WO2018121677A1 - 同步检测黄曲霉毒素和甲萘威混合污染的时间分辨荧光免疫层析试剂盒、制备方法及应用 - Google Patents

同步检测黄曲霉毒素和甲萘威混合污染的时间分辨荧光免疫层析试剂盒、制备方法及应用 Download PDF

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WO2018121677A1
WO2018121677A1 PCT/CN2017/119440 CN2017119440W WO2018121677A1 WO 2018121677 A1 WO2018121677 A1 WO 2018121677A1 CN 2017119440 W CN2017119440 W CN 2017119440W WO 2018121677 A1 WO2018121677 A1 WO 2018121677A1
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time
carbaryl
aflatoxin
immunochromatographic
test strip
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PCT/CN2017/119440
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English (en)
French (fr)
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李培武
唐晓倩
张奇
张兆威
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中国农业科学院油料作物研究所
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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
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    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—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
    • 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
    • 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
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/60—Synthetic polymers other than synthetic polypeptides as analytes

Definitions

  • the invention particularly relates to a time-resolved fluorescent immunochromatography kit for synchronously detecting mixed contamination of aflatoxin and carbaryl, a preparation method and application thereof.
  • Aflatoxin is a major class of mycotoxin contaminants. It is highly toxic and polluted and is classified as a type of carcinogen by the World Health Organization. Its toxicity is carcinogenic, mutagenic, and teratogenic to animals, causing DNA damage and immunosuppression. Aflatoxin contamination can occur in all aspects of planting, harvesting, storage, transportation, consumption, etc.
  • the contaminated products are mainly cereals and nuts, and are also detected in other products such as meat, eggs and milk.
  • Carbaryl is a kind of carbamate pesticide widely used in crops such as vegetables, fruits, grains, etc. It has good control effect on pests. However, due to its abuse and illegal use in agricultural production, it makes it It poses a major threat to people's dietary safety.
  • the detection methods of aflatoxin and carbaryl are mainly liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry. These methods have good stability, high sensitivity and good accuracy, but the pre-processing steps are complicated and the cost of sample detection is high.
  • Immunochromatography overcomes the above-mentioned deficiencies. Based on the specific reaction of antigen-antibody, the antigen is fixed by nitrocellulose, so that the free target in the chromatography process competes with the antigen on the detection line to bind the labeled antibody. Detecting the amount of marker on the line calculates the amount of target in the sample.
  • the problem to be solved by the present invention is to provide an immunochromatography time-resolved fluorescent immunochromatography kit capable of synchronously detecting mixed contamination of aflatoxin and carbaryl, a preparation method thereof and an application thereof.
  • the time-resolved fluorescent immunochromatography kit can be used for synchronous detection of aflatoxin and carbaryl in the sample, and has the characteristics of simple operation, rapidity and high sensitivity.
  • the technical solution adopted by the present invention is:
  • Immunochromatographic time-resolved fluorescent immunochromatography kit for simultaneous detection of aflatoxin and carbaryl mixed contamination, which comprises immunochromatographic time-resolved fluorescent test strips and anti-aflatoxin monoclonal containing ruthenium-labeled a sample reaction bottle of an antibody, a ⁇ -labeled carbaryl monoclonal antibody lyophilized product, wherein: the immunochromatographic time-resolved fluorescent test strip comprises a cardboard, and one side of the cardboard is pasted with an absorbent pad and a test pad from top to bottom.
  • a sample pad adjacent pads are overlapped and connected at a joint, the test pad is based on a nitrocellulose membrane, and a lateral quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control
  • the line was coated with a rabbit anti-mouse polyclonal antibody, and the detection line was located under the quality control line, and the number was 2, and the test line was coated with aflatoxin-bovine serum albumin conjugate and carbaryl- An ovalbumin conjugate; the anti-naphthoquinone monoclonal antibody is secreted by the hybridoma cell line Jnw1D2 deposited under the accession number CCTCC No. C201654.
  • the hybridoma cell line was deposited with the China Center for Type Culture Collection (CCTCC) on March 29, 2016. The deposit address is China, Wuhan, Wuhan University, and the accession number is CCTCC NO: C201654.
  • the tritiated anti-naphthoquinone monoclonal antibody is prepared according to the following method: shaking the carbofuran monoclonal antibody and the activated guanidine labeling reagent in a boric acid buffer for more than 2 hours, and centrifuging The supernatant was removed and blocked to obtain the target product; the tritiated anti-aflatoxin monoclonal antibody was prepared by shaking the aflatoxin monoclonal antibody and the activated guanidine labeling reagent in boric acid buffer. After reacting for more than 2 hours, centrifuge to remove the supernatant and block to obtain the target product.
  • the activation method is a sputum labeling reagent, ultrasonically dispersed in a boric acid buffer solution, and then slowly added to the carbodiimide solution, shaken and activated, centrifuged to remove the supernatant, and reconstituted with a boric acid buffer solution. .
  • the activation time is 15-30 min.
  • the blocking solution is a boric acid buffer containing 0.5-1% BSA.
  • the mass ratio of the guanidine labeling reagent to the aflatoxin monoclonal antibody is 1: (0.001-0.1)
  • the mass ratio of the guanidine labeling reagent to the carbaryl antibody is 1: (0.001-0.1).
  • the sputum-labeled anti-aflatoxin monoclonal antibody lyophilized product is obtained by adding a ruthenium-labeled monoclonal antibody to an antibody protection solution and lyophilizing;
  • the sputum-labeled anti-methionine monoclonal antibody lyophilized product is a lyophilized anti-menacarb monoclonal antibody added to the antibody protection solution;
  • the antibody protective solution is 0.01%-0.30 vt% (volume fraction) of Tween-20, 0.5-1.5 wt% sucrose and 0.1-1 wt% bovine serum albumin (BSA) aqueous solution;
  • BSA bovine serum albumin
  • the absorbent pad in the immunochromatographic time-resolved fluorescent test strip has a length of 15-35 mm and a width of 3-5 mm; the sample pad has a length of 12-18 mm and a width of 2-5 mm, and the overlapping length of the adjacent pads is 1-3 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 immunochromatography time-resolved fluorescent test strip is 6-15 mm, and the spacing between each adjacent two detection lines It is 1.5-4.5mm, and the distance between the detection line and the quality control line near the quality control line is 4-10mm; the sample reaction bottle is 1-5mL bayonet bottle.
  • the amount of aflatoxin-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 0.4-0.8 ⁇ g per cm detection line.
  • the required amount of the carbaryl-egg albumin conjugate is 0.8-1.0 ⁇ g;
  • the content of the sputum-labeled anti-aflatoxin monoclonal antibody lyophilized product in the sample reaction bottle is 0.1-0.3 ⁇ g,
  • the content of the quinone-labeled anti-menacarb monoclonal antibody lyophilized product in the sample reaction bottle is 0.2-0.4 ⁇ g;
  • the preparation method of the time-resolved fluorescent test strip is as follows:
  • the aflatoxin-bovine serum albumin conjugate, the carbaryl-ovalbumin conjugate was formulated into a coating solution having a concentration of 0.25-2 mg/mL, and the film was applied to a nitrocellulose membrane by a dicing method. Packing separately, obtaining 2 test lines, and then drying at 37-40 ° C for 30-60 minutes;
  • the rabbit anti-mouse polyclonal antibody was formulated into a coating solution with a concentration of 0.1-0.45 mg/mL, and coated on the nitrocellulose membrane by a filming method to obtain a quality control line, which was required for each centimeter of the quality control line.
  • the rabbit anti-mouse polyclonal antibody is coated in an amount of 0.4-0.8 ⁇ g, and then dried at 37-40 ° C for 30-60 minutes;
  • the glass fiber membrane is placed in a blocking solution to be wetted, taken out, and dried at 37-40 ° C for 4-10 hours to obtain a sample pad, which is then stored in a desiccator at room temperature;
  • Adhesive pads, test pads, and sample pads are sequentially pasted from top to bottom on one side of the paperboard, and adjacent pads are overlapped at the joints to obtain an immunochromatographic time-resolved fluorescent test strip;
  • 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, dihydrogen phosphate Potassium 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 blocking solution used in the preparation of the immunochromatographic time-resolved fluorescent test strip is: 0.5-2 g of egg albumin per 100 mL, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, and twelve water. 0.29 g of disodium hydrogen phosphate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate;
  • the above-mentioned immunochromatography time-resolved fluorescent immunochromatography kit is used for detecting the content of aflatoxin and carbaryl: the sample to be tested is pretreated to obtain the sample solution to be tested, and then added to the sample reaction bottle, mixed, and inserted. The time-resolved fluorescent test strips were reacted at 37 ° C for 6 minutes, and then detected by a time-resolved fluorescence tester to obtain the detection line (T) fluorescence intensity and the quality control line (C) fluorescence intensity on the immunochromatographic time-resolved fluorescent test strip.
  • T detection line
  • C quality control line
  • the content of aflatoxin and carbaryl in the sample solution is finally converted to the content of aflatoxin and carbaryl in the sample to be tested;
  • 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 proportional to the concentration of aflatoxin and carbaryl. owned:
  • the immunochromatography time-resolved fluorescent kit provided by the invention can realize synchronous and rapid detection of two mycotoxins of aflatoxin and carbaryl on a test strip, and the antibodies used are all monoclonal antibodies, and the specificity is good. High sensitivity, no interference between the two, simple and fast.
  • the minimum detection limit of aflatoxin in the detection solution is 0.02 ng/mL for the immunochromatography time-resolved fluorescence kit provided by the invention, and the minimum detection limit for the carbaryl is 0.01 ng/mL, the detection limit can meet the EU Limited requirements in food.
  • sample preparation method is simple. Sample pretreatment only needs to add methanol water extract to the sample for ultrasonic extraction for 5-10 minutes, then let stand for 5-10 minutes, and take the supernatant to dilute it for testing. The whole sample pretreatment process is simple and fast.
  • 1 is a schematic view showing the structure of aflatoxin and carbaryl time-resolved fluorescent immunochromatographic test strips provided by the present invention.
  • 1 absorbent pad, 2 test pad, 3 sample pad, 4 quality control line, 5 carbaryl detection line, 6 aflatoxin detection line.
  • mice Six 6-week-old BALB/c mice were purchased, and bovine serum albumin (BSA) was coupled with 6-(1-naphthyloxycarboxamide)-hexanoic acid (CNH) to obtain the carbinol complete antigen CNH-BSA.
  • BSA bovine serum albumin
  • CNH 6-(1-naphthyloxycarboxamide)-hexanoic acid
  • 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 boosted and the immunization dose was twice that of the previous amount.
  • mice to be fused to the neck were removed under aseptic conditions.
  • the spleen cells were isolated and mixed with the murine myeloma cells SP2/0 at a ratio of 5:1, and the mixed cells were washed with RPMI-1640 basal medium, centrifuged at 1200 rpm for 5 min.
  • 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 first step was to use an indirect ELISA method to screen positive holes that were resistant to carbaryl and not to carrier protein BSA.
  • the second step used indirect competitive ELISA to detect positive wells screened in the first step.
  • RNA obtained in Step 1 as a template, oligo (dT) 15 as primers for reverse transcription in accordance with the reverse transcriptase SuperScript TM -2II specification, first strand cDNA synthesis; primer oligo (dT) 15 purchased by Invitrogen;
  • heavy chain variable region primers are 5'-ACG ACG TTG TAA AAC GAC GGC-3' (21mer) and light chain variable region primers are 5'-ACG ACG TTG TAA AAC GAC GGC-3 '(21mer) and 5'-CAG GGG CCA GTG GAT AGA CAG ATG G-3' (21mer).
  • the obtained gene sequence result the heavy chain variable region coding gene sequence is 339 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 obtained gene sequence by 113 amino acids. Composition, the sequence is shown in SEQ ID NO: 3.
  • the light chain variable region coding gene sequence is 315 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 105 amino acids, and the sequence is SEQ ID. NO: 4 is shown.
  • the obtained anti-menadione monoclonal antibody hybridoma cell line Jnw1D2 was injected into BALB/c mice previously treated with Freund's incomplete adjuvant, and the ascites of the mouse was collected, and the antibody was purified by caprylic acid-ammonium sulfate method.
  • the operation was as follows: the mouse ascites was filtered with double-layer filter paper, centrifuged at 12000 r/min for 15 min or more at 4 ° C, and the supernatant was aspirated, and the obtained ascites was mixed with 4 volumes of acetate buffer, and n-octanoic acid was slowly added under stirring.
  • n-octanoic acid required per milliliter of ascites is 30-35 ⁇ L, mixed at room temperature for 30-60 min, and allowed to 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.
  • the lyophilized powder is collected, which is a purified monoclonal antibody against carbaryl;
  • 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 subtype of the anti-carbaryl monoclonal antibody secreted by the hybridoma cell line Jnw1D2 was identified as IgG2b using a commercially available subtype identification kit.
  • the titer of the antibody was determined by enzyme-linked immunosorbent assay (ELISA) to be 1.6 ⁇ 10 4 .
  • the 50% inhibitory concentration of carbaryl has an IC 50 of 0.668 ng/kg, and has no cross-reaction with carbofuran, aldicarb, and methomyl.
  • the anti-aflatoxin monoclonal antibody is secreted by the hybridoma cell line 1C11 with the accession number CCTCC NO.C201018, and is prepared in advance according to the method reported in the patent No. CN201010245095.5, which is prepared by the hybridization obtained.
  • the tumor cell line 1C11 was injected with BALB/c mice previously treated with Freund's incomplete adjuvant, and the ascites of the mice was collected, and the anti-aflatoxin monoclonal antibody was obtained after purification.
  • the purification method is octanoic acid-ammonium sulfate method
  • the specific operation is: filtering the mouse ascites with double-layer filter paper, filtering the ascites at 4 ° C, centrifugation at 12000 r / min for more than 15 min, aspirating the supernatant, the supernatant and 4 times the volume Mix the acetate buffer, slowly add n-octanoic acid while 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 4°C, 12000r/min After centrifugation for more than 30 min, 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
  • 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 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.
  • the 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate , 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, add water to a volume of 100mL;
  • Time-resolved fluorescent immunochromatography kit for quantitative detection of aflatoxin and carbaryl including fluorescent test strips, anti-aflatoxin monoclonal antibody containing sputum label, sputum-labeled carbaryl monoclonal antibody, sample reaction vial
  • the fluorescent test strip is as shown in FIG. 1 , and comprises a cardboard board.
  • One side of the paperboard is pasted with an absorbent pad, a test pad and a sample pad from top to bottom, and the adjacent pads are overlapped at the joint, and the overlap length is 1mm, immunochromatography time-resolved fluorescent test strips, the absorbent pad is 18mm long and 4mm wide; the test pad is 25mm long and 4mm wide; the sample pad is 15mm long and 4mm wide, and the overlapping length of adjacent pads is 1mm;
  • the detection pad is based on a nitrocellulose membrane, and a lateral quality control line and two 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 coating amount is 0.4 ⁇ g/cm, the quality control line is located below the quality control line, the number is 2, and the test line is coated with aflatoxin-bovine serum albumin conjugate and carbaryl-ovalbumin The conjugates were coated with 0.4 ⁇ g/cm and 1 ⁇ g/cm,
  • the distance between the detection line of the toxin-bovine serum albumin conjugate and the upper edge of the nitrocellulose membrane was 8 mm, and the distance between the quality control line and the detection line coated with aflatoxin-bovine serum albumin conjugate was 4 mm, two The spacing of the test lines is 4 mm.
  • the absorbent paper is cut to a size of 18 mm and a width of 4 mm to obtain an absorbent pad;
  • the carbaryl coating antigen (carbaryl-ovalbumin conjugate, obtained by coupling 6-(1-naphthyloxycarboxamide)-hexanoic acid (CNH) to ovalbumin) was prepared in coating buffer.
  • the coating amount of the antigen was 1.0 ⁇ g, and then dried at 37 ° C for 60 minutes;
  • the aflatoxin-coated antigen (aflatoxin-bovine serum albumin) was formulated into a coating solution having a concentration of 0.25 mg/mL with a coating buffer; at a position of 8 mm from the nitrocellulose membrane, a mask was used. The method is carried out laterally on the nitrocellulose membrane to obtain a detection line, and the coating amount of the coated antigen per cm of the detection line is 0.4 ⁇ g, and then dried at 37 ° C for 60 minutes;
  • the coating buffer solution comprises: 0.1 g of bovine serum albumin BSA, 0.002 g of sodium azide, 0.08 g of sodium chloride, 0.029 g of disodium hydrogen phosphate dodecahydrate, and 0.002 g of potassium chloride per 10 mL. Potassium dihydrogen phosphate 0.002g;
  • the rabbit anti-mouse polyclonal antibody was formulated into a coating solution with a concentration of 0.25 mg/mL in a coating buffer; it was laterally drawn by a film at a position 4 mm from the detection line coated with the aflatoxin-coated antigen.
  • the coating was coated on a nitrocellulose membrane to obtain a quality control line.
  • the amount of the rabbit anti-mouse polyclonal antibody required per centimeter of the control line was 0.4 ⁇ g, and then dried at 37 ° C for 2 hours;
  • the coating buffer is:
  • Each 10mL contains 0.1g of bovine serum albumin, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride, and 0.002g of potassium dihydrogen phosphate;
  • the nitrocellulose membrane is 25 mm long and 4 mm wide.
  • the glass fiber membrane is cut into a size of 15 mm and a width of 4 mm, and is placed in a blocking solution to be wetted, taken out, and dried at 37 ° C for 6 hours to obtain a sample pad, which is then stored in a desiccator at room temperature;
  • the blocking solution is 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.3 g of sodium dihydrogen phosphate dihydrate, 1.0 g of Tween-20, 1.0 g of polyvinylpyrrolidone (PVPK-30), 0.25 g of EDTA, 0.5 g of bovine serum.
  • Albumin BSA 0.02 g sodium azide, water to a volume of 100 mL;
  • the absorbent pad, the test pad and the sample pad are sequentially pasted on the side of the cardboard from top to bottom, and the adjacent pads are overlapped at the joint, and the overlapping length is 1 mm, that is, the fluorescent test strip is obtained.
  • the supernatant was again centrifuged, reconstituted by adding 1 mL of boric acid buffer containing 0.5% BSA, vortexed and mixed, sonicated for 10 min, and shaken at 25 ° C for 2 h to obtain the target product ⁇ -labeled anti-aflatoxin monoclonal antibody.
  • the above-mentioned cockroach labeling reagent can be purchased from Shanghai Youyou Biotechnology Co., Ltd., but is not limited thereto.
  • the supernatant was again centrifuged, reconstituted by adding 1 mL of boric acid buffer containing 0.5% BSA, vortexed and mixed, sonicated for 10 min, and shaken at 25 ° C for 2 h to obtain the target product ⁇ -labeled monoclonal antibody against carbaryl.
  • the above-mentioned cockroach labeling reagent can be purchased from Shanghai Youyou Biotechnology Co., Ltd., but is not limited thereto.
  • the above-mentioned ruthenium-labeled anti-aflatoxin monoclonal antibody is at a ratio of 1:300 (V:V), and the above-mentioned ruthenium-labeled anti-carbaryl monoclonal antibody is 1:250 (V:V)
  • the ratio was added to a certain volume of the antibody protection solution, and lyophilized with a lyophilizer.
  • the antimony-labeled anti-aflatoxin monoclonal antibody lyophilized product 0.1 ⁇ g and the sputum-labeled anti-carbaryl monoclonal antibody lyophilized product 0.3 ⁇ g were placed.
  • the antibody protective solution was 0.01 vt% Tween-20, 0.5 wt% sucrose, and 1 wt% bovine serum albumin (BSA).
  • Aflatoxin/carbaryl was added in a ratio of 1:10 to prepare a mixed standard solution of each aflatoxin and carbaryl concentration gradient.
  • Aflatoxin concentrations of 1.5 ng/mL, 0.5 ng/mL, 0.15 ng/mL, 0.05 ng/mL, 0.015 ng/mL, 0.005 ng/mL of aflatoxin standard solution and 15 ng/mL, 5 ng/mL , 1.5 ng / mL, 0.5 ng / mL, 0.15 ng / mL, 0.05 ng / mL;
  • the detection instrument is a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. Detecting the ratio of the fluorescence intensity of each fluorescent test strip detection line to the fluorescence intensity of the quality control line (T/C);
  • the concentration of aflatoxin and carbaryl standard is taken as the abscissa, and the ratio of the corresponding detection line of the standard solution to the fluorescence intensity of the quality control line, that is, the T/C value is the ordinate, and the relationship curve is obtained.
  • the effective detection range of the method is 0.1-ng/mL of carbaryl; aflatoxin 0.02-0.85 ng/mL.
  • the detection instrument is a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm.
  • the ratio of the time-resolved fluorescence intensity of each fluorescent test strip detection line to the time-resolved fluorescence intensity of the quality control line (T/C) is detected; and then substituted into the above-mentioned fluorescent test strip detection line for time-resolved fluorescence intensity and quality control line
  • the ratio of time-resolved fluorescence intensity (T/C) to the concentration of the standard substance is obtained, and the concentration of aflatoxin and carbaryl in the sample solution is obtained, and then the aflatoxin and the sample in the sample can be obtained according to the dilution factor. Nathene content.
  • the recoveries of aflatoxin in the grain samples were as follows: 106.5%, 90.2%, 86.1%; the recoveries of carbaryl were: 104.1%, 89.5%, 83.7%
  • Time-resolved fluorescent immunochromatography kit for quantitative detection of aflatoxin and carbaryl, including fluorescent test strips, anti-aflatoxin monoclonal antibody containing sputum label, sputum-labeled carbaryl monoclonal antibody, sample reaction vial
  • the fluorescent test strip comprises cardboard, and one side of the cardboard is pasted with an absorbent pad, a test pad and a sample pad from top to bottom, and the adjacent pads are overlapped at the joint, the overlap length is 2 mm, and the immunochromatography time is
  • the absorption pad in the fluorescent test strip is 18 mm long and 3 mm wide; the test pad is 25 mm long and 3 mm wide; the sample pad is 15 mm long and 3 mm wide, and the overlapping length of adjacent pads is 2 mm; the test pad is nitrocellulose
  • the membrane is a base pad, and a lateral quality control line and two detection lines are arranged from top to bottom on the nitrocellulose membrane, and the quality control line is coated with a rabbit anti-mouse
  • the detection line is located below the quality control line, and the number is 2, and the detection line is coated with aflatoxin-bovine serum albumin conjugate and carbaryl-ovalbumin conjugate.
  • the amount is 0.6 ⁇ g/cm and 0.6 ⁇ g/cm, respectively, close to the quality control line detection line and The distance between the upper edges of the nitrocellulose membrane was 12 mm, the distance between the quality control line and the first detection line was 3 mm, and the distance between the two detection lines was 3 mm.
  • the antibody protective solution is 0.1vt% (volume fraction) of Tween-20, 1wt% sucrose and 0.5wt% bovine serum albumin (BSA) aqueous solution; ⁇ labeled anti-aflatoxin monoclonal antibody preparation ⁇ mark reagent
  • the mass ratio of the effective amount to the aflatoxin monoclonal antibody is 1:0.1
  • the mass ratio of the guanidine labeling reagent to the carbamazepine monoclonal antibody in an effective amount to the carbaryl antibody is 1:0.1.
  • the spiked recovery test of aflatoxin and carbaryl in the nut sample take 5g of nut sample, add 20mL of 80% methanol solution, homogenize for 5 minutes, let stand, pass double-layer filter paper, collect filtrate, 1:1 The ratio is diluted. Accurately add aflatoxin (carbaryl) standards 0.005 ng/mL, 0.01 ng/mL, and 0.1 ng/mL (0.05 ng/mL, 0.1 ng/mL, and 1 ng/mL), respectively, and take the above-mentioned samples to be tested. 300 ⁇ L of the test solution was added to a sample reaction flask containing a ruthenium-labeled monoclonal antibody, and the mixture was mixed.
  • the detection instrument is a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm.
  • the ratio of the time-resolved fluorescence intensity of each fluorescent test strip detection line to the time-resolved fluorescence intensity of the quality control line (T/C) is detected; and then substituted into the above-mentioned fluorescent test strip detection line for time-resolved fluorescence intensity and quality control line
  • the ratio of time-resolved fluorescence intensity (T/C) to the concentration of the standard substance is obtained, and the concentration of aflatoxin and carbaryl in the sample solution is obtained, and then the aflatoxin and the sample in the sample can be obtained according to the dilution factor. Nathene content.
  • the recoveries of aflatoxin in the nut samples were as follows: 103.0%, 92.0%, 85.4%; the recoveries of carbaryl were: 108.5%, 91.5%, 80.9%.

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Abstract

一种同步检测黄曲霉毒素和甲萘威混合污染的时间分辨荧光免疫层析试剂盒、制备方法及其应用。它包括免疫层析时间分辨荧光试纸条和含有铕标记的抗黄曲霉毒素单克隆抗体、铕标记的甲萘威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的检测线上分别上包被黄曲霉毒素-牛血清白蛋白偶联物和甲萘威-卵清白蛋白偶联物；所述的抗甲萘威单克隆抗体为由保藏编号为CCTCC NO.C201654的杂交瘤细胞株Jnw1D2分泌产生的。其可用于样品中黄曲霉毒素、甲萘威含量的同步检测，具有操作简单、快速、灵敏度高的特点。

Description

同步检测黄曲霉毒素和甲萘威混合污染的时间分辨荧光免疫层析试剂盒、制备方法及应用 技术领域
本发明具体涉及一种同步检测黄曲霉毒素和甲萘威混合污染的时间分辨荧光免疫层析试剂盒、制备方法及其应用。
背景技术
黄曲霉毒素是一类主要的真菌毒素污染物，毒性强、污染广，被世界卫生组织划分为一类致癌物。其毒性表现在对动物致癌、致突变、致畸，可引起DNA损伤和免疫抑制。黄曲霉毒素污染可发生在种植、收获和贮藏、运输、消费等各环节，污染的产品多以谷物、坚果为主，在其他产品如肉、蛋、奶中也均有检出。甲萘威是一种被广泛用于蔬菜、水果、谷物等农作物的一类氨基甲酸酯类农药，对害虫具有较好的防治效果，然而由于其在农业生产上的滥用和违禁使用，使其对人们的饮食安全造成重大威胁，我国规定甲萘威在谷物中限量0.5-1μg/mL，蔬菜中限量为1μg/mL。黄曲霉毒素与甲萘威在谷物中往往会造成同时污染，需要研制一种方便快捷的检测技术对两类污染物进行同时检测。
目前，黄曲霉毒素与甲萘威的检测方法主要有液相色谱法、气相色谱-质谱联用法、液相色谱-质谱联用法等。这些方法稳定性好、灵敏度高、准确性好，但是前处理步骤复杂，样品检测成本高。免疫层析法克服了上述不足，其以抗原抗体特异性反应为基础，用硝酸纤维素将抗原固定，使层析过程中游离靶标物与检测线上的抗原竞争结合已标记抗体，通过结合在检测线上标记物的量计算样品中靶标物的含量。
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、甲萘威混合污染的免疫层析时间分辨荧光免疫层析试剂盒、制备方法及其应用。该时间分辨荧光免疫层析试剂盒可用于样品中黄曲霉毒素、甲萘威含量的同步检测，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、甲萘威混合污染的免疫层析时间分辨荧光免疫层析试剂盒，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抗黄曲霉毒素单克隆抗体、铕标记的甲萘威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纸板，纸板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检测线，所述质控线包被有兔抗鼠多克隆抗体，所述检测线位于质控线下方，个数为2条，检测 线上分别上包被黄曲霉毒素-牛血清白蛋白偶联物和甲萘威-卵清白蛋白偶联物；所述的抗甲萘威单克隆抗体为由保藏编号为CCTCC NO.C201654的杂交瘤细胞株Jnw1D2分泌产生的。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:C201654。
按上述方案，所述铕标记的抗甲萘威单克隆抗体是按照以下方法制备得到的：将甲萘威单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中振摇反应2h以上，离心去上清，封闭得到目标产物；所述铕标记的抗黄曲霉毒素单克隆抗体是按照以下方法制备得到的：将黄曲霉毒素单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中振摇反应2h以上，离心去上清，封闭得到目标产物
按上述方案，所述的活化方法为取铕标记试剂，用硼酸缓冲液中超声分散，然后加缓慢加入碳二亚胺溶液，振荡活化，离心去上清液，用硼酸缓冲液复溶，备用。所述的活化时间为15-30min。
按上述方案，所述的封闭液为含0.5-1％BSA的硼酸缓冲液。
按上述方案，所述铕标记试剂以有效量计与黄曲霉毒素单克隆抗体的质量比为1:(0.001-0.1)，铕标记试剂以有效量计与甲萘威抗体的质量比为1:(0.001-0.1)。
按上述方案，所述铕标记的抗黄曲霉毒素单克隆抗体冻干品是将铕标记的单克隆抗体加入抗体保护液中冻干的；
所述铕标记的抗甲萘威单克隆抗体冻干品是将铕标记的抗甲萘威单克隆抗体加入抗体保护液中冻干的；
所述的抗体保护液为0.01％-0.30vt％(体积分数)的吐温-20、0.5-1.5wt％蔗糖和0.1-1wt％牛血清蛋白(BSA)水溶液；
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15-35mm，宽3-5mm；样品垫长12-18mm，宽2-5mm，相邻各垫的交叠长度为1-3mm；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6-15mm，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的间距为4-10mm；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-牛血清白蛋白偶联物的包被量为0.4～0.8μg，每厘米检测线所需的甲萘威-卵清白蛋白偶联物的包被量为0.8-1.0μg；所述样品反应瓶中铕标记的抗黄曲霉毒素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抗甲萘威单克隆抗体冻干品的含量为0.2-0.4μg；
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)将吸水纸剪裁为吸水垫；
(2)检测垫的制备：
将黄曲霉毒素-牛血清白蛋白偶联物、甲萘威-卵清白蛋白偶联物配制成浓度为0.25-2mg/mL的包被液，用划膜方式将其于硝酸纤维素膜上进行分别间隔包被，得到2条检测线，然后于37-40℃条件下干燥30-60分钟；
将兔抗鼠多克隆抗体配成浓度为0.1-0.45mg/mL的包被液，用划膜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0.4～0.8μg，然后于37-40℃条件下干燥30-60分钟；
(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4-10小时，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：
在纸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-牛血清白蛋白偶联物包被液、甲萘威-卵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中含有卵清白蛋白0.5-2g，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免疫层析试剂盒在黄曲霉毒素、甲萘威含量检测中的应用：将待测样品经前处理获得待测样品溶液后，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分钟后，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、甲萘威浓度的关系曲线，获得待测样品溶液中黄曲霉毒素、甲萘威的含量，最后经换算即得待测样 品中黄曲霉毒素、甲萘威的含量；
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、甲萘威浓度的关系曲线是采用以下方法得到的：
(1)配制得到一系列梯度浓度的黄曲霉毒素标准品溶液；配制得到一系列浓度的甲萘威标准品溶液。
(2)将适量上述各梯度浓度的黄曲霉毒素、甲萘威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10分钟，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)的荧光强度值，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)与黄曲霉毒素浓度的关系曲线；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)与甲萘威浓度的关系曲线；
本发明的有益效果：
(1)快速、同步检测黄曲霉毒素、甲萘威。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、甲萘威两种真菌毒素的同步、快速检测，使用的抗体均为单克隆抗体，特异性好、灵敏度高，两者之间无干扰，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的最低检测限为0.02ng/mL，对甲萘威的最低检测限为0.01ng/mL，该检测限能满足欧盟对食品中的限量要求。
(3)样品前处理方法简单。样品前处理只需要将甲醇水提取液加入样品中超声提取5-10分钟，然后静置5-10分钟，取上清液稀释即可进行检测，整个样品前处理过程简单、快速。
附图说明
图1为本发明提供的黄曲霉毒素、甲萘威时间分辨荧光免疫层析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甲萘威检测线、6黄曲霉毒素检测线。
具体实施方式
实施例1 抗甲萘威单克隆抗体的获得
杂交瘤细胞株Jnw1D2的筛选
1.动物免疫
购买6周龄BALB/c小鼠6只，用6-(1-萘氧基甲酰胺)-己酸(CNH)偶联牛血清白蛋白(BSA)，获得甲萘威完全抗原CNH-BSA，免疫小鼠。第一次免疫将甲萘威完全抗原与等体积 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甲萘威完全抗原乳化，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加强免疫，免疫剂量为之前量的2倍。
2.细胞融合
于加强免疫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1mL PEG，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 HAT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，筛选出抗甲萘威而不抗载体蛋白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，用甲萘威作为竞争原，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IC50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Jnw1D2，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:C201654。
4.抗甲萘威单克隆抗体杂交瘤细胞株Jnw1D2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Jnw1D2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GENEBANK中小鼠抗体基因序列的保守位点设计引物，以cDNA为模板扩增抗体轻、重链可变区基因。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’-ACG ACG TTG TAA AAC GAC GGC-3’(21mer)和轻链可变区引物为5’-ACG ACG TTG TAA AAC GAC GGC-3’(21mer)和5’-CAG GGG CCA GTG GAT AGA CAG ATG G-3’(21mer)。
得到的基因序列结果：重链可变区编码基因序列长339bp，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113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315bp,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105个氨基酸组成，序列如SEQ ID NO:4所示。
5.抗甲萘威单克隆抗体的制备纯化、亚型和特性鉴定
将获得的抗甲萘威单克隆抗体杂交瘤细胞株Jnw1D2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，即为纯化好的抗甲萘威单克隆抗体；
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Jnw1D2分泌的抗甲萘威单克隆抗体的亚型为IgG2b。通过酶联免疫吸附法(ELISA)测得抗体的效价可达1.6×10 4。对甲萘威的50％抑制浓度IC 50为0.668ng/kg，与克百威、涕灭威、灭多威等无交叉反应。
实施例2 抗黄曲霉毒素单克隆抗体的获得
抗黄曲霉毒素单克隆抗体由保藏编号为CCTCC NO.C201018的杂交瘤细胞株1C11分泌产生，具体根据授权号为CN201010245095.5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1C11注射预先用福氏不完全佐剂处理过的BALB/c小鼠，收集该小鼠的腹水，纯化处理后获得抗黄曲霉毒素单克隆抗体。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，即得纯化好的抗黄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0.01mol/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；
实施例3 同步检测黄曲霉毒素、甲萘威时间分辨荧光试剂盒及其应用
定量检测黄曲霉毒素、甲萘威的时间分辨荧光免疫层析试剂盒，包括荧光试纸条、含有铕标记的抗黄曲霉毒素单克隆抗体、铕标记的甲萘威单克隆抗体、样品反应瓶，所述的荧光试纸条如图1所示，包括纸板，纸板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，免疫层析时间分辨荧光试纸条中的吸水垫长18mm，宽4mm；检测垫长25mm，宽4mm；样品垫长15mm，宽4mm，相邻各垫的交叠长度为1mm；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2条检测线，所述质控线包被有兔抗鼠多克隆抗体，包被量为0.4μg/cm，质控线所述检测线位于质控线下方，个数为2条，检测线上分别上包被黄曲霉毒素-牛血清白蛋白偶联物和甲萘威-卵清白蛋白偶联物，包被量分别为0.4μg/cm和1μg/cm，包被黄曲霉毒素-牛血清白蛋白偶联物的检测线与硝酸纤维素膜上沿的间距为8mm，质控线和包被黄曲霉毒素-牛血清白蛋白偶联物的检测线的间距4mm，两条检测线的间距4mm。
所述荧光试纸条的获得：
(1)吸水垫的制备
将吸水纸剪裁成长18mm，宽4mm的规格，即得吸水垫；
(2)检测垫的制备
检测线的包被：
将甲萘威包被抗原(甲萘威-卵清白蛋白偶联物，用6-(1-萘氧基甲酰胺)-己酸(CNH)偶联卵清白蛋白获得)用包被缓冲液配制成浓度为1mg/mL的包被液；于距硝酸纤维素膜上沿12mm的位置，用划膜方式将其横向包被于硝酸纤维素膜上，得到检测线，每厘米检测线所需包被抗原的包被量为1.0μg，然后于37℃条件下干燥60分钟；
将黄曲霉毒素包被抗原(黄曲霉毒素-牛血清白蛋白)用包被缓冲液配制成浓度为0.25mg/mL的包被液；于距硝酸纤维素膜上沿8mm的位置，用划膜方式将其横向包被于硝酸纤维素膜上，得到检测线，每厘米检测线所需包被抗原的包被量为0.4μg，然后于37℃条件下干燥60分钟；
所述的包被缓冲液为：每10mL中含有牛血清白蛋白BSA 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；
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0.25mg/mL的包被液；于距靠近包被黄曲霉毒素包被抗原的检测线4mm的位置，用划膜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0.4μg，然后于37℃条件下干燥2小时；
所述的包被缓冲液为：
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；
所述的硝酸纤维素膜长25mm，宽4mm。
(3)样品垫的制备：
将玻璃纤维膜剪裁成长15mm，宽4mm的规格，放入封闭液中浸湿，取出，于37℃条件下干燥6小时，得样品垫，然后置干燥器中室温保存；
所述的封闭液为2.9g十二水磷酸氢二钠，0.3g二水合磷酸二氢钠，1.0g Tween-20，1.0g聚乙烯吡咯烷酮(PVPK-30)，0.25g EDTA，0.5g牛血清白蛋白BSA，0.02g叠氮钠，加水定容至100mL所得；
(4)荧光试纸条的组装：
在纸板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，即得荧光试纸条。
所述铕标记的抗黄曲霉毒素单克隆抗体的获得：
取0.2mol/L pH 8.18的硼酸缓冲液800μL，加入200μL铕标记试剂(粒径100nm，固形物含量1％)，振荡混匀。超声3S。加入40μL 15mg/mL EDC溶液，振荡混匀15min。13000r/min，10℃，离心10min，去上清液，加入1mL硼酸缓冲液复溶，振荡混匀，超声3S。加入20μg黄曲霉毒素单克隆抗体，混匀，250r/min、25℃下摇床过夜。再次离心去上清，加入1mL含0.5％BSA的硼酸缓冲液复溶，振荡混匀，超声10min，25℃下摇床2h，得目标产物铕标记的抗黄曲霉毒素单克隆抗体。上述铕标记试剂可购于上海优你生物科技有限公司，但不限于此。
所述铕标记的抗甲萘威单克隆抗体的获得：
取0.2mol/L pH 8.18的硼酸缓冲液800μL，加入200μL铕标记试剂(粒径100nm，固形物含量1％)，振荡混匀。超声3S。加入40μL 15mg/mL EDC溶液，振荡混匀15min。13000r/min，10℃，离心10min，去上清液，加入1mL硼酸缓冲液复溶，振荡混匀，超声3S。加入20μg抗甲萘威单克隆抗体，混匀，250r/min、25℃下摇床过夜。再次离心去上清，加入1mL含0.5％BSA的硼酸缓冲液复溶，振荡混匀，超声10min，25℃下摇床2h，得目标产物铕标记的抗甲萘威单克隆抗体。上述铕标记试剂可购于上海优你生物科技有限公司，但不限于此。
所述样品反应瓶的获得:将上述铕标记抗黄曲霉毒素单克隆抗体以1：300(V:V)的比例，上述铕标记抗甲萘威单克隆抗体以1：250(V:V)的比例加入到一定体积的抗体保护液中用冻干机冻干，取铕标记抗黄曲霉毒素单克隆抗体冻干品0.1μg和铕标记抗甲萘威单克隆抗体冻干品0.3μg放入样品反应瓶中。所述的抗体保护液为0.01vt％的吐温-20、0.5wt％蔗糖和1wt％牛血清蛋白(BSA)。
上述的时间分辨荧光免疫层析试纸条黄曲霉毒素、甲萘威定量检测中的应用：
1.荧光试纸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)与黄曲霉毒素、甲萘威浓度的关系曲线的建立：
(1)对经高效液相色谱法(HPLC)检测为黄曲霉毒素和甲萘威为阴性的谷物进行样品前处理。取谷物样品25g，添加80％甲醇水溶液100mL，均质提取5分钟，静置，过双层滤纸，搜集滤液，以1：4的比例进行稀释。
(2)对上述谷物样品稀释液进行标准品物质加标，制备混合标准品溶液。将黄曲霉毒素/甲萘威以1：10的比例添加配得各黄曲霉毒素和甲萘威浓度梯度的混合标准品溶液。黄曲霉毒素浓度分别为1.5ng/mL、0.5ng/mL、0.15ng/mL、0.05ng/mL、0.015ng/mL、0.005ng/mL的黄曲霉毒素标准品溶液和15ng/mL、5ng/mL、1.5ng/mL、0.5ng/mL、0.15ng/mL、0.05ng/mL；
(3)取黄曲霉毒素与甲萘威混合标准品溶液300μL加入样品瓶，加入到含有铕标记的单克隆抗体的样品反应瓶中，混匀，将荧光试纸条样品垫一端插入反应瓶中，37℃反应10分钟，上机检测。检测仪器为时间分辨荧光免疫分析仪，激发波长365nm，发射波长615nm。检测得到各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)；
(4)分别以黄曲霉毒素和甲萘威标准品浓度为横坐标，各浓度标准品溶液相应的检测线与质控线荧光强度的比值即T/C值为纵坐标，拟合得到关系曲线。该方法的有效检测范围为甲萘威0.1-1ng/mL；黄曲霉毒素0.02-0.85ng/mL。
2.谷物样品中黄曲霉毒素和甲萘威含量的加标回收实验：
取谷物样品25g，添加80％甲醇水溶液100mL，均质提取5分钟，静置，过双层滤纸，搜集滤液，以1：1的比例进行稀释。向其中分别准确添加黄曲霉毒素(甲萘威)标准品0.1ng/mL、0.5ng/mL和0.8ng/mL，取上述待测样品检测液300μL加入到含有铕标记的单克隆抗体的样品反应瓶中，混匀，将荧光试纸条样品垫一端插入反应瓶中，37℃反应10min，上机检测。检测仪器为时间分辨荧光免疫分析仪，激发波长365nm，发射波长615nm。检测得到各荧光试纸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)；然后将其代入上述得到的荧光试纸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)与标准品物质浓度的关系曲线，得到样品溶液中的黄曲霉毒素与甲萘威物质浓度，然后根据稀释倍数即可求得样品中黄曲霉毒素与甲萘威物质含量。测得谷物样品中的黄曲霉毒素加标回收率依次为：106.5％，90.2％，86.1％；甲萘威加标回收率依次为：104.1％，89.5％，83.7％。
实施例4
定量检测黄曲霉毒素、甲萘威的时间分辨荧光免疫层析试剂盒，包括荧光试纸条、含有铕标记的抗黄曲霉毒素单克隆抗体、铕标记的甲萘威单克隆抗体、样品反应瓶，所述的荧光试纸条包括纸板，纸板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2mm，免疫层析时间分辨荧光试纸条中的吸水垫长18mm，宽3mm；检测垫长25mm，宽3mm；样品垫长15mm，宽3mm，相邻各垫的交叠长度为2mm；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2条检测线，所述质控线包被有兔抗鼠多克隆抗体，包被量为0.6μg/cm，质控线所述检测线位于质控线下方，个数为2条，检测线上分别上包被黄曲霉毒素-牛血清白蛋白偶联物和甲萘威-卵清白蛋白偶联物，包被量分别为0.6μg/cm和0.6μg/cm，靠近质控线的检测线与硝酸纤维素膜上沿的间距为12mm，质控线和第一条检测线的间距3mm，两条检测线的间距3mm。
所述的样品反应瓶中铕标记抗黄曲霉毒素单克隆抗体冻干品0.2μg和铕标记抗甲萘威单克隆抗体冻干品0.2μg放入样品反应瓶中。所述的抗体保护液为0.1vt％(体积分数)的吐温-20、1wt％蔗糖和0.5wt％牛血清蛋白(BSA)水溶液；铕标记抗黄曲霉毒素单克隆抗体制备中铕标记试剂以有效量计与黄曲霉毒素单克隆抗体的质量比为1:0.1，铕标记抗甲萘威单克隆抗体制备中铕标记试剂以有效量计与甲萘威抗体的质量比为1:0.1。
坚果样品中黄曲霉毒素和甲萘威含量的加标回收实验：取坚果样品5g，添加80％甲醇水溶液20mL，均质提取5分钟，静置，过双层滤纸，搜集滤液，以1：1的比例进行稀释。向其中分别准确添加黄曲霉毒素(甲萘威)标准品0.005ng/mL、0.01ng/mL和0.1ng/mL(0.05ng/mL、0.1ng/mL和1ng/mL)，取上述待测样品检测液300μL加入到含有铕标记的单克隆抗体的样品反应瓶中，混匀，将荧光试纸条样品垫一端插入反应瓶中，37℃反应10min，上机检测。检测仪器为时间分辨荧光免疫分析仪，激发波长365nm，发射波长615nm。检测得到各荧光试纸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)；然后将其代入上述得到的荧光试纸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)与标准品物质浓度的关系曲线，得到样品溶液中的黄曲霉毒素与甲萘威物质浓度，然后根据稀释倍数即可求得样品中黄曲霉毒素与甲萘威物质含量。测得坚果样品中的黄曲霉毒素加标回收率依次为：103.0％，92.0％，85.4％；甲萘威加标回收率依次为：108.5％，91.5％，80.9％。

Claims (10)

  1. 同步检测黄曲霉毒素、甲萘威混合污染的免疫层析时间分辨荧光免疫层析试剂盒，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抗黄曲霉毒素单克隆抗体、铕标记的甲萘威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纸板，纸板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检测线，所述质控线包被有兔抗鼠多克隆抗体，所述检测线位于质控线下方，个数为2条，检测线上分别上包被黄曲霉毒素-牛血清白蛋白偶联物和甲萘威-卵清白蛋白偶联物；所述的抗甲萘威单克隆抗体为由保藏编号为CCTCC NO.C201654的杂交瘤细胞株Jnw1D2分泌产生的。
  2. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，其特征在于：所述铕标记的抗甲萘威单克隆抗体是按照以下方法制备得到的：将甲萘威单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中振摇反应2h以上，离心去上清，封闭得到目标产物；所述铕标记的抗黄曲霉毒素单克隆抗体是按照以下方法制备得到的：将黄曲霉毒素单克隆抗体和活化后的铕标记试剂在硼酸缓冲液中振摇反应2h以上，离心去上清，封闭得到目标产物
  3. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，其特征在于：所述的活化方法为取铕标记试剂，用硼酸缓冲液中超声分散，然后加缓慢加入碳二亚胺溶液，振荡活化，离心去上清液，用硼酸缓冲液复溶，备用；所述的活化时间为15-30min；所述的封闭液为含0.5-1％BSA的硼酸缓冲液。
  4. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，其特征在于：所述铕标记试剂以有效量计与黄曲霉毒素单克隆抗体的质量比为1:(0.001-0.1)，铕标记试剂以有效量计与甲萘威抗体的质量比为1:(0.001-0.1)。
  5. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，其特征在于：所述铕标记的抗黄曲霉毒素单克隆抗体冻干品是将铕标记的单克隆抗体加入抗体保护液中冻干的；
    所述铕标记的抗甲萘威单克隆抗体冻干品是将铕标记的抗甲萘威单克隆抗体加入抗体保护液中冻干的；
    所述的抗体保护液为0.01％-0.30vt％(体积分数)的吐温-20、0.5-1.5wt％蔗糖和0.1-1wt％牛血清蛋白(BSA)水溶液；
  6. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，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15-35mm，宽3-5mm；样品垫长12-18mm，宽2-5mm，相邻各垫的交叠长度为1-3mm；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6-15mm，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的间距为4-10mm；所述的样品反应瓶为1-5mL的卡口瓶。
  7. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒，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-牛血清白蛋白偶联物的包被量为0.4～0.8μg，每厘米检测线所需的甲萘威-卵清白蛋白偶联物的包被量为0.8-1.0μg；所述样品反应瓶中铕标记的抗黄曲霉毒素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抗甲萘威单克隆抗体冻干品的含量为0.2-0.4μg。
  8. 根据权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒的制备方法，其特征在于：步骤如下：
    (1)将吸水纸剪裁为吸水垫；
    (2)检测垫的制备：
    将黄曲霉毒素-牛血清白蛋白偶联物、甲萘威-卵清白蛋白偶联物配制成浓度为0.25-2mg/mL的包被液，用划膜方式将其于硝酸纤维素膜上进行分别间隔包被，得到2条检测线，然后于37-40℃条件下干燥30-60分钟；
    将兔抗鼠多克隆抗体配成浓度为0.1-0.45mg/mL的包被液，用划膜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0.4～0.8μg，然后于37-40℃条件下干燥30-60分钟；
    (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4-10小时，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：
    在纸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- 牛血清白蛋白偶联物包被液、甲萘威-卵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中含有卵清白蛋白0.5-2g，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；
  9. 权利要求1所述的免疫层析时间分辨荧光免疫层析试剂盒在黄曲霉毒素、甲萘威含量检测中的应用，其特征在于：将待测样品经前处理获得待测样品溶液后，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分钟后，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、甲萘威浓度的关系曲线，获得待测样品溶液中黄曲霉毒素、甲萘威的含量，最后经换算即得待测样品中黄曲霉毒素、甲萘威的含量。
  10. 根据权利要求9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、甲萘威浓度的关系曲线是采用以下方法得到的：
    (1)配制得到一系列梯度浓度的黄曲霉毒素标准品溶液；配制得到一系列浓度的甲萘威标准品溶液。
    (2)将适量上述各梯度浓度的黄曲霉毒素、甲萘威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10分钟，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)的荧光强度值，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)与黄曲霉毒素浓度的关系曲线；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)与甲萘威浓度的关系曲线。
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