromatographic time-resolved fluorescent test strip (T/C) Obtaining aflatoxin B1, fumonisin B1, Aspergillus oryzae in the sample solution to be tested, respectively, with the curves of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone, and ochratoxin concentration The content of toxin A, zearalenone, variegyr toxin, and finally converted to aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone, variegyr toxin in the sample to be tested Contain the amount.
  • the ratio of the fluorescence intensity of the immunochromatographic time-resolved fluorescent test strip detection line to the fluorescence intensity of the quality control line is respectively associated with aflatoxin B1, fumonisin B1, ochratoxin A,
  • the relationship between zearalenone and variegyr concentration is obtained by the following method:
  • the minimum detection limit of aflatoxin B1 in the detection solution is 0.06 ng/mL
  • the lowest detection limit of fumonisin B1 is 0.2 ng/mL
  • the lowest detection value of ochratoxin A is provided by the immunochromatography time-resolved fluorescence kit provided by the invention.
  • the detection limit is 0.5 ng/mL
  • the minimum detection limit of zearalenone is 1 ng/mL
  • the minimum detection limit of ochratoxin is 0.3 ng/mL.
  • the detection limit can meet the five mycotoxins in the food of the European Union. Limited requirements.
  • 1 is an immunochromatography time-resolved fluorescence test of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone, and ochratoxin immunochromatography time-resolved fluorescence test kit provided by the present invention.
  • the aflatoxin universal monoclonal antibody is secreted by the hybridoma cell line 3G1 with the accession number CCTCC NO.C201014, and is prepared in advance according to the method reported in the patent No. ZL201210117614.9 by the method of obtaining the hybridization
  • the tumor cell line 3G1 was injected with BALB/c mice previously treated with Freund's incomplete adjuvant, and the ascites of the mice was collected, and the aflatoxin B1 monoclonal antibody was obtained after purification.
  • the sodium oxide solution adjusts the pH of the mixture to 7.4, pre-cooling at 4 ° C, slowly adding ammonium sulfate to a final concentration of ammonium sulfate of 0.277 g / mL, standing at 4 ° C for more than 2 h, then centrifuging at 4 ° C, 12000 r / min for more than 30 min The supernatant was discarded, and the resulting pellet was resuspended in 0.01 mol/L phosphate buffer volume of 1/10 of the original ascites fluid, placed in a dialysis bag, dialyzed against pure water, and the fully dialyzed protein solution was frozen at -70 ° C in a refrigerator. Then, freeze-drying with a freeze vacuum dryer, collecting the lyophilized powder, which is purified.
  • Aflatoxin B1 monoclonal antibody the antibody is placed in a refrigerator at -20 ° C for use;
  • the acetate buffer solution is 0.29 g sodium acetate, 0.141 mL acetic acid is added to water to 100 mL, and the 0.1 mol/L phosphate buffer solution is 0.8 g sodium chloride and 0.29 g hydrogen phosphate dodecahydrate.
  • the ochratoxin A monoclonal antibody is secreted by the hybridoma cell line 1H2 with the accession number CCTCC NO.C201329, and is prepared in advance according to the method reported in the patent application No. 201310115921.8 by the hybridoma cell line 1H2.
  • the abdomen water of the mice was injected into the abdomen of BALB/c mice previously treated with Freund's incomplete adjuvant, and purified to obtain an ochratoxin A monoclonal antibody.
  • the purification method is a octanoic acid-ammonium sulfate method, and the specific steps are: filtering the mouse ascites with a double-layer filter paper, centrifuging at 12000 r/min for 15 min or more at 4 ° C, aspirating the supernatant, and clearing the obtained abdomen water with 4 times volume of vinegar.
  • Mix the acid salt buffer slowly add n-octanoic acid under stirring, the volume of n-octanoic acid per ml of ascites is 30-35 ⁇ L, mix at room temperature for 30-60min, stand at 4°C for more than 2h, then centrifuge at 4°C, 12000r/min for more than 30min.
  • the precipitate was discarded, and the obtained supernatant was filtered through a double-layer filter paper, and a 1/10 filtrate volume of a phosphate buffer having a molar concentration of 0.1 mol/L and a pH of 7.4 was added, and 2 mol/L of sodium hydroxide was used.
  • the solution was adjusted to pH 7.4, pre-cooled at 4 ° C, slowly added ammonium sulfate to a final concentration of ammonium sulfate of 0.277 g / mL, allowed to stand at 4 ° C for more than 2 h, then centrifuged at 4 ° C, 12000 r / min for more than 30 min, discarded
  • the supernatant was resuspended in a phosphate buffer solution of 0.01 mol/L and pH 7.4 in the original ascites volume, placed in a dialysis bag, dialyzed against pure water, and the fully dialyzed protein solution was placed - Freeze in a 70 °C refrigerator, then freeze-dry with a freeze dryer to collect the lyophilized powder.
  • Ochratoxin A monoclonal antibody the antibody is a standby set -20 °C refrigerator;
  • the acetate buffer solution is 0.29 g sodium acetate, 0.141 mL acetic acid is added to water to 100 mL, and the 0.01 mol/L phosphate buffer solution is 0.8 g sodium chloride and 0.29 g hydrogen phosphate dodecahydrate.
  • Disodium, 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g add water to make up to 100mL;
  • the 0.1mol / L phosphate buffer is 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g, water to a volume of 100mL;
  • N-octanoic acid the volume of n-octanoic acid required per milliliter of ascites is 30-35 ⁇ L, mixed at room temperature for 30-60min, allowed to stand at 4°C for more than 2h, then centrifuged at 4°C, 12000r/min for more than 30min, discard the precipitate, and the supernatant obtained will be obtained.
  • a 1/10 filtrate volume of a phosphate buffer having a molar concentration of 0.1 mol/L and a pH of 7.4 was added, and the pH of the mixture was adjusted to 7.4 with a 2 mol/L sodium hydroxide solution.
  • the final concentration of ammonium sulfate to ammonium sulfate is 0.277g/mL, and it is allowed to stand at 4°C for more than 2h, then centrifuged at 4°C and 12000r/min for more than 30min, and the supernatant is discarded.
  • the obtained precipitate is 0.01mol/L of the original ascites volume of 1/10.
  • the phosphate buffer solution with pH 7.4 was resuspended, placed in a dialysis bag, dialyzed against pure water, and the fully dialyzed protein solution was frozen in a -70 ° C refrigerator, and then freeze-dried in a freeze dryer to collect the lyophilized powder.
  • the purified zearalenone monoclonal antibody is obtained, and the antibody is placed in a refrigerator at -20 ° C for use;
  • the variegated aspergillin monoclonal antibody is secreted by the hybridoma cell line ST03 with the accession number CCTCC No. C2013187, and is prepared in advance according to the method reported in the patent application No. 201410115952.8 by the preparation method: the obtained hybridoma cell Strain ST03 was injected into the abdomen of Balb/c mice previously treated with Freund's incomplete adjuvant, and the ascites of the mice was collected and purified to obtain a monoclonal antibody against ochratoxin.
  • the purification is a octanoic acid-ammonium sulfate purification method, the specific operation is: filtering the mouse ascites with a double-layer filter paper, centrifuging at 12000 r/min for 15 min or more at 4 ° C, aspirating the supernatant, and clearing the obtained abdomen water with 4 times volume of vinegar.
  • the pH of the mixture was 7.4, pre-cooled at 4 ° C, slowly added ammonium sulfate to a final concentration of ammonium sulfate of 0.277 g / mL, and allowed to stand at 4 ° C for more than 2 h, then centrifuged at 4 ° C, 12000 r / min for more than 30 min, and discarded the supernatant.
  • the obtained pellet was resuspended in 0.01 mol/L phosphate buffer solution of 1/10 of the original ascites volume, placed in a dialysis bag, dialyzed against pure water, and the fully dialyzed protein solution was frozen in a -70 ° C refrigerator, followed by freezing.
  • the dryer is lyophilized, and the lyophilized powder is collected to obtain the purified variegated toxin.
  • Monoclonal antibody the antibody is placed in a refrigerator at -20 ° C for use;
  • the commercially available fumonisin B 1 standard was purchased for complete antigen synthesis.
  • the specific synthesis procedure was as follows: 2 mg of FB 1 standard powder and 2 mg of EDC were respectively dissolved in 500 ⁇ L of 0.01 mol/L PBS solution to obtain EDC solution and FB 1 .
  • 4 mg/mL (solution of 0.01 mol/L PBS) in EDC was added dropwise to the dissolved FB 1 solution, and gently stirred at room temperature for 10 minutes.
  • a BSA solution of 5 mg/mL (solution of 0.01 mol/L PBS) was added dropwise to the above mixture, and the reaction was stirred at room temperature for 4 hours. Dialysis for 3 days.
  • the routine UV scanning method was used to identify the FB 1 -BSA complete antigen preparation.
  • mice Six 6-week-old BALB/c mice were purchased and the fumonisin complete antigen FB 1 -BSA synthesized in the laboratory was immunized.
  • the first immunization embedding the fumonisin complete antigen with an equal volume of Freund's complete adjuvant and subcutaneously injecting multiple injections into the neck of the mouse.
  • the second immunization was carried out 3 weeks later, and was emulsified with an equal volume of fumonisin complete antigen using Freund's incomplete adjuvant, and injected subcutaneously in the neck of the mouse.
  • the third and fourth immunizations were separated from the previous immunization by two weeks, and the immunization method was the same as the second.
  • the four immunization doses were the same, only 100 ⁇ g/head.
  • the mice were collected from the tail vein, serum was separated, and the serum titer of the mice was monitored by indirect ELISA.
  • the serum sensitivity of the mice was determined by indirect competitive ELISA. The titer and sensitivity were relatively high.
  • the mice corresponding to the serum were given the last booster, and the immunization dose was twice that of the previous amount.
  • PEG polyethylene glycol
  • the complete medium containing 1% HAT contains 20% (by volume) fetal bovine serum, 75% (by volume) RPMI-1640 base medium, 1% (by weight) L-glutamine, 1% (% by volume) HEPES, 1% (by volume) double antibody (10000 units per milliliter penicillin and 10000 micrograms per ml of streptomycin), 2% (by volume) growth factor (HFCS) and 1% (weight percent) secondary yellow ⁇ -aminopterin-thymidine, HAT and methylcellulose, were purchased from sigma-Aldrich.
  • the cells were cultured in HAT liquid until the cells grew to 2/3. At the bottom of the well, the culture supernatant is aspirated for detection.
  • a two-step screening method was used. The first step was to select positive holes that were resistant to fumonisin but not to the carrier protein BSA by indirect ELISA. The second step was to detect the positive wells screened in the first step by indirect competitive ELISA.
  • heavy chain variable region primers are 5'-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3' (28mer
  • S, M, R and W are the merging bases
  • M A/C
  • R A/G
  • S G/C
  • W A/T
  • the light chain variable region primer is 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24mer).
  • the obtained gene sequence result the heavy chain variable region coding gene sequence is 379 bp in length, and the sequence is as shown in SEQ ID NO: 1, and the heavy chain variable region encoded by the gene sequence is deduced from the 126 amino acids according to the obtained gene sequence. Composition, the sequence is shown in SEQ ID NO: 3.
  • the light chain variable region coding gene sequence is 348 bp in length, and the sequence is as shown in SEQ ID NO: 2.
  • the light chain variable region encoded by the gene sequence is composed of 116 amino acids, and the sequence is SEQ ID. NO: 4 is shown.
  • n-octanoic acid Slowly add n-octanoic acid, the volume of n-octanoic acid required per milliliter of ascites is 30-35 ⁇ L, mix at room temperature for 30-60 min, and let stand at 4 ° C for more than 2 h. After centrifugation at 12000 r/min for more than 30 min at 4 ° C, the precipitate was discarded, and the obtained supernatant was filtered through a double-layer filter paper, and then a 1/10 filtrate volume of a phosphate buffer solution having a molar concentration of 0.1 mol/L and a pH of 7.4 was added.
  • the pH of the mixture was adjusted to 7.4 with 2 mol/L sodium hydroxide solution, and ammonium sulfate was slowly added to the final concentration of ammonium sulfate to 0.277 g/mL in an ice bath, and allowed to stand at 4 ° C for more than 2 h, then 12000 r/min, 4 ° C. After centrifugation for more than 30 min, the supernatant was discarded, and the obtained pellet was resuspended in a phosphate buffer of 0.01 mol/L and pH 7.4 in a volume of 1/10 volume of ascites, and placed in a dialysis bag, and dialyzed against 0.01 mol/L PBS.
  • the PB was dialyzed for two days, the protein solution in the dialysis bag was taken out, centrifuged, the supernatant was collected, the precipitate was discarded, pre-frozen at -70 ° C, and then lyophilized in a lyophilizer. Collecting the lyophilized powder, which is a purified anti-fumonic acid B 1 monoclonal antibody;
  • the acetate buffer solution is 0.29 g sodium acetate, 0.141 mL acetic acid is added to water to 100 mL, and the 0.01 mol/L phosphate buffer solution is 0.8 g sodium chloride and 0.29 g hydrogen phosphate dodecahydrate.
  • the fluorescent microspheres were taken, added to 1 mL of 0.2 mol/L boric acid buffer solution of pH 8.2, sonicated for 300 seconds at 300 w, then slowly added 40 uL of 15 mg/mL carbodiimide, shaken at room temperature for 20 min, and centrifuged at 17000 g for 15 min. The precipitate was collected and reconstituted with 0.2 mol/L boric acid buffer of pH 8.2.
  • the activated fluorescent microspheres were added to 1 mg/ml antibody (aflatoxin B1 monoclonal antibody 35 ul, ochratoxin A monoclonal antibody 45 ul, Zea mays
  • the reaction was shaken at 4 ° C for 12 h, centrifuged at 12000 g for 10 min, and contained 0.2 mol/L of 1% BSA.
  • the absorbent paper is cut into a length of 16 mm and a width of 4 mm, that is, an absorbent pad is obtained;
  • the aflatoxin B1-bovine serum albumin conjugate was formulated into a 0.25 mg/mL solution with a coating buffer, and coated with nitrocellulose by a line spray method at a position of 6 mm from the nitrocellulose membrane.
  • a detection line I is obtained on the membrane, and the amount of aflatoxin B1-bovine serum albumin conjugate required per cm of detection line I is 100 ng; the fumonisin B1-bovine serum albumin conjugate is packaged
  • the buffer solution was prepared into a coating solution of 0.25 mg/mL, and was coated on a nitrocellulose membrane by a line spray method at a position 4 mm from the detection line to obtain a detection line II, which was required per cm of the detection line II.
  • the amount of the fumonisin B1-bovine serum albumin conjugate was 200 ng; the ochratoxin A-bovine serum albumin conjugate was formulated into a coating solution of 0.45 mg/mL with a coating buffer.
  • the position of the detection line II4 mm was coated on a nitrocellulose membrane by a line spray method to obtain a detection line III, and the amount of the ochratoxin A-bovine serum albumin conjugate required per cm of the detection line III was 160 ng; the zearalenone-bovine serum albumin conjugate was formulated into a coating solution of 0.35 mg/mL with a coating buffer.
  • the detection line III coated with a nitrocellulose membrane by line spray to obtain the detection line IV, the required zearalenone-bovine serum albumin conjugate per cm of detection line IV
  • the coating amount was 200 ng; the variegated toxin-bovine serum albumin conjugate was formulated into a coating solution of 0.4 mg/mL with a coating buffer, and was sprayed by a line spray at a position 4 mm from the detection line IV.
  • Coating on a nitrocellulose membrane to obtain a detection line V the amount of the required ochratoxin-bovine serum albumin conjugate per cm of detection line V is 200 ng; and then drying at 37 ° C for 120 min;
  • the coating buffer is: 0.002g sodium azide, 0.08g sodium chloride, 0.029g disodium hydrogen phosphate dodecahydrate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate, and the volume is adjusted to 10mL with water.
  • the nitrocellulose membrane was 33 mm long and 4 mm wide.
  • the glass fiber membrane is cut into a length of 12 mm and a width of 4 mm, and is placed in a blocking solution to be wetted, taken out, and dried at 40 ° C for 4 hours to obtain a sample pad, which is then stored in a desiccator at room temperature;
  • the blocking solution is 1 g ovalbumin, 2 g sucrose, 0.02 g sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate, Add water to make up to 100mL;
  • Adhesive pads, test pads and sample pads are sequentially pasted from top to bottom on one side of the PVC substrate, and adjacent pads are overlapped at the joints, and the overlap length is 1 mm, that is, see FIG. 1;
  • sample reaction vial prepared sputum labeled aflatoxin B1 monoclonal antibody, tritiated fumonisin B1 monoclonal antibody, tritiated ochratoxin A monoclonal antibody, sputum labeled corn red Monoclonal methone monoclonal antibody, tritiated H. albicans monoclonal antibody; reconstituted in 1.5% (m/v) trehalose, 2% (m/v) bovine serum albumin 0.01 mol/L pH 8.2 In the phosphate buffer solution, each sample is placed in a sample reaction flask, placed in a freeze dryer, and lyophilized for use.
  • Aflatoxin B1, ochratoxin A, zearalenone, and fumonisin B1 were respectively assigned to the following concentration gradients of aflatoxin B1, ochratoxin A, zearalenone, and fumonisin B1.
  • 6 solutions aflatoxin B1 (0ng/mL, 0.05ng/mL, 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/mL), fumonisin B1 (0ng/mL, 0.05ng) /mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.0 ng/mL), ochratoxin A (0 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2 ng/ mL, 4 ng/mL), zearalenone (0 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2.5 ng/mL,
  • the standard solution was repeated 5 times at each concentration point, and the test strip was tested with a multi-toxin test, and detected by a time-resolved fluorescence detector: 150 ⁇ L of the above standard solution was added to the sample reaction bottle, mixed, and inserted into the immunochromatographic time-resolved fluorescence.
  • the test strip was reacted at 37 ° C for 6 min, and the residual liquid of the sample pad was blotted with absorbent paper, and immediately detected by a time-resolved fluorescence immunoassay analyzer (excitation wavelength: 365 nm, measurement wavelength: 615 nm), and the fluorescence signal intensity of the detection region was read. Value, calculate the 5 repeated averages.
  • the standard curve is drawn by the natural logarithm of the standard series concentration value (Lnc) and the fluorescence signal intensity value of the T line/the fluorescence signal intensity value of the C line (T/C).
  • the standard curve parameters of the test object are shown in the following table:
  • test sample Take 20g of the test sample to be added to 100mL 70% (volume fraction) methanol solution, homogenize for 2min, let stand, filter with double-layer filter paper, collect 1mL of filtrate, add 5mL sample dilution to dilute the filtrate, mix; take the feed to be tested 150 ⁇ L of the sample test solution was added to the sample reaction flask, mixed, and inserted into the immunochromatographic time-resolved fluorescent test strip.
  • the residual liquid of the sample pad was blotted with absorbent paper and immediately detected by a time-resolved fluorescence immunoassay analyzer ( Excitation wavelength: 365 nm, measurement wavelength: 615 nm), and obtain the ratio of the time-resolved fluorescence intensity of the five detection lines of each immunochromatographic time-resolved fluorescent test strip to the time-resolved fluorescence intensity of the quality control line (T/C), and then respectively Substituting the ratio of the fluorescence intensity of the immunochromatographic time-resolved fluorescent test strip to the fluorescence intensity of the quality control line (T/C) and the concentration of aflatoxin B1, the concentration of fumonisin B1, the concentration of ochratoxin A, corn
  • the relationship between the concentration of gibberellin and the concentration of ochratoxin, the content of aflatoxin B1 in the feed sample was 6.4 ⁇ g/kg, the content of fumonisin B1 was 4.1
  • the absorption pad of the immunochromatographic time-resolved fluorescent test strip has a length of 14 mm and a width of 3 mm; the test pad has a length of 30 mm and a width of 3 mm; the sample pad has a length of 16 mm and a width of 3 mm, and the overlapping length of the adjacent pads It is 2 mm; the distance between the detection line on the detection pad near the quality control line and the upper edge of the nitrocellulose membrane in the fluorescent test strip is 8 mm, and the spacing between each adjacent two detection lines is 4 mm.
  • the amount of aflatoxin B1-bovine serum albumin conjugate required per cm of detection line on the detection pad in the immunochromatographic time-resolved fluorescent test strip is 200 ng, and the fumonisin required per cm of detection line
  • the coating amount of B1-bovine serum albumin conjugate was 250 ng, and the amount of ochratoxin A-bovine serum albumin conjugate required per cm of detection line was 200 ng, and the corn required per cm of detection line
  • the coating amount of zearalenone-bovine serum albumin conjugate was 250 ng, and the amount of variegated toxin-bovine serum albumin conjugate required per cm of detection line was 300 ng; per centimeter of quality control line
  • the required amount of the rabbit anti-mouse polyclonal antibody is 150 ng; the content of the sputum-labeled aflatoxin B1 monoclonal antibody lyophilizate in the sample reaction bottle is 0.2 ⁇ g, and the sample reaction bottle is labeled
  • the content of zearalenone monoclonal antibody lyophilized product is 0.2 ⁇ g.
  • Sample reaction flask europium labeled monoclonal antibody toxin, Aspergillus versicolor content lyophilized product was 0.3 g;
  • a sample containing a variety of toxins confirmed by high performance liquid chromatography (aflatoxin B1 content of 5.1 ⁇ g / kg, fumonisin B1 content of 3.9 ⁇ g / kg, ochratoxin content of 1.5 ⁇ g / kg, Zea mays
  • the ketene content was 41.2 ⁇ g/kg and the variegated toxin content was 5.3 ⁇ g/kg.
  • the residual liquid of the sample pad was blotted with absorbent paper and immediately detected by a time-resolved fluorescence immunoassay analyzer ( Excitation wavelength: 365 nm, measurement wavelength: 615 nm), the ratio of the time-resolved fluorescence intensity of the five detection lines of each immunochromatographic time-resolved fluorescent test strip to the time-resolved fluorescence intensity of the quality control line (T/C) was obtained, and then respectively Substituting the ratio of the fluorescence intensity of the immunochromatographic time-resolved fluorescent test strip to the fluorescence intensity of the quality control line (T/C) and the concentration of aflatoxin B1, the concentration of fumonisin B1, the concentration of ochratoxin A, corn The relationship between the concentration of gibberellin and the concentration of ochratoxin, the content of aflatoxin B1 in the feed sample was 5.3 ⁇ g/kg, the content of fumonisin B1 was 3.7

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Abstract

同步检测黄曲霉毒素B1等五种真菌毒素混合污染的免疫层析时间分辨荧光试剂盒及应用。试剂盒包括免疫层析时间分辨荧光试纸条和含有铕标记的各单克隆抗体冻干品的样品反应瓶；其中荧光试纸条包括PVC衬底，衬底的一面从上到下依次粘贴吸水垫(1)、检测垫(2)和样品垫(3)，相邻各垫在连接处交叠连接，检测垫(2)以硝酸纤维素膜为基垫，自上而下设置横向质控线(5)和5条检测线(5、6、7、8、9)，分别包被各毒素的牛血清白蛋白偶联物，伏马毒素B1单克隆抗体为由保藏编号为CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生。试剂盒可用于黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染的同步检测。

Description

同步检测黄曲霉毒素B1等五种真菌毒素混合污染的免疫层析时间分辨荧光试剂盒及应用 技术领域
本发明涉及真菌毒素免疫层析时间分辨荧光试剂盒，具体涉及一种同步检测黄曲霉毒素B1等五种真菌毒素混合污染的免疫层析时间分辨荧光试剂盒及应用。
背景技术
真菌毒素是真菌在粮油或饲料中生长代谢所产生的一系列有毒有害物质，目前在自然界中已发现的真菌毒素多达400多种。按照主要产毒菌种，真菌毒素可以分为曲霉菌毒素(如黄曲霉毒素、杂色曲霉毒素等)、青霉菌毒素和镰刀菌毒素(如玉米赤霉烯酮等)等几大类。真菌毒素具有致癌、致畸、致突变的作用，可引起人体的急性或慢性中毒，严重危害人体健康。真菌毒素污染农作物、食品及饲料，给人类和家畜的生命健康带来巨大威胁。为了保障粮食卫生安全，我国对粮食中的真菌毒素制定了相关的限量标准(GB2761—2011)。当前污染农产品食品及饲料等的主要真菌毒素包括黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素。黄曲霉毒素主要出现在谷物和豆类植物中，若禽类接触到被黄曲霉毒素污染后的饲料，则在牛奶、奶制品、鸡蛋和肉类中也会检测到黄曲霉毒素。黄曲霉毒素主要通过食物消化感染，长期的皮肤接触也会造成慢性感染，引发急性或者慢性肝损伤、急性肝炎、肝细胞脂肪变性，最终导致肝癌。伏马毒素是由串珠镰刀菌产生的水溶性代谢产物，主要由多氢醇和丙三酸组成双酯类化合物，现已发现11种伏马毒素，其中伏马毒素B1危害最严重，主要感染玉米、高粱、大米等农作物。伏马毒素是一种致癌剂，引发脑水肿，脑坏死以及肝脏毒性，严重损伤免疫系统。赭曲霉毒素主要感染谷类农作物以及豆类植物，如玉米、大麦、小麦、咖啡豆、豌豆、可可豆等。赭曲霉毒素在机体内胃肠道，如十二指肠、空肠段被吸收，具有强肾毒性。玉米赤霉烯酮污染广泛，在世界各大洲的谷物以及农副产品中都能都检测到，多污染玉米、燕麦、小麦、大麦、小米等农产品食品，以及奶制品、牛肉等食品。杂色曲霉毒素主要是由黄曲霉、杂色曲霉、构巢曲霉、细皱曲霉等真菌产生的次级代谢产物，其毒性仅次于黄曲霉毒素，可诱发肝癌、肺癌及其他肿瘤
目前，真菌毒素的检测方法主要有薄层色谱法、酶联免疫法、液相色谱法、液相色谱-质谱联用法。薄层色谱法简捷、便利，但重现性差、精度低；酶联免疫法特异性强、前处理简单，但假阳性率高，不能作为确证方法；液相色谱、液相色谱-质谱联用法稳定性 好、灵敏度较高，但样品前处理过程复杂，检测时间长，所使用的仪器昂贵，对实验环境和检测人员要求高，难以实现快速检测。免疫层析荧光检测技术是20世纪80年代初期开发的一种免疫分析方法，它是在单克隆抗体技术、荧光标记免疫技术和新材料技术基础上发展起来的快速检测技术。该技术以微孔膜为固相载体，将已知的特异性抗原固定于硝酸纤维素膜上作为检测线。当加入待测样品后，样品经毛细管的扩散作用首先与免疫探针相结合，并继续层析至检测线，标记物与待测物的复合物被检测线的抗原截获，从而呈现荧光条带。该法具有快速、灵敏度高、特异性强、稳定性好、操作简单等优点，结果判断直观可靠，容易被基层单位人员掌握，适合现场快速筛查和基层大面积推广，特别适合农产品食品及饲料等领域迫切需要的能快速、同步检测真菌毒素混合污染的检测技术，以实现对农产品食品及饲料中真菌毒素混合污染的同步、快速监测。
发明内容
本发明所要解决的问题是提供一种能同步检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染的免疫层析时间分辨荧光试剂盒、制备方法及其应用。该免疫层析时间分辨荧光试剂盒可用于黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素含量的同步检测，具有操作简单、快速、灵敏度高的特点。
为解决上述技术问题，本发明所采用的技术方案为：
同步检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染的免疫层析时间分辨荧光试剂盒，其特征在于：它包括免疫层析时间分辨荧光试纸条和含有铕标记的黄曲霉毒素B1单克隆抗体、铕标记的伏马毒素B1单克隆抗体、铕标记的赭曲霉毒素A单克隆抗体、铕标记的玉米赤霉烯酮单克隆抗体、铕标记的杂色曲霉单克隆抗体冻干品的样品反应瓶。其中：所述的免疫层析时间分辨荧光试纸条包括PVC衬底，衬底的一面从上到下依次粘贴吸水垫、检测垫和样品垫，相邻各垫在连接处交叠连接，所述检测垫以硝酸纤维素膜为基垫，硝酸纤维素膜上自上而下设置横向质控线和5条检测线，所述质控线包被有兔抗鼠多克隆抗体，所述5条线上分别包被黄曲霉毒素B1-牛血清白蛋白偶联物、伏马毒素B1-牛血清白蛋白偶联物、赭曲霉毒素A-牛血清白蛋白偶联物、玉米赤霉烯酮-牛血清白蛋白偶联物、杂色曲霉毒素-牛血清白蛋白偶联物，所述的伏马毒素B1单克隆抗体为由保藏编号CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生的单克隆抗体。该杂交瘤细胞株已于2016年3月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO:C201636。
按上述方案，所述铕标记的单克隆抗体是按照以下方法制备得到：
铕标记的黄曲霉毒素B1单克隆抗体将黄曲霉毒素B1单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中混合、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的黄曲霉毒素B1单克隆抗体；1mL活化后的铕标记试剂可偶联黄曲霉毒素B1单克隆抗体：30μg-80μg。
铕标记的赭曲霉毒素A单克隆抗体将赭曲霉毒素A单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的赭曲霉毒素A单克隆抗体；1mL活化后的铕标记试剂可偶联赭曲霉毒素A单克隆抗体：40μg-90μg。
铕标记的玉米赤霉烯酮单克隆抗体将玉米赤霉烯酮单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的玉米赤霉烯酮单克隆抗体；1mL活化后的铕标记试剂可偶联玉米赤霉烯酮单克隆抗体：30μg-90μg。
铕标记的伏马毒素B1单克隆抗体将伏马毒素B1单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的伏马毒素B1单克隆抗体；1mL活化后的铕标记试剂可偶联伏马毒素B1单克隆抗体：30μg-80μg。
铕标记的杂色曲霉单克隆抗体将杂色曲霉单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的杂色曲霉单克隆抗体；1mL活化后的铕标记试剂可偶联杂色曲霉单克隆抗体：30μg-80μg。
按上述方案，所述的活化方法为取铕标记试剂，用pH 8.2 0.2mol/L的硼酸缓冲液中超声分散，然后缓慢加入碳二亚胺溶液，室温振荡活化，离心去上清液，用pH 8.2 0.2mol/L的硼酸缓冲液复溶，备用，所述的活化时间为15-30min。
按上述方案，上述配制好的铕标记的黄曲霉毒素B1单克隆抗体、铕标记的伏马毒素B1单克隆抗体、铕标记的赭曲霉毒素A单克隆抗体、铕标记的玉米赤霉烯酮单克隆抗体、铕标记的杂色曲霉单克隆抗体复溶于含1.5％(m/v)海藻糖、2％(m/v)牛血清白蛋白的0.01mol/L pH 8.2的磷酸缓冲液中备用，使用时，将其放入样品反应瓶中，置于冷冻干燥机中冻干，得到各铕标记的单克隆抗体冻干品，备用。
按上述方案，所述免疫层析时间分辨荧光试纸条中的吸水垫长10-16mm，宽3-5mm；检测垫长25-33mm，宽3-5mm；样品垫长12-18mm，宽3-5mm，相邻各垫的交叠长度为1-2mm；所述免疫层析时间分辨荧光试纸条中检测垫上靠近质控线的检测线与硝酸纤维素膜上沿 的间距为5-10mm，每相邻两条检测线之间的间距为1.5-4.5mm，靠近质控线的检测线与质控线3-6mm；所述的样品反应瓶为1-5mL的卡口瓶。
按上述方案，所述免疫层析时间分辨荧光试纸条中检测垫上每厘米检测线所需的黄曲霉毒素B1-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的伏马毒素B1-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的赭曲霉毒素A-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的玉米赤霉烯酮-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的杂色曲霉毒素-牛血清白蛋白偶联物的包被量为100-400ng；每厘米质控线所需的兔抗鼠多克隆抗体的包被量为80-200ng。
所述样品反应瓶中铕标记的黄曲霉毒素B1单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的伏马毒素B1单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的赭曲霉毒素A单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的玉米赤霉烯酮单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的杂色曲霉毒素单克隆抗体冻干品的含量为0.1-0.3μg。
按上述方案，所述的同步检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染的免疫层析时间分辨荧光速测试剂盒还包括样品稀释液和样品稀释液吸管，所述的样品稀释液为体积分数为0.01％-0.30％的吐温-20水溶液。
按上述方案，所述的时间分辨荧光试纸条的制备方法如下：
(1)将吸水纸剪裁得吸水垫；
(2)检测垫的制备：
将黄曲霉毒素B1-牛血清白蛋白偶联物、伏马毒素B1-牛血清白蛋白偶联物、赭曲霉毒素A-牛血清白蛋白偶联物、玉米赤霉烯酮-牛血清白蛋白偶联物、杂色曲霉毒素-牛血清白蛋白偶联物配制成浓度为0.08-0.50mg/mL的包被液，用线喷方式将其于硝酸纤维素膜上进行分别间隔包被，得到5条检测线，然后于37-40℃条件下干燥60-120min；
将兔抗鼠多克隆抗体配成浓度为0.1-0.5mg/mL的包被液，用线喷方式将其横向包被于硝酸纤维素膜上，得质控线然后于37-40℃条件下干燥60-120min；
(3)样品垫的制备：
将玻璃纤维膜放入封闭液中浸湿，取出，于37-40℃条件下干燥4-6h，得样品垫，然后置干燥器中室温保存；
(4)免疫层析时间分辨荧光试纸条的组装：
在PVC底板的一面从上到下依次粘贴吸水垫、检测垫、样品垫，相邻各垫在连接处交 叠连接，即得免疫层析时间分辨荧光试纸条。
按上述方案，所述免疫层析时间分辨荧光试纸条的制备中配制黄曲霉毒素B1-牛血清白蛋白偶联物包被液、伏马毒素B1-牛血清白蛋白偶联物、赭曲霉毒素A-牛血清白蛋白偶联物包被液、玉米赤霉烯酮-牛血清白蛋白偶联物包被液、杂色曲霉毒素-牛血清白蛋白偶联物包被液中所使用的包被缓冲液为：每10mL中含有牛血清白蛋白0.1g，叠氮化钠0.002g，氯化钠0.08g，十二水磷酸氢二钠0.029g，氯化钾0.002g，磷酸二氢钾0.002g；
配制兔抗鼠多克隆抗体包被液中所使用的包被缓冲液为：每10mL中含有叠氮化钠0.002g，氯化钠0.08g，十二水磷酸氢二钠0.029g，氯化钾0.002g，磷酸二氢钾0.002g；
所述免疫层析时间分辨荧光试纸条的制备中使用的封闭液为：每100mL中含有卵清蛋白0.5g，蔗糖2g，叠氮化钠0.02g，氯化钠0.8g，十二水磷酸氢二钠0.29g，氯化钾0.02g，磷酸二氢钾0.02g。
上述免疫层析时间分辨荧光速测试剂盒在黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素含量检测中的应用：将待测样品经前处理获得待测样品溶液，加入样品反应瓶中，混匀，插入时间分辨荧光试纸条，37℃反应6-10min后，用时间分辨荧光测试仪进行检测，获得免疫层析时间分辨荧光试纸条上检测线(T)荧光强度与质控线(C)荧光强度的比值；基于预先获得的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)分别与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线，获得待测样品溶液中黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素的含量，最后经换算即得待测样品中黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素的含量。
按上述方案，所述的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)分别与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线是采用以下方法得到的：
(1)配制得到系列梯度浓度的黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮和杂色曲霉毒素标准溶液；
(2)将适量上述梯度的黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素标准品溶液分别加入到样品反应瓶中，混匀，插入免疫层析时间分辨荧光试纸条，37℃反应6min，用时间分辨荧光免疫分析仪检测得到各免疫层析时间分辨荧光试纸条上检测线(T)和质控线(C)的荧光强度，由此获得各免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)；
(3)经拟合得到免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线。
本发明的有益效果：
(1)快速、同步定量检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮和杂色曲霉毒素。本发明提供的免疫层析时间分辨荧光试剂盒能在一条试纸条上实现对黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮和杂色曲霉毒素的同步、快速定量检测，使用的抗体均为单克隆抗体，特异性好、灵敏度高，各真菌毒素的检测之间无干扰，简单、快速。
(2)灵敏度高。本发明提供的免疫层析时间分辨荧光试剂盒对检测溶液中黄曲霉毒素B1的最低检测限为0.06ng/mL，伏马毒素B1的最低检测限为0.2ng/mL，赭曲霉毒素A的最低检测限为0.5ng/mL，玉米赤霉烯酮的最低检测限为1ng/mL，杂色曲霉毒素的最低检测限为0.3ng/mL，该检测限能满足欧盟对食品中这5种真菌毒素的限量要求。
附图说明
图1为本发明提供的黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素免疫层析时间分辨荧光速测试剂盒中免疫层析时间分辨荧光试纸条的结构示意图。图中：1吸水垫、2检测垫、3样品垫、4质控线、5黄曲霉毒素B1检测线、6伏马毒素B1检测线、7赭曲霉毒素A检测线、8玉米赤霉烯酮检测线、9杂色曲霉毒素检测线。
具体实施方式
实施例1 黄曲霉毒素B1单克隆抗体的获得
黄曲霉毒素通用单克隆抗体由保藏编号为CCTCC NO.C201014的杂交瘤细胞株3G1分泌产生，具体根据授权号为ZL201210117614.9的专利中报道的方法预先制得，制备方法为：将获得的杂交瘤细胞株3G1注射预先用福氏不完全佐剂处理过的BALB/c小鼠，收集小鼠的腹水，纯化处理后获得黄曲霉毒素B1单克隆抗体。其中，纯化方法为辛酸-硫酸铵法，具体操作为：用双层滤纸过滤小鼠腹水，过滤后的腹水于4℃，12000r/min离心15min以上，吸取上清，将上清与4倍体积的醋酸盐缓冲液混合，边搅拌边缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔 浓度为0.1mol/L和pH值7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH值至7.4，4℃预冷，缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃上清，将所得沉淀用原腹水体积1/10的0.01mol/L磷酸盐缓冲液重悬，装入透析袋，用纯水透析，将充分透析好的蛋白溶液置-70℃冰箱冷冻，然后用冷冻真空干燥机冻干，收集冻干粉，即得纯化好的黄曲霉毒素B1单克隆抗体，将抗体置-20℃冰箱中备用；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容至100mL所得；所述的0.1mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，磷酸二氢钾0.02g，加水定容至100mL所得；
实施例2 赭曲霉毒素A单克隆抗体的获得
赭曲霉毒素A单克隆抗体由保藏编号为CCTCC NO.C201329的杂交瘤细胞株1H2分泌产生，具体根据申请号为201310115921.8的专利中报道的方法预先制得，制备方法为：将杂交瘤细胞株1H2注射到预先用福氏不完全佐剂处理过的BALB/c小鼠的腹部，收集小鼠的腹水，纯化即得赭曲霉毒素A单克隆抗体。所述的纯化方法为辛酸-硫酸铵法，具体步骤为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH值为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH值至7.4，4℃预冷，缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃上清，将所得沉淀用原腹水体积1/10的0.01mol/L、pH值为7.4的磷酸盐缓冲液重悬，装入透析袋，用纯水透析，将充分透析好的蛋白溶液置-70℃冰箱冷冻，之后用冷冻干燥机冻干，收集冻干粉，即得纯化好的赭曲霉毒素A单克隆抗体，将抗体置-20℃冰箱中备用；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.01mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，磷酸二氢钾0.02g，加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为8g氯化钠，2.9g十二水磷酸氢二钠，0.2g氯化钾，磷酸二氢钾0.2g，加水定容到100mL所得；
实施例3 玉米赤霉烯酮单克隆抗体的获得
玉米赤霉烯酮单克隆抗体由保藏编号为CCTCC NO.C201328的杂交瘤细胞株2D3分泌产 生，具体根据申请号为201310115825.3的专利中报道的方法预先制得，制备方法为：将杂交瘤细胞株2D3注射到预先用福氏不完全佐剂处理过的BALB/c小鼠的腹部，收集小鼠的腹水，纯化即得玉米赤霉烯酮单克隆抗体；所述的纯化方法为辛酸-硫酸铵法，具体步骤为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L、pH值为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH值至7.4，4℃预冷，缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃上清，将所得沉淀用原腹水体积1/10的0.01mol/L、pH值为7.4的磷酸盐缓冲液重悬，装入透析袋，用纯水透析，将充分透析好的蛋白溶液置-70℃冰箱冷冻，之后用冷冻干燥机冻干，收集冻干粉，即得纯化好的玉米赤霉烯酮单克隆抗体，将抗体置-20℃冰箱中备用；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.01mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，磷酸二氢钾0.02g，加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为8g氯化钠，2.9g十二水磷酸氢二钠，0.2g氯化钾，磷酸二氢钾0.2g，加水定容到100mL所得；
实施例4杂色曲霉毒素单克隆抗体的获得
杂色曲霉毒素单克隆抗体由保藏编号为CCTCC NO.C2013187的杂交瘤细胞株ST03分泌产生，具体根据申请号为201410115952.8的专利中报道的方法预先制得，制备方法为：将获得的杂交瘤细胞株ST03注射到预先用福氏不完全佐剂处理过的Balb/c小鼠的腹部，收集小鼠的腹水，纯化后得到杂色曲霉毒素单克隆抗体。所述的纯化为辛酸-硫酸铵纯化法，具体操作为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为33μL，室温混合30-60min，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH值为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH值至7.4，4℃预冷，缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后4℃，12000r/min离心30min以上，弃上清，将所得沉淀用原腹水体积1/10的0.01mol/L磷酸盐缓冲液重悬，装入透析袋，用纯水透析，将充分透析好的蛋白溶液置-70℃冰箱冷冻，之 后用冷冻干燥机冻干，收集冻干粉，即得纯化好的杂色曲霉毒素单克隆抗体，将抗体置-20℃冰箱中备用；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，磷酸二氢钾0.02g，加水定容到100mL所得。
实施例5 伏马毒素B1单克隆抗体的获得
杂交瘤细胞株Fm7A11的筛选
1.抗原合成及动物免疫
购买市售的伏马毒素B 1标准品进行完全抗原合成，具体合成步骤如下:将2mg的FB 1标准品粉末与2mg的EDC分别溶于500μL的0.01mol/LPBS溶液，得到EDC溶液和FB 1溶液，将4mg/mL(溶液为0.01mol/LPBS)的EDC溶液逐滴加入溶解好的FB 1溶液中，室温下轻轻搅拌10分钟。将5mg/mL(溶液为0.01mol/LPBS)的BSA溶液逐滴加入到上述混合液中，室温搅拌反应4小时。透析3天。最后进行常规紫外扫描法鉴定，鉴定结果表明FB 1-BSA完全抗原制备成功。
购买6周龄BALB/c小鼠6只，免疫实验室合成的伏马毒素完全抗原FB 1-BSA。第一次免疫将伏马毒素完全抗原与等体积的弗氏完全佐剂乳化后，于小鼠颈背部皮下多点注射。第二次免疫于3周后进行，采用弗氏不完全佐剂与等体积的伏马毒素完全抗原乳化，于小鼠颈背部皮下多点注射。第三次与第四次免疫分别与上一次免疫间隔两周，免疫方式与第二次相同。四次免疫剂量相同，仅为100μg/只。第三次免疫后第7天，小鼠尾静脉采血，分离血清，采用间接ELISA法监测小鼠血清效价，并用间接竞争ELISA法测定小鼠血清灵敏度，选择效价、灵敏度均相对较高的血清对应的小鼠进行最后一次加强免疫，免疫剂量为之前量的2倍。
2.细胞融合
于最后一次加强免疫3天后，采用50％(重量百分数)的聚乙二醇即PEG(分子量为1450)作融合剂，按常规方法进行细胞融合，具体步骤：无菌条件下脱颈处死待融合小鼠，分离脾细胞，与鼠源骨髓瘤细胞SP2/0以5：1的个数比混合，用RPMI-1640基础培养液洗混合细胞，1200rpm，离心5min。弃去上清，控干，加入1mLPEG，融合1分钟，缓慢加入RPMI-1640基础培养液，离心，弃上清，沉淀即为融合细胞，用20mL完全培养基重悬，将悬起的细胞加入到80mL半固体培养基中，混匀后加到6孔细胞培养板上，2mL/孔，置于37℃二氧化碳培养箱培养。
所述的含1％HAT的细胞完全培养基含有20％(体积百分数)胎牛血清，75％(体积百分数)RPMI-1640基础培养液，1％(重量百分数)L-谷氨酰胺，1％(体积百分数)HEPES，1％(体积百分数)双抗(10000单位每毫升青霉素和10000微克每毫升链霉素)，2％(体积百分数)生长因子(HFCS)和1％(重量百分数)次黄嘌呤-氨基蝶岭-胸腺嘧啶核苷即HAT和甲基纤维素购于sigma-Aldrich公司。
3.细胞株的筛选及克隆
待细胞融合后2-3周，细胞集落长至肉眼可见时，用微量移液器将克隆从培养基中挑出，转移至96孔细胞培养板采用HAT液体培养，待细胞长至2/3孔底时，吸取培养上清进行检测。采用两步筛选法，第一步采用间接ELISA方法，筛选出抗伏马毒素而不抗载体蛋白BSA的阳性孔；第二步采用间接竞争ELISA法对第一步筛选出的阳性孔进行检测，用伏马毒素B 1作为竞争原，选择吸光值和灵敏度均较高的孔(吸光值较高指竞争原为0的孔即阳性对照孔的最终测定值较高，灵敏度较高指抑制率为50％时的竞争原浓度亦IC 50值较小)，采用有限稀释法进行亚克隆，亚克隆后采用同样的两步法进行检测，如此重复亚克隆4-5次后，获得杂交瘤细胞株Fm7A11。该杂交瘤细胞株已于2016年3月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO:C201636。
抗伏马毒素B 1单克隆抗体杂交瘤细胞株Fm7A11抗体可变区序列测定
(1)提取总RNA：采用天根公司的总RNA提取试剂盒并按照说明书提取可产生杂交瘤细胞株Fm7A11的总RNA；
(2)合成cDNA：以步骤1获得的总RNA为模板，oligo(dT)15为引物，按照SuperScript TM-2II反转录酶说明书进行反转录，合成cDNA第一链；引物oligo(dT)15由Invitrogen购得；
(3)PCR法克隆可变区基因：根据GENBANK中小鼠抗体基因序列的保守位点设计引物，以CDNA为模版扩增抗体重链、轻链可变区基因。PCR程序为：94℃30s、58℃45s、72℃1min，扩增30个循环，最后72℃延伸10min。PCR产物经过1％(重量百分数)的琼脂糖凝胶电泳分离后，用试剂盒纯化回收DNA片段，连接在载体pMD18-T中，转化大肠杆菌DH5α感受态细胞，挑取阳性克隆，送至上海桑尼生物科技有限公司进行测序。其中引物的序列分别为：重链可变区引物为5’-CAG GTS MAR CTG MAG GAG TCW G-3’(22mer)和5’-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3’(28mer),其中S、M、R和W为兼并碱基，M＝A/C，R＝A/G，S＝G/C，W＝A/T，轻链可变区引物为5’-GAC ATC AAG ATG ACC CAG TCT CCA-3’ (24mer)和5’-CCG TTT TAT TTC CAG CTT GGT CCC-3’(24mer)。
得到的基因序列结果：重链可变区编码基因序列长379bp，序列如SEQ ID NO:1所示，根据所获得的基因序列推导出该基因序列所编码的重链可变区由126个氨基酸组成，序列如SEQ ID NO:3所示。轻链可变区编码基因序列长348bp,序列如SEQ ID NO:2所示，根据所获得的基因序列推导出该基因序列所编码的轻链可变区由116个氨基酸组成，序列如SEQ ID NO:4所示。
5.抗伏马毒素B 1单克隆抗体的制备纯化、亚型和特性鉴定
将实施例1获得的抗伏马毒素B 1单克隆抗体杂交瘤细胞株Fm7A11注射预先用弗氏不完全佐剂处理过的BALB/c小鼠，收集该小鼠的腹水，采用辛酸-硫酸铵法纯化抗体，具体操作为：用双层滤纸过滤小鼠腹水，4℃，12000r/min离心15min以上，吸取上清，将所得腹水上清与4倍体积的醋酸盐缓冲液混合，搅拌下缓慢加入正辛酸，每毫升腹水所需的正辛酸体积为30-35μL，室温混合30-60min，4℃静置2h以上。12000r/min，4℃离心30min以上，弃沉淀，将得到的上清液用双层滤纸过滤后，加入1/10滤液体积的摩尔浓度为0.1mol/L和pH为7.4的磷酸盐缓冲液，用2mol/L的氢氧化钠溶液调节该混合液的pH至7.4，冰浴中缓慢加入硫酸铵至硫酸铵终浓度为0.277g/mL，4℃静置2h以上，然后12000r/min，4℃离心30min以上，弃上清，将所得沉淀用原腹水体积1/10体积的摩尔浓度为0.01mol/L、pH为7.4的磷酸盐缓冲液重悬，装入透析袋，用0.01mol/LPBS透析两天，再改用PB透析两天，将透析袋中蛋白溶液取出，离心，收集上清，弃沉淀，放入-70℃预冻后放入冻干机中冻干。收集冻干粉，即为纯化好的抗伏马毒素B 1单克隆抗体；
所述的醋酸盐缓冲液为0.29g醋酸钠，0.141mL醋酸加水定容到100mL所得；所述的0.01mol/L的磷酸盐缓冲液为0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，0.02g磷酸二氢钾，加水定容到100mL所得；所述的0.1mol/L的磷酸盐缓冲液为8g氯化钠，2.9g十二水磷酸氢二钠，0.2g氯化钾，0.2g磷酸二氢钾，加水定容到100mL所得。
用市售亚型鉴定试剂盒鉴定杂交瘤细胞株Fm7A11分泌的抗伏马毒素B1单克隆抗体的亚型为IgG2b。
用常规非竞争酶联免疫吸附法(ELISA)测得小鼠腹水纯化得到的抗体效价可达到3.2×10 5，即抗体稀释3.2×10 5倍时溶液测定结果为阳性。用常规间接竞争ELISA测定其对伏马毒素B 1灵敏度为0.32ng/mL。对伏马毒素B 2、B 3的交叉反应率为4.3％和12.8％。与黄曲霉毒素、玉米赤霉烯酮、T-2毒素、赭曲霉毒素、呕吐毒素的交叉反应率均小于0.1％。
实施例6
取铕荧光微球，加入1mL 0.2mol/L pH8.2的硼酸缓冲液中，300w超声处理40秒，然后缓慢加入40uL 15mg/mL的碳二亚胺，室温振摇20min后，17000g离心15min，收集沉淀，用0.2mol/L pH 8.2的硼酸缓冲液复溶，将活化的荧光微球加入1mg/ml抗体(黄曲霉毒素B1单克隆抗体35ul、赭曲霉毒素A单克隆抗体45ul、玉米赤霉烯酮单克隆抗体55ul、伏马毒素B1单克隆抗体40ul、杂色曲霉毒素单克隆抗体50ul)，4℃振摇搅拌反应12h后，12000g离心10min，含1％BSA的0.2mol/L pH8.2的硼酸缓冲液复溶，4℃振摇搅拌反应4h，12000g离心10min收集沉淀，复溶于含1.5％(m/v)海藻糖、2％(m/v)牛血清白蛋白的0.2mol/L pH8.2的硼酸缓冲液中，即得铕标记的真菌毒素抗体，置于4℃保存备用。
同步检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染同步检测的免疫层析时间分辨荧光试剂盒及其应用
黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染同步检测免疫层析时间分辨荧光速测试剂盒，它包括免疫层析时间分辨荧光试纸条、含有铕标记的抗黄曲霉毒素B1单克隆抗体、铕标记的伏马毒素B1单克隆抗体、铕标记的赭曲霉毒素A单克隆抗体冻干品、铕标记的玉米赤霉烯酮单克隆抗体冻干品、铕标记的杂色曲霉毒素单克隆抗体冻干品的样品反应瓶、样品稀释液及样品稀释液吸管，所述的免疫层析时间分辨荧光试纸条包括PVC衬底，PVC衬底的一面从上到下依次粘贴吸水垫、检测垫和样品垫，相邻各垫在连接处交叠连接，交叠长度为1mm；
(1)吸水垫的制备
将吸水纸剪裁成长16mm，宽4mm的规格，即得吸水垫；
2)检测垫的制备
检测线的包被：
将黄曲霉毒素B1-牛血清白蛋白偶联物用包被缓冲液配制成0.25mg/mL的溶液，于距硝酸纤维素膜上沿6mm的位置，用线喷方式将其包被于硝酸纤维素膜上得到检测线I，每厘米检测线I上所需黄曲霉毒素B1-牛血清白蛋白偶联物的包被量为100ng；将伏马毒素B1-牛血清白蛋白偶联物用包被缓冲液配制成0.25mg/mL的包被液，于距检测线I 4mm的位置，用线喷方式将其包被于硝酸纤维素膜上得到检测线II，每厘米检测线II上所需伏马毒素B1-牛血清白蛋白偶联物的包被量为200ng；将赭曲霉毒素A-牛血清白蛋白偶联物用包被缓冲液配制成0.45mg/mL的包被液，于距检测线II4mm的位置，用线喷方式将其包被于硝酸纤维素膜上得到检测线III，每厘米检测线III上所需赭曲霉毒素A-牛血清白蛋白偶联物的包被量为160ng；将玉米赤霉烯酮-牛血清白蛋白偶联物用包被缓冲液配制成0.35mg/mL的包被液， 于距检测线III 4mm的位置，用线喷方式将其包被于硝酸纤维素膜上得到检测线IV，每厘米检测线IV上所需玉米赤霉烯酮-牛血清白蛋白偶联物的包被量为200ng；将杂色曲霉毒素-牛血清白蛋白偶联物用包被缓冲液配制成0.4mg/mL的包被液，于距检测线IV 4mm的位置，用线喷方式将其包被于硝酸纤维素膜上得到检测线V，每厘米检测线V上所需杂色曲霉毒素-牛血清白蛋白偶联物的包被量为200ng；然后于37℃条件下干燥120min；
质控线的包被：
将兔抗鼠多克隆抗体用包被缓冲液配成浓度为0.4mg/mL的包被液；于距检测线I 4.5mm的位置，用线喷方式将其横向包被于硝酸纤维素膜上，得质控线，每厘米质控线所需的兔抗鼠多克隆抗体的包被量为120ng，然后于37℃条件下干燥120min；
所述的包被缓冲液为：0.002g叠氮化钠，0.08g氯化钠，0.029g十二水磷酸氢二钠，0.002g氯化钾，0.002g磷酸二氢钾，加水定容至10mL所得；
所述的硝酸纤维素膜长33mm，宽4mm。
(3)样品垫的制备：
将玻璃纤维膜剪裁成长12mm，宽4mm的规格，放入封闭液中浸湿，取出，于40℃条件下干燥4小时，得样品垫，然后置干燥器中室温保存；
所述的封闭液为1g卵清白蛋白，2g蔗糖，0.02g叠氮化钠，0.8g氯化钠，0.29g十二水磷酸氢二钠，0.02g氯化钾，0.02g磷酸二氢钾，加水定容至100mL所得；
(4)免疫层析时间分辨荧光试纸条的组装：
在PVC衬底的一面从上到下依次粘贴吸水垫、检测垫和样品垫，相邻各垫在连接处交叠连接，交叠长度为1mm，即得，见图1；
所述的样品反应瓶的获得：配制好的铕标记的黄曲霉毒素B1单克隆抗体、铕标记的伏马毒素B1单克隆抗体、铕标记的赭曲霉毒素A单克隆抗体、铕标记的玉米赤霉烯酮单克隆抗体、铕标记的杂色曲霉单克隆抗体；复溶于含1.5％(m/v)海藻糖、2％(m/v)牛血清白蛋白的0.01mol/L pH 8.2的磷酸缓冲液中，各取一定量放入样品反应瓶中，置于冷冻干燥机中冻干，备用。所述样品反应瓶中铕标记的黄曲霉毒素B1单克隆抗体冻干品的含量为0.2μg，所述样品反应瓶中铕标记的伏马毒素B1单克隆抗体冻干品的含量为0.3μg，所述样品反应瓶中铕标记的赭曲霉毒素A单克隆抗体冻干品的含量为0.25μg，所述样品反应瓶中铕标记的玉米赤霉烯酮单克隆抗体冻干品的含量为0.2μg，所述样品反应瓶中铕标记的杂色曲霉毒素单克隆抗体冻干品的含量为0.3μg；
所述黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合 污染同步检测免疫层析时间分辨荧光检测试剂盒在饲料样品黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素检测中的应用：
取经确认的阴性饲料样品20g加入100mL 70％(体积分数)的甲醇溶液，均质2min，静置、用双层滤纸过滤，收集滤液1mL，加入5mL样品稀释液稀释滤液，混匀，得到空白基质溶液；
配置黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮、伏马毒素B1分别对应为以下浓度梯度的黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮、伏马毒素B1混合标准溶液6个，黄曲霉毒素B1(0ng/mL、0.05ng/mL、0.1ng/mL、0.25ng/mL、0.5ng/mL、1.0ng/mL)、伏马毒素B1(0ng/mL、0.05ng/mL、0.1ng/mL、0.25ng/mL、0.5ng/mL、1.0ng/mL)、赭曲霉毒素A(0ng/mL、0.25ng/mL、0.5ng/mL、1.0ng/mL、2ng/mL、4ng/mL)、玉米赤霉烯酮(0ng/mL、0.5ng/mL、1.0ng/mL、2.5ng/mL、5.0ng/mL、10ng/mL)、杂色曲霉毒素(0ng/mL、0.05ng/mL、0.1ng/mL、0.25ng/mL、0.5ng/mL、1.0ng/mL)配置成梯度的混合标准溶液。标准溶液每个浓度点重复5次，用多毒素检测试纸条进行检测，用时间分辨荧光检测仪检测：将上述标准品溶液150μL加入样品反应瓶中，混匀，插入免疫层析时间分辨荧光试纸条，37℃反应6min后，用吸水纸吸干样品垫残留液体，立即用时间分辨荧光免疫分析仪检测，(激发波长：365nm，测定波长：615nm)，读取检测区域的荧光信号强度值，计算5次重复平均值。以标准系列浓度值自然对数(Lnc)和T线的荧光信号强度值/C线的荧光信号强度值(T/C)绘制标准曲线，标准曲线公式如Y＝a*lnc+b，5种检测对象的标准曲线参数如下表所示：
  a b R 2 检测限/ng/mL
黄曲霉毒素B1 -1.425 3.524 0.991 0.06
伏马毒素B1 -1.750 3.448 0.989 0.2
赭曲霉毒素A -1.594 4.788 0.989 0.5
玉米赤霉烯酮 -1.750 5.751 0.979 1.0
杂色曲霉 -1.643 3.364 0.976 0.3
取待检饲料样品20g加入100mL 70％(体积分数)的甲醇溶液，均质2min，静置、用双层滤纸过滤，收集滤液1mL，加入5mL样品稀释液稀释滤液，混匀；取待检饲料样品检测液150μL加入样品反应瓶中，混匀，插入免疫层析时间分辨荧光试纸条，37℃反应6min后，用吸水纸吸干样品垫残留液体，立即用时间分辨荧光免疫分析仪检测(激发波长：365nm， 测定波长：615nm)，获得各免疫层析时间分辨荧光试纸条5条检测线时间分辨荧光强度与质控线时间分辨荧光强度的比值(T/C)，然后将其分别代入上述得到的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)与黄曲霉毒素B1浓度、伏马毒素B1浓度、赭曲霉毒素A浓度、玉米赤霉烯酮浓度、杂色曲霉毒素浓度的关系曲线，得该饲料样品中的黄曲霉毒素B1含量为6.4μg/kg、伏马毒素B1含量为4.1μg/kg、赭曲霉毒素含量为0μg/kg、玉米赤霉烯酮含量为0μg/kg、杂色曲霉毒素含量为3.3μg/kg。
实施例7
与实施例6不同的是：免疫层析时间分辨荧光试纸条中吸水垫长14mm，宽3mm；检测垫长30mm，宽3mm；样品垫长16mm，宽3mm，相邻各垫的交叠长度为2mm；所述荧光试纸条中检测垫上靠近质控线的检测线与硝酸纤维素膜上沿的间距为8mm，每相邻两条检测线之间的间距为4mm。所述免疫层析时间分辨荧光试纸条中检测垫上每厘米检测线所需的黄曲霉毒素B1-牛血清白蛋白偶联物的包被量为200ng，每厘米检测线所需的伏马毒素B1-牛血清白蛋白偶联物的包被量为250ng，每厘米检测线所需的赭曲霉毒素A-牛血清白蛋白偶联物的包被量为200ng，每厘米检测线所需的玉米赤霉烯酮-牛血清白蛋白偶联物的包被量为250ng，每厘米检测线所需的杂色曲霉毒素-牛血清白蛋白偶联物的包被量为300ng；每厘米质控线所需的兔抗鼠多克隆抗体的包被量为150ng；所述样品反应瓶中铕标记的黄曲霉毒素B1单克隆抗体冻干品的含量为0.2μg，所述样品反应瓶中铕标记的伏马毒素B1单克隆抗体冻干品的含量为0.3μg，所述样品反应瓶中铕标记的赭曲霉毒素A单克隆抗体冻干品的含量为0.3μg，所述样品反应瓶中铕标记的玉米赤霉烯酮单克隆抗体冻干品的含量为0.2μg，所述样品反应瓶中铕标记的杂色曲霉毒素单克隆抗体冻干品的含量为0.3μg；
取经高效液相色谱确认的含多种毒素的饲料样品(黄曲霉毒素B1含量为5.1μg/kg、伏马毒素B1含量为3.9μg/kg、赭曲霉毒素含量为1.5μg/kg、玉米赤霉烯酮含量为41.2μg/kg、杂色曲霉毒素含量为5.3μg/kg)。
取待检饲料样品10g加入30mL 70％(体积分数)的甲醇溶液，均质2min，静置、用双层滤纸过滤，收集滤液1mL，加入9mL样品稀释液稀释滤液，混匀；取待检饲料样品检测液150μL加入样品反应瓶中，混匀，插入免疫层析时间分辨荧光试纸条，37℃反应6min后，用吸水纸吸干样品垫残留液体，立即用时间分辨荧光免疫分析仪检测(激发波长：365nm，测定波长：615nm)，获得各免疫层析时间分辨荧光试纸条5条检测线时间分辨荧光强度与质控线时间分辨荧光强度的比值(T/C)，然后将其分别代入上述得到的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)与黄曲霉毒素B1浓度、伏 马毒素B1浓度、赭曲霉毒素A浓度、玉米赤霉烯酮浓度、杂色曲霉毒素浓度的关系曲线，得该饲料样品中的黄曲霉毒素B1含量为5.3μg/kg、伏马毒素B1含量为3.7μg/kg、赭曲霉毒素含量为1.8μg/kg、玉米赤霉烯酮含量为41.8μg/kg、杂色曲霉毒素含量为5.7μg/kg。

Claims (10)

  1. 同步检测黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素混合污染的免疫层析时间分辨荧光试剂盒，其特征在于：它包括免疫层析时间分辨荧光试纸条和含有铕标记的黄曲霉毒素B1单克隆抗体、铕标记的伏马毒素B1单克隆抗体、铕标记的赭曲霉毒素A单克隆抗体、铕标记的玉米赤霉烯酮单克隆抗体、铕标记的杂色曲霉单克隆抗体冻干品的样品反应瓶，其中：所述的免疫层析时间分辨荧光试纸条包括PVC衬底，衬底的一面从上到下依次粘贴吸水垫、检测垫和样品垫，相邻各垫在连接处交叠连接，所述检测垫以硝酸纤维素膜为基垫，硝酸纤维素膜上自上而下设置横向质控线和5条检测线，所述质控线包被有兔抗鼠多克隆抗体，所述5条线上分别包被黄曲霉毒素B1-牛血清白蛋白偶联物、伏马毒素B1-牛血清白蛋白偶联物、赭曲霉毒素A-牛血清白蛋白偶联物、玉米赤霉烯酮-牛血清白蛋白偶联物、杂色曲霉毒素-牛血清白蛋白偶联物，所述的伏马毒素B1单克隆抗体为由保藏编号CCTCC NO.C201636的杂交瘤细胞株Fm7A11分泌产生的单克隆抗体。
  2. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述铕标记的单克隆抗体是按照以下方法制备得到：
    铕标记的黄曲霉毒素B1单克隆抗体将黄曲霉毒素B1单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中混合、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的黄曲霉毒素B1单克隆抗体；1mL活化后的铕标记试剂可偶联黄曲霉毒素B1单克隆抗体：30μg-80μg；
    铕标记的赭曲霉毒素A单克隆抗体将赭曲霉毒素A单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的赭曲霉毒素A单克隆抗体；1mL活化后的铕标记试剂可偶联赭曲霉毒素A单克隆抗体：40μg-90μg；
    铕标记的玉米赤霉烯酮单克隆抗体将玉米赤霉烯酮单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的玉米赤霉烯酮单克隆抗体；1mL活化后的铕标记试剂可偶联玉米赤霉烯酮单克隆抗体：30μg-90μg；
    铕标记的伏马毒素B1单克隆抗体将伏马毒素B1单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的伏马毒素B1单克隆抗体；1mL活化后的铕标记试剂可偶联伏马毒素B1单克隆抗体：30μg-80μg；
    铕标记的杂色曲霉单克隆抗体将杂色曲霉单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中溶解、振荡反应，然后经离心、复溶、封闭步骤得到目标产物铕标记的杂色曲霉单克隆抗体；1mL活化后的铕标记试剂可偶联杂色曲霉单克隆抗体：30μg-80μg。
  3. 根据权利要求2所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述的活化方法为取铕标记试剂，用pH 8.2 0.2mol/L的硼酸缓冲液中超声分散，然后缓慢加入碳二亚胺溶液，室温振荡活化，离心去上清液，用pH 8.2 0.2mol/L的硼酸缓冲液复溶，备用，所述的活化时间为15-30min。
  4. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述免疫层析时间分辨荧光试纸条中的吸水垫长10-16mm，宽3-5mm；检测垫长25-33mm，宽3-5mm；样品垫长12-18mm，宽3-5mm，相邻各垫的交叠长度为1-2mm；所述免疫层析时间分辨荧光试纸条中检测垫上靠近质控线的检测线与硝酸纤维素膜上沿的间距为5-10mm，每相邻两条检测线之间的间距为1.5-4.5mm，靠近质控线的检测线与质控线3-6mm；所述的样品反应瓶为1-5mL的卡口瓶。
  5. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述免疫层析时间分辨荧光试纸条中检测垫上每厘米检测线所需的黄曲霉毒素B1-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的伏马毒素B1-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的赭曲霉毒素A-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的玉米赤霉烯酮-牛血清白蛋白偶联物的包被量为100-400ng，每厘米检测线所需的杂色曲霉毒素-牛血清白蛋白偶联物的包被量为100-400ng；每厘米质控线所需的兔抗鼠多克隆抗体的包被量为80-200ng。
  6. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述样品反应瓶中铕标记的黄曲霉毒素B1单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的伏马毒素B1单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的赭曲霉毒素A单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的玉米赤霉烯酮单克隆抗体冻干品的含量为0.1-0.3μg，所述样品反应瓶中铕标记的杂色曲霉毒素单克隆抗体冻干品的含量为0.1-0.3μg。
  7. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：其还包括样品稀释液和样品稀释液吸管，所述的样品稀释液为体积分数为0.01％-0.30％的吐温-20水溶液。
  8. 根据权利要求1所述的免疫层析时间分辨荧光试剂盒，其特征在于：所述的时间分辨荧光试纸条的制备方法如下：
    (1)将吸水纸剪裁得吸水垫；
    (2)检测垫的制备：
    将黄曲霉毒素B1-牛血清白蛋白偶联物、伏马毒素B1-牛血清白蛋白偶联物、赭曲霉毒素A-牛血清白蛋白偶联物、玉米赤霉烯酮-牛血清白蛋白偶联物、杂色曲霉毒素-牛血清白蛋白 偶联物配制成浓度为0.08-0.50mg/mL的包被液，用线喷方式将其于硝酸纤维素膜上进行分别间隔包被，得到5条检测线，然后于37-40℃条件下干燥60-120min；
    将兔抗鼠多克隆抗体配成浓度为0.1-0.5mg/mL的包被液，用线喷方式将其横向包被于硝酸纤维素膜上，得质控线然后于37-40℃条件下干燥60-120min；
    (3)样品垫的制备：
    将玻璃纤维膜放入封闭液中浸湿，取出，于37-40℃条件下干燥4-6h，得样品垫，然后置干燥器中室温保存；
    (4)免疫层析时间分辨荧光试纸条的组装：
    在PVC底板的一面从上到下依次粘贴吸水垫、检测垫、样品垫，相邻各垫在连接处交叠连接，即得免疫层析时间分辨荧光试纸条。
  9. 权利要求1所述的免疫层析时间分辨荧光速测试剂盒在黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素含量检测中的应用，其特征在于：将待测样品经前处理获得待测样品溶液，加入样品反应瓶中，混匀，插入时间分辨荧光试纸条，37℃反应6-10min后，用时间分辨荧光测试仪进行检测，获得免疫层析时间分辨荧光试纸条上检测线(T)荧光强度与质控线(C)荧光强度的比值；基于预先获得的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)分别与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线，获得待测样品溶液中黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素的含量，最后经换算即得待测样品中黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素的含量。
  10. 根据权利要求8所述的应用，其特征在于：所述的免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)分别与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线是采用以下方法得到的：
    (1)配制得到系列梯度浓度的黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮和杂色曲霉毒素标准溶液；
    (2)将适量上述梯度的黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素标准品溶液分别加入到样品反应瓶中，混匀，插入免疫层析时间分辨荧光试纸条，37℃反应6min，用时间分辨荧光免疫分析仪检测得到各免疫层析时间分辨荧光试纸条上检测线(T)和质控线(C)的荧光强度，由此获得各免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比值(T/C)；
    (3)经拟合得到免疫层析时间分辨荧光试纸条检测线荧光强度与质控线荧光强度的比 值(T/C)与黄曲霉毒素B1、伏马毒素B1、赭曲霉毒素A、玉米赤霉烯酮、杂色曲霉毒素浓度的关系曲线。
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