WO2013155883A1 - 杂交瘤细胞株 3g1 及其产生的抗黄曲霉毒素b1单克隆抗体 - Google Patents

杂交瘤细胞株 3g1 及其产生的抗黄曲霉毒素b1单克隆抗体 Download PDF

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WO2013155883A1
WO2013155883A1 PCT/CN2013/070614 CN2013070614W WO2013155883A1 WO 2013155883 A1 WO2013155883 A1 WO 2013155883A1 CN 2013070614 W CN2013070614 W CN 2013070614W WO 2013155883 A1 WO2013155883 A1 WO 2013155883A1
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aflatoxin
monoclonal antibody
hybridoma cell
cell line
ser
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PCT/CN2013/070614
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French (fr)
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李培武
李鑫
张奇
丁小霞
张文
李冉
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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    • 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
    • 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

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  • the present invention relates to a hybridoma cell line 3G1 and a monoclonal antibody against aflatoxin B1 produced thereby.
  • Aflatoxin is a secondary metabolite produced by the secretion of Aspergillus flavus and Aspergillus parasiticus, and is a natural toxic compound that can cause various damage to humans and animals.
  • Aflatoxin has been found in more than 20 species, including aflatoxin B1 ( AFB1) is the most toxic, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic.
  • AFB1 aflatoxin B1
  • AFB1 aflatoxin B1
  • AFM1 AFM1
  • AFB1 It can also contaminate a variety of raw materials, including fruits, dried fruits, vegetables, condiments, oil crops, tobacco and Chinese herbal medicines, etc., which can be seen: AFB1
  • the food chain is extremely polluted and can be found in almost every part of the food chain.
  • AFB1 in agricultural and food products in tropical and subtropical regions The detection rate is higher and the pollution is more serious.
  • the pollution of grain, oil and food such as peanuts, corn and peanut oil is the most serious.
  • the international trade disputes of agricultural products caused by AFB1 exceeding the standard also occur frequently. 1999, 2000 After the arrival of peanuts exported to the EU in China in 2001 and 2001, more than 40% of the total aflatoxins and AFB1 were detected by the EU.
  • the existing aflatoxin detection methods mainly include thin layer chromatography, precision instrumental analysis and immunological analysis.
  • thin layer chromatography is the most commonly used detection method for aflatoxin detection. This method does not require special equipment and equipment. It can be carried out in general laboratories, but there are large reagent consumption, cumbersome operation, easy interference, and poor accuracy. It can not be accurately quantified, and it is not harmful to the experimental personnel and the surrounding environment. It is not suitable for rapid on-site detection.
  • the precision instrumental analysis method includes fluorescence spectrophotometry and high performance liquid chromatography. These methods have high sensitivity and accurate detection results, but the required instruments and equipment are expensive, and the sample preparation process is cumbersome, requiring a high degree of purification of aflatoxin samples.
  • the problem to be solved by the present invention is to provide a hybridoma cell line 3G1 and a monoclonal antibody against aflatoxin B1 produced thereby.
  • the present invention provides a hybridoma cell line 3G1 which has been on July 13, 2010 Nissin is located in the China Center for Type Culture Collection (CCTCC). The deposit address is China, Wuhan, Wuhan University, and the accession number is CCTCC NO. C201014. The classification is mouse hybridoma cells. 3G1. It has the heavy chain variable region coding gene sequence of the anti-aflatoxin B1 monoclonal antibody shown in SEQ ID NO. 1 in the sequence listing and SEQ ID NO. 2 in the sequence listing. The anti-aflatoxin B1 monoclonal antibody light chain variable region encoding gene sequence is shown.
  • the heavy chain variable region thereof has the amino acid sequence shown by SEQ ID NO. 3 in the Sequence Listing; the light chain variable region has the amino acid sequence shown by SEQ ID NO. 4 in the Sequence Listing.
  • the monoclonal antibody against aflatoxin B1 can recognize aflatoxin B1, aflatoxin B1 50% inhibitory concentration IC 50 of 1.6ng / mL.
  • the hybridoma cell line 3G1 provided by the present invention is obtained by a two-step screening method, wherein the specific step is: converting aflatoxin B1 into its hemiacetal form AFB 2a under the action of H 2 SO 4 , and then reacting with the BSA amino group.
  • the fused cells were screened in two steps by ELISA: the first step was to screen positive clones that were only resistant to aflatoxin but not to carrier protein BSA by indirect ELISA; the second step was to screen out the first step by indirect competitive ELISA.
  • the positive clone culture medium was tested, and AFB1 was used as the competition source. Positive clones with low absorbance and high sensitivity were selected for continuous cloning by limiting dilution method.
  • the same two-step screening method was used for antibody detection about 10 days after cloning. After 2-3 clones, the hybridoma cell line 3G1 was obtained by final screening.
  • the preparation method of the monoclonal antibody against aflatoxin B1 provided by the invention is as follows: the obtained hybridoma cell line 1C8 BALB/c mice pretreated with Freund's incomplete adjuvant were injected, and the ascites of the mice was collected, and the anti-aflatoxin B1 monoclonal antibody was obtained after purification.
  • the purification method is an octanoic acid-ammonium sulfate method, and the specific operation is: filtering the mouse ascites with a double-layer filter paper, and filtering the ascites. Centrifuge at 4 °C, 12000r/min for more than 15min, aspirate the supernatant, mix the supernatant with 4 volumes of acetate buffer, slowly add n-octanoic acid while stirring, and the volume of n-octanoic acid 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 30min Above, 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 having a molar concentration of 0.1 mol/L and pH
  • the sodium hydroxide solution adjusted the pH of the mixture to 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 4 ° C, Centrifuge at 12000r/min for more than 30min, discard the supernatant, and use the original ascites volume of 1/10 0.01mol/L.
  • the phosphate buffer solution was resuspended, placed in a dialysis bag, dialyzed against pure water, and the fully dialyzed protein solution was frozen in a refrigerator at -70 °C, then lyophilized with a freeze vacuum dryer to collect the lyophilized powder, which was purified.
  • Anti-Aflatoxin B1 Monoclonal antibody put the antibody in the refrigerator at -20 °C for use;
  • Acetate buffer 0.29g sodium acetate was added to 0.141mL acetic acid, and the volume was adjusted to 100mL with pure water.
  • phosphate buffer 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g Potassium chloride, potassium dihydrogen phosphate 0.02g, add water to 100mL.
  • the hybridoma cell line 3G1 provided by the present invention can be used for preparing a high titer anti-aflatoxin B1 monoclonal antibody, and an anti-aflatoxin B1 mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method for measuring the titer Up to 6.40 ⁇ 10 6 .
  • ELISA antibody enzyme-linked immunosorbent assay
  • the anti-aflatoxin B1 monoclonal antibody provided by the present invention can be applied to determine the content of aflatoxin B1.
  • FIG. 1 is a front elevational view of an aflatoxin B1 immunochromatographic test strip prepared in Example 4 of the present invention.
  • Figure 2 is a left side view of the aflatoxin B1 immunochromatographic test strip prepared in Example 4 of the present invention.
  • 1 cardboard 1 Absorbent pad; 3 test pad; 4 gold pad; 5 sample pad.
  • Fig. 3 is a graph showing the result of the detection of a sample of aflatoxin B1 immunochromatographic test strip provided in Example 4 of the present invention.
  • Example 1 Screening of hybridoma cell line 3G1
  • the commercially available aflatoxin B1 standard was purchased for complete antigen synthesis.
  • the specific synthetic steps were as follows: 4 mg of AFB1 was dissolved in 2 mL of acetone, 40 ⁇ L of 10% H 2 SO 4 was added , and the mixture was stirred at 56 ° C for 4 h; After evaporation to dryness, 5 mL of H 2 O was added, and then extracted twice with 25 mL of chloroform, and then the organic layer was washed with 20 mL of H 2 O, and the organic layer was evaporated.
  • mice Six 6-week-old female BALB/c mice were purchased and the self-synthesized aflatoxin B1 complete antigen AFB 2a -BSA was immunized.
  • the first immunization mixed the complete antigen AFB 2a -BSA with an equal amount of Freund's complete adjuvant, and then the mice were injected subcutaneously at the neck and back.
  • the second immunization was performed 4 weeks after the first immunization, and was emulsified with an equal volume of complete antigen AFB 2a -BSA by Freund's incomplete adjuvant, and the mice were intraperitoneally injected.
  • the third immunization and the second immunization interval were 4 weeks, the immunization method and dose were the same as the second immunization, and the fourth immunization was performed 3 weeks after the third immunization.
  • the immunization method and the immunization dose were the same as the second immunization.
  • the dose per immunization was 50 ⁇ g per mouse.
  • One week after the first three immunizations blood was collected from the tail vein, serum was separated, and serum antibody titers were monitored by indirect ELISA.
  • One week after the fourth immunization blood was collected from the tail vein, serum was separated, and the serum antibody titer of the mice was monitored by indirect ELISA.
  • the serum sensitivity of the mice was determined by indirect competitive ELISA, and the serum with relatively high titer and sensitivity was selected. The mice were given the last booster and the immunization dose was twice as high as before.
  • AFB1 was purchased from Sigma-Aldrich.
  • mice were sacrificed by cervical dislocation and the spleen was taken under sterile conditions to isolate the spleen cells and the murine myeloma cells SP2/0. Mix the 5:1 ratio, wash the mixed cells with RPMI-1640 base medium, then fuse with 50% PEG, fuse for 1 minute, then top up RPMI-1640 The basal medium, the centrifuge, the supernatant, the mouse spleen cells and the murine myeloma cells. The fused cells formed by SP2/0 were resuspended in 72 mL of RPMI-1640 basal medium, and the resuspended cells were added dropwise.
  • RPMI-1640 base medium containing 20% (by volume) fetal bovine serum, 2% (% by weight) growth factor and 1% (by weight) hypoxanthine - aminopterin - thymidine.
  • the above SP2/0 was purchased from Shanghai Fanke Biotechnology Co., Ltd.; RPMI-1640
  • the basal medium was purchased from Hyclone; 1% hypoxanthine-aminopterin-thymidine (HAT) was purchased from Sigma-Aldrich.
  • the culture wells with hybridoma cell growth were screened by ELISA.
  • the screening was performed in two steps. The first step was to screen positive holes against aflatoxin B1 but not against carrier protein BSA by indirect ELISA. The second step was indirect.
  • the competitive ELISA method was used to detect the positive wells screened in the first step.
  • the aflatoxin B1 was used as the competition source, and the pores with higher absorbance and sensitivity were selected. (The higher absorbance value refers to the pores with the competition of 0, which is the negative control well.
  • the final measured value is higher, the higher sensitivity means that the competitive original concentration at the inhibition rate is 50%, that is, the IC 50 value is smaller.
  • the clone is cloned by the limiting dilution method, and the same two-step method is used for detection about 10 days after cloning. After repeating the cloning 2-3 times, the hybridoma cell line 3G1 was obtained.
  • Hybridoma cell lines can be produced by using Tiangen's total RNA extraction kit and extracting according to the instructions. 3G1 total RNA ;
  • RNA obtained in step 1 was used as a template, and oligo (dT) 15 was used as a primer, and reverse transcription was carried out according to the SuperScript TM -2 II reverse transcriptase specification to synthesize the first strand of cDNA; primer oligo (dT) 15 ) purchased by Invitrogen;
  • the obtained gene sequence result the heavy chain variable region coding gene sequence is 347 bp in length, and the sequence is SEQ ID NO: 1.
  • the heavy chain variable region encoded by the gene sequence was deduced from the obtained gene sequence to be composed of 115 amino acids, and the sequence is shown in SEQ ID NO: 3.
  • Light chain variable region coding gene sequence length 338 bp the sequence is as shown in SEQ ID NO: 2, and according to the obtained gene sequence, the light chain variable region encoded by the gene sequence is composed of 110 amino acids, and the sequence is SEQ ID. NO: 4 is shown.
  • Anti-Aflatoxin B1 monoclonal antibody hybridoma cell line 3G1 obtained in Example 2 BALB/c mice pretreated with Freund's incomplete adjuvant were injected, and the ascites of the mice was collected.
  • the antibody was purified by caprylic acid-ammonium sulfate method.
  • the specific operation was as follows: the mouse ascites was filtered with double-layer filter paper at 4 °C. Centrifuge at 12000r/min for 15min, aspirate the supernatant, mix the obtained ascites with 4 volumes of acetate buffer, and slowly add n-octanoic acid with stirring. The volume of n-octanoic acid per milliliter of ascites is 33.
  • the acetate buffer is obtained by adding 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL of water; 0.1mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g, and water to 100mL Income.
  • the subtype of the anti-aflatoxin B1 monoclonal antibody secreted by the hybridoma cell line 3G1 was identified as IgG 2a using a commercially available subtype identification kit.
  • the titer of 3G1 mouse ascites antibody was 6.4 ⁇ 10 6 by conventional non-competitive enzyme-linked immunosorbent assay (ELISA), which was positive when the mouse ascites antibody was diluted 6.4 ⁇ 10 6 times.
  • ELISA enzyme-linked immunosorbent assay
  • the sensitivity to aflatoxin B1 was 1.6 ng/mL by conventional indirect competitive ELISA, the cross-reaction rate with aflatoxin B2 was 6.4%, and the cross-reaction rate of aflatoxin G1 and G2 was less than 1%.
  • the immunochromatographic test strip comprises the following steps:
  • the absorbent paper is cut into a size of 16mm wide and 3mm, which means that the absorbent pad is obtained;
  • Aflatoxin artificially synthesized complete antigen AFB2a-BSA was formulated into 0.1mg/mL coating solution A
  • the coating liquid A was laterally coated on the nitrocellulose membrane by a point spray method at a position of 15 mm from the nitrocellulose membrane to obtain a detection line, and the complete antigen required per cm of detection line AFB2a-BSA
  • the coating amount is 75 ng and then dried at 37 ° C for 15 minutes;
  • the coating liquid A is: 10 mg aflatoxin artificially synthesized complete antigen AFB2a-BSA, 1g Bovine serum albumin, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, bring water to a volume 100mL of the obtained;
  • Rabbit anti-mouse polyclonal antibody was formulated into 0.2 mg/mL coating solution B; 5 mm from the detection line The position of the coated liquid B was coated on the nitrocellulose membrane by a point spray method to obtain a quality control line. The amount of the rabbit anti-mouse polyclonal antibody required per centimeter of the control line was 100 ng, and then Dry at 37 °C 15 minutes;
  • the coating solution B is 20 mg rabbit anti-mouse polyclonal antibody, 0.02 g sodium azide, 0.8 g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, add water to a volume of 100mL;
  • the nitrocellulose membrane is 25 mm long and 3 mm wide;
  • the glass fiber membrane is cut into a size of 13mm and a width of 3mm, and is placed in the sealing liquid A to be wetted and taken out. Drying at °C for 6 hours, the sample pad is obtained, and then stored in a desiccator at room temperature;
  • the blocking solution A is 2g bovine serum albumin, 2.5g sucrose, 0.02g sodium azide, 0.8g Sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, add water to a volume of 100mL;
  • the glass fiber membrane is cut into a size of 9 mm wide and 3 mm wide, and is placed in the sealing liquid B to be wetted and taken out. Drying at °C for 16 hours, on a dry glass fiber membrane, nano-gold-labeled anti-aflatoxin B1 was sprayed by spot spray. The monoclonal antibody solution is sprayed laterally, and the nano-gold-labeled anti-aflatoxin B1 monoclonal antibody is 600 ng per cm of the spray length, then vacuum-dried for 6 hours, and stored in a desiccator at room temperature;
  • the blocking solution B is 2g bovine serum albumin, 0.1mL Triton X-100, 0.3g Polyvinylpyrrolidone, 2.5g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g Potassium dihydrogen phosphate, add water to a volume of 100mL;
  • the specific labeling method of the nano gold-labeled anti-aflatoxin B1 monoclonal antibody solution is: measuring 50.0 mL mass concentration as 0.01% nano gold solution, adjust the pH value of the solution with 0.1 mol/L potassium carbonate solution to 5.5; slowly add 2.5 mL 0.1 mg/mL while stirring.
  • Aqueous anti-aflatoxin B1 monoclonal antibody solution stirring for 30 min; adding a mass concentration of 10% aqueous solution of bovine serum albumin to bovine serum albumin at a final concentration of 1%, continue to stir After 30 min at 4 °C, centrifuge at 1500 r/min for 15 min, take the supernatant and discard the precipitate; centrifuge the supernatant at 12000 r/min for 30 min. Discard the supernatant, add 50.0mL of the labeled washing preservation solution; centrifuge at 12000r/min for 30 minutes, discard the supernatant, and resuspend the pellet with the marker washing preservation solution to obtain 5.0 mL.
  • the concentrate was placed in a refrigerator at 4 °C, and the mass concentration of the nano-gold-labeled anti-aflatoxin B1 monoclonal antibody solution was 0.06 mg/mL;
  • the nano gold in the nano gold solution has a particle diameter of 15 nm;
  • the 0.1 mol/L potassium carbonate aqueous solution is: 13.8 g of potassium carbonate dissolved in pure water to a volume of 1000 mL, Filtration of 0.22 ⁇ m membrane; the 0.1mg/mL anti-aflatoxin B1 monoclonal antibody aqueous solution is 1mg anti-aflatoxin B1 monoclonal antibody dissolved in 10mL Made in pure water; the 10% bovine serum albumin aqueous solution is 10 g of bovine serum albumin dissolved in 100 mL of pure water and filtered through a 0.22 ⁇ m filter; the labeled washing and preservation solution is: 2.0g polyethylene glycol -20000, 0.2g sodium azide, 0.1235g boric acid, pure water to 1000mL, 0.22 ⁇ m filter membrane obtained;
  • the blank peanut sample was added as a negative control to the sample pad of another aflatoxin B1 immunochromatographic test strip, which was used as a control test strip, and the result was read after 15 minutes; test result: 1#
  • the quality control line of the test strip shows a red line, and the test line does not develop color, it is judged as a positive result, indicating that the content of aflatoxin B1 in the sample to be tested is greater than or equal to 10 ng / mL, see Figure 3-1 ;
  • the quality control line of the test strip shows a red line, and the color of the test line is lighter than the test strip test line, which is judged as a positive result, indicating that the content of aflatoxin B1 in the sample to be tested is higher than or equal to 1 ng/ mL And less than 10 ng/mL, see Figure 3-2.
  • Hybridoma cell line 3G1 and its anti-aflatoxin B1 monoclonal antibody Hybridoma cell line 3G1 and its anti-aflatoxin B1 monoclonal antibody

Abstract

提供了一种杂交瘤细胞株3G1及其产生的抗黄曲霉毒素B1单克隆抗体。所述的杂交瘤细胞株3G1，其保藏号为CCTCC NO.201014，可以用于制备高效价抗黄曲霉毒素B1单克隆抗体，酶联免疫吸附分析（ELISA）法测得的效价可达6.4X106。所述抗黄曲霉毒素B1单克隆抗体灵敏度高、特异性好，对黄曲霉毒素B1的50%抑制浓度 IC50为1.6ng/mL，与黄曲霉毒素B2交叉反应率为6.4%，与黄曲霉毒素G1和黄曲霉毒素G2的交叉反应率均小于1%，可用于测定黄曲霉毒素B1含量。

Description

杂交瘤细胞株 3G1 及其产生的抗黄曲霉毒素B1单克隆抗体 技术领域
本发明涉及杂交瘤细胞株 3G1 及其产生的抗黄曲霉毒素 B1 单克隆抗体。
背景技术
黄曲霉毒素主要是由黄曲霉和寄生曲霉分泌产生的次生代谢产物，是能引起人畜各种损害的天然有毒化合物。黄曲霉毒素目前已发现 20 余种，其中黄曲霉毒素 B1 （ AFB1 ）的毒性最强，它的毒性是氰化钾的 10 倍，砒霜的 68 倍。历史上发生过很多 AFB1 污染食物链引发的人类中毒事件。奶牛摄入了 AFB1 污染了的谷物后，经体内代谢后形成另一种致癌物质黄曲霉毒素 M1 （ AFM1 ）， AFM1 进入牛奶中则会严重威胁人类健康。另外， AFB1 还能污染多种原料，包括水果、干果、蔬菜、调味品、油料作物、烟草及中草药等，由此可见： AFB1 对食物链的污染极其广泛，几乎在食物链的每个环节都能发现存在。除此，在热带和亚热带地区农产品和食品中 AFB1 的检出率更高，污染更严重，花生、玉米和花生油等粮油食品污染最为严重，同时， AFB1 超标而导致的农产品国际贸易纠纷事件也时常发生， 1999 年、 2000 年、 2001 年我国出口欧盟的花生到岸后， 40% 以上被欧盟检出黄曲霉毒素总量和 AFB1 分量超标，遭到退货或被迫转口贸易，致使我国农产品进出口贸易蒙受巨大损失，影响了我国农产品国际市场声誉。因此，加强花生等农产品及制品中 AFB1 的检测、特别是速测，及时了解和掌握农产品中 AFB1 的污染情况对保障我国食品消费安全具有重要意义。
现有的黄曲霉毒素检测方法主要包括薄层层析法、精密仪器分析法和免疫学分析法。其中薄层层析法是黄曲霉毒素检测最常用的检测方法，该方法不需要特殊的仪器设备，一般实验室都可开展，但存在试剂消耗量大、操作繁琐、结果易受干扰、准确性差，不能准确定量，且对实验人员和周围环境污染危害较大等不足，不适于现场快速检测。精密仪器分析法包括荧光分光光度法和高效液相色谱法等，这个些方法灵敏度高，检测结果准确，但所需的仪器设备昂贵，样品前处理过程繁琐，要求黄曲霉毒素样品纯化程度高，检测过程耗时，对实验环境要求高，难以实现快速检测。近年来，用于黄曲霉毒素检测的免疫分析技术逐渐发展起来，克服了前两者的一些缺点，具有特异性强、灵敏度高、样品前处理简单、检测成本低、对实验人员和周围环境的污染危害小等优点，适于现场批量检测等。免疫分析是利用抗原和抗体的特异性的结合反应和抗体、抗原上的标记物的生物、物理或化学放大作用来对超微量残留物进行定性、定量检测，所以要研究建立针对黄曲霉毒素 B1 的任何一种免疫学检测技术，都必须先获得抗黄曲霉毒素 B1 的抗体。
技术问题
本发明所要解决的问题是提供杂交瘤细胞株 3G1 及其产生的抗黄曲霉毒素 B1 单克隆抗体。
技术解决方案
本发明提供了杂交瘤细胞株 3G1 ，该细胞株已于 2010 年 7 月 13 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. C201014 ，分类命名为小鼠杂交瘤细胞 3G1 。其具有序列表中 SEQ ID NO.1 所示的抗黄曲霉毒素 B1 单克隆抗体重链可变区编码基因序列和序列表中 SEQ ID NO.2 所示的抗黄曲霉毒素 B1 单克隆抗体轻链可变区编码基因序列。
抗黄曲霉毒素 B1 单克隆抗体，它由保藏编号为 CCTCC NO. C201014 的杂交瘤细胞株 3G1 分泌产生。其重链可变区具有序列表中 SEQ ID NO.3 所示的氨基酸序列；轻链可变区具有序列表中 SEQ ID NO.4 所示的氨基酸序列。该抗黄曲霉毒素 B1 单克隆抗体可以识别黄曲霉毒素 B1 ，对黄曲霉毒素 B1 的 50% 抑制浓度 IC50 为 1.6ng/mL 。
抗黄曲霉毒素 B1 单克隆抗体在黄曲霉毒素 B1 测定中的应用。
本发明提供的杂交瘤细胞株 3G1 是采用两步筛选法获得的，其具体步骤为：将黄曲霉毒素 B1 在 H2SO4 作用下转化为其半缩醛形式 AFB2a ，然后与 BSA 氨基发生醛胺缩合反应，在 NaBH4 还原作用下 C=N 被还原为 C-N ，生成 AFB2a-BSA 复合物完全抗原 AFB1-BSA ，用其免疫 BALB/c 小鼠 4-6 次，最后一次加强免疫用 2 倍于前一次免疫剂量的 AFB2a-BSA ，免疫 3 天后进行细胞融合。采用 ELISA 方法分两步进行筛选融合细胞：第一步采用间接 ELISA 法筛选出仅能抗黄曲霉毒素而不抗载体蛋白 BSA 的阳性克隆；第二步采用间接竞争 ELISA 法对第一步筛选出的阳性克隆培养液进行检测，采用 AFB1 作为竞争原，选择吸光值低、灵敏度较高的阳性克隆进行有限稀释法继续克隆，克隆后 10 天左右采用同样的两步筛选法进行抗体检测，如此重复克隆 2-3 次后，最终筛选获得杂交瘤细胞株 3G1 。
本发明提供的抗黄曲霉毒素 B1 单克隆抗体的制备方法，步骤如下：将获得的杂交瘤细胞株 1C8 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 B1 单克隆抗体。
按上述方案，所述的纯化方法为辛酸 - 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 ～ 35 μ L ，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 pH7.4 的磷酸盐缓冲液，用 2 mol/L 的氢氧化钠溶液调节该混合液的 pH 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 B1 单克隆抗体，将抗体置 -20 ℃冰箱中备用；
醋酸盐缓冲液： 0.29g 醋酸钠加入 0.141mL 醋酸，纯水定容至 100mL ；
0.1mol/L 磷酸盐缓冲液： 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 。
 有益效果
本发明的有益效果在于：
(1) 本发明提供的杂交瘤细胞株 3G1 可以用于制备高效价抗黄曲霉毒素 B1 单克隆抗体，抗黄曲霉毒素 B1 小鼠腹水抗体酶联免疫吸附分析（ ELISA ）法测得的效价可达 6.40 × 106 。
(2) 本发明提供的抗黄曲霉毒素 B1 单克隆抗体灵敏度高、特异性好，对黄曲霉毒素 B1 的 50% 抑制浓度 IC50 为 1.6ng/mL ，与黄曲霉毒素 B2 交叉反应率为 6.4% ，与黄曲霉毒素 G1 和黄曲霉毒素 G2 的交叉反应率均小于 1% 。
(3) 本发明提供的抗黄曲霉毒素 B1 单克隆抗体可应用于测定黄曲霉毒素 B1 含量。
 附图说明
图 1 为本发明实施例 4 制备的黄曲霉毒素 B1 免疫层析试纸条的正视图。图中： 1 纸板、 2 吸水垫； 3 检测垫； 4 金标垫； 5 样品垫； 6 质控线； 7 检测线。
图 2 为本发明实施例 4 制备的黄曲霉毒素 B1 免疫层析试纸条的左视图。图中： 1 纸板； 2 吸水垫； 3 检测垫； 4 金标垫； 5 样品垫。
图 3 为应用本发明实施例 4 提供的黄曲霉毒素 B1 免疫层析试纸条检测样品的结果判定图。图中： 8 对照试纸条； 9 检测试纸条； 6 质控线； 7 检测线。
本发明的实施方式
实施例 1 ：杂交瘤细胞株 3G1 的筛选
1. 抗原合成及动物免疫
购买市售黄曲霉毒素 B1 标准品进行完全抗原合成，具体合成步骤如下：将 4 mg AFB1 溶于 2 mL 丙酮，加入 40 μ L 10% H2SO4 ，混合物在 56 ℃ 搅拌反应 4 h ；产物蒸干后，加入 5 mL 的 H2O ，用 25 mL 氯仿提取两次，然后用 20 mL H2O 洗涤有机层，保留有机层；蒸去有机溶剂，得黄色固体产物。取 1.0 mg 产物，向其中加入 2 mL 0.5% BSA 溶液（ 4 mL PBS （磷酸盐缓冲液， pH7.4 ）溶解 20 mg BSA ） 37 ℃反应 30 min ；加入 100 μ L 6.5 mM NaBH4 ， 4 ℃反应 30 min ；加入 50 μ L 0.1mol/L HCl ，除去过量的 NaBH4 。在 4 ℃ 条件下， PBS 溶液（磷酸盐缓冲液， pH7.4 ）透析 3d ，除去 AFB1 及 AFB2a ，最后进行常规紫外扫描法鉴定，鉴定结果表明制备得到了黄曲霉毒素 B1 完全抗原 AFB2a-BSA 。
购买 6 周龄雌性 BALB/c 小鼠 6 只，免疫自行合成的黄曲霉毒素 B1 完全抗原 AFB2a-BSA 。第一次免疫将完全抗原 AFB2a-BSA 与等量福氏完全佐剂混合乳化，然后小鼠颈背部皮下多点注射。第二次免疫于首免 4 周后进行，采用福氏不完全佐剂与等体积完全抗原 AFB2a-BSA 乳化，小鼠腹腔注射。第三次免疫与第二次免疫间隔时间 4 周，免疫方式及剂量与第二次相同，第四次免疫于第三次免疫 3 周后进行，免疫方式及免疫剂量与第二次免疫相同，每次免疫剂量为每鼠 50 μ g 。前 3 次每次免疫后一周，尾静脉采血，分离血清，采用间接 ELISA 法监测小鼠血清抗体效价。第 4 次免疫后一周，尾静脉采血，分离血清，采用间接 ELISA 法监测小鼠血清抗体效价，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，免疫剂量为之前的 2 倍。 AFB1 购于 Sigma-Aldrich 公司。
2. 细胞融合
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，具体步骤如下：
（ 1 ）颈椎脱臼法处死免疫小鼠，在无菌条件下取脾脏，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，然后用 50%PEG 融合，融合时间 1 分钟，然后加满 RPMI-1640 基础培养液，离心，移去上清，小鼠脾细胞和鼠源骨髓瘤细胞 SP2/0 形成的融合细胞用 72mL RPMI-1640 基础培养液重悬，将重悬起来的细胞滴加到 96 孔细胞培养板内， 2 滴 / 孔，置 37 ℃二氧化碳培养箱培养，所述的 RPMI-1640 基础培养液为含有 20% （体积百分数）胎牛血清， 2% （重量百分数）生长因子和 1% （重量百分数）次黄嘌呤 - 氨基蝶呤 - 胸腺嘧啶核苷。上述 SP2/0 购于上海泛柯生物科技有限公司； RPMI-1640 基础培养液购于 Hyclone 公司； 1% 次黄嘌呤 - 氨基蝶呤 - 胸腺嘧啶核苷（ HAT ）购于 Sigma-Aldrich 公司。
3. 细胞株的筛选及克隆
待细胞融合后第 12 天左右，细胞集落长到占孔底 1/2 面积大小，培养液变黄，即可进行抗体检测。采用 ELISA 方法对有杂交瘤细胞生长的培养孔进行筛选，筛选分两步进行，第一步采用间接 ELISA 法筛选出抗黄曲霉毒素 B1 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，用黄曲霉毒素 B1 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 的孔即阴性对照孔的最终测定值较高，灵敏度较高指抑制率为 50% 时的竞争原浓度亦即 IC50 值较小），采用有限稀释法进行克隆，克隆后 10 天左右采用同样的两步法进行检测，如此重复克隆 2-3 次后，获得杂交瘤细胞株 3G1 。
实施例 2 ：抗黄曲霉毒素 B1 单克隆抗体杂交瘤细胞株系 3G1 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系 3G1 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 SuperScriptTM-2 Ⅱ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 为模版扩增抗体轻、重链可变区基因。 PCR 程序为： 94 ℃ 30s 、 55 ℃ 1min 、 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，连接到载体 pMD18-T 中，转化大肠杆菌 DH5 α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 ， -AGG TSM ARC TGC AGS AGT CWG G-3 ， （ 22mer ）和 5 ， -TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3 ， （ 32mer ）其中 S 、 M 、 R 和 W 为兼并碱基， M=A/C ， R=A/G ， S=C/G ， W=A/T ，轻链可变区引物为 5 ， -GAC ATT GAG CTC ACC CAG CTT GGT GCC -3 ， （ 24mer ）和 5 ， -CCG TTT CAG CTC 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 347bp ，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 115 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 338bp ，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 110 个氨基酸组成，序列如 SEQ ID NO:4 所示。
实施例 3 ：抗黄曲霉毒素 B1 单克隆抗体的制备纯化、亚型和特性鉴定
将实施例 2 获得的抗黄曲霉毒素 B1 单克隆抗体杂交瘤细胞株系 3G1 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，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 ，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 33 μ L ，室温混合 30min ， 4 ℃静置 2h ，然后 4 ℃， 12000r/min 离心 30min ，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 2 mol/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 ，然后 4 ℃， 12000r/min 离心 30min ，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，对纯水透析，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，即得纯化好的抗黄曲霉毒素 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。
用市售亚型鉴定试剂盒鉴定杂交瘤细胞株 3G1 分泌的抗黄曲霉毒素 B1 单克隆抗体的亚型为 IgG2a 。
用常规非竞争酶联免疫吸附分析（ ELISA ）法测得 3G1 的鼠腹水抗体的效价可达 6.4 × 106 ，即鼠腹水抗体稀释 6.4 × 106 倍时的溶液测定结果为阳性。用常规间接竞争 ELISA 法鉴定其对黄曲霉毒素 B1 的灵敏度为 1.6ng/mL ，与黄曲霉毒素 B2 交叉反应率为 6.4% ，黄曲霉毒素 G1 与 G2 的交叉反应率均小于 1% 。
实施例 4 ：抗体应用
将杂交瘤细胞株 3G1 分泌的抗黄曲霉毒素 B1 单克隆抗体用于制备黄曲霉毒素 B1 免疫层析试纸条，制备方法包括以下步骤：
(1) 吸水垫的制备
将吸水纸剪裁成长 16mm 宽 3mm 的规格，即得吸水垫；
(2) 检测垫的制备
检测线的包被：
将黄曲霉毒素人工合成完全抗原 AFB2a-BSA 配制成 0.1mg/mL 的包被液 A ；于距硝酸纤维素膜上沿 15mm 的位置，用点喷方式将包被液 A 横向包被于硝酸纤维素膜上，得到检测线，每厘米检测线所需完全抗原 AFB2a-BSA 的包被量为 75ng ，然后于 37 ℃条件下干燥 15 分钟；
所述的包被液 A 为： 10mg 黄曲霉毒素人工合成完全抗原 AFB2a-BSA ， 1g 牛血清白蛋白， 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；
质控线的包被：
将兔抗鼠多克隆抗体配成 0.2mg/mL 的包被液 B ；于距检测线 5mm 的位置，用点喷方式将包被液 B 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 100ng ，然后于 37 ℃条件下干燥 15 分钟；
所述的包被液 B 为将 20mg 兔抗鼠多克隆抗体， 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；
所述的硝酸纤维素膜长 25mm ，宽 3mm ；
(3) 样品垫的制备：
将玻璃纤维膜剪裁成长 13mm 宽 3mm 的规格，放入封闭液 A 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 A 为 2g 牛血清白蛋白， 2.5g 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) 金标垫的制备：
将玻璃纤维膜剪裁成长 9mm 宽 3mm 的规格，放入封闭液 B 中浸湿，取出，于 37 ℃条件下干燥 16 小时，于干燥好的玻璃纤维膜上，用点喷方式将纳米金标记的抗黄曲霉毒素 B1 单克隆抗体溶液横向喷涂，每厘米喷涂长度所需纳米金标记的抗黄曲霉毒素 B1 单克隆抗体为 600ng ，然后真空冷冻干燥 6h ，置干燥器中室温保存；
所述的封闭液 B 为 2g 牛血清白蛋白， 0.1mL 曲拉通 X-100 ， 0.3g 聚乙烯吡咯烷酮， 2.5g 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；
所述的纳米金标记的抗黄曲霉毒素 B1 单克隆抗体溶液的具体标记方法为：量取 50.0mL 质量浓度为 0.01% 的纳米金溶液，用 0.1 mol/L 碳酸钾水溶液调节溶液 pH 值为 5.5 ；在搅拌的状态下缓慢加入 2.5mL 0.1mg/mL 的抗黄曲霉毒素 B1 单克隆抗体水溶液，继续搅拌 30min ；加入质量浓度为 10% 牛血清白蛋白水溶液至牛血清白蛋白的终质量浓度为 1% ，继续搅拌 30min ；于 4 ℃放置 2h 后， 1500r/min 离心 15min ，取上清液，弃沉淀；将上清液 12000r/min 离心 30 min ，弃去上清液，加入 50.0mL 标记洗涤保存液；再以 12000r/min 离心 30 min ，弃去上清液，将沉淀用标记洗涤保存液重悬，得到 5.0mL 浓缩物，置 4 ℃冰箱备用，其中纳米金标记的抗黄曲霉毒素 B1 单克隆抗体溶液的质量浓度为 0.06mg/mL ；
所述纳米金溶液中纳米金的粒径为 15nm ；
所述的 0.1 mol/L 碳酸钾水溶液为： 13.8g 碳酸钾溶于纯水定容至 1000mL ， 0.22µm 滤膜过滤所得；所述的 0.1mg/mL 抗黄曲霉毒素 B1 单克隆抗体水溶液为 1mg 抗黄曲霉毒素 B1 单克隆抗体溶解在 10 mL 纯水中制成；所述的 10% 牛血清白蛋白水溶液为 10g 牛血清白蛋白溶解在 100mL 纯水中， 0.22µm 滤膜过滤所得；所述的标记洗涤保存液为： 2.0g 聚乙二醇 -20000 ， 0.2g 叠氮钠， 0.1235 克硼酸，纯水定容至 1000mL ， 0.22µm 滤膜过滤所得；
(5) 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、金标垫和样品垫，相邻各垫在连接处交叠连接，交叠长度为 1mm ，即得黄曲霉毒素 B1 免疫层析试纸条，见图 1 和图 2 。
上述黄曲霉毒素 B1 免疫层析试纸条的应用：
分别取已知不含黄曲霉毒素 B1 的 1# 和 2# 花生样品进行粉碎，然后采用 10% 甲醇水（ V 甲醇 /V 水 =1 ∶ 9 ）搅拌混匀，吸取 1# 和 2# 待测花生样品各 1mL ，取 100 μ L 稀释好的样品溶液分别加入不同浓度的黄曲霉毒素 B1 标准品作为加标样品，然后各 100 μ L 加入黄曲霉毒素 B1 免疫层析试纸条的样品垫，作为检测组；同时取 100 μ L 不含有黄曲霉毒素 B1 的空白花生样品作为阴性对照加入另一黄曲霉毒素 B1 免疫层析试纸条的样品垫，将其作为对照试纸条， 15 分钟后读取结果；检测结果： 1# 检测试纸条的质控线显示出红色线条，而检测线不显色，则判为阳性结果，表明待测样品中的黄曲霉毒素 B1 的含量大于或等于 10ng/mL ，见图 3-1 ； 2# 检测试纸条的质控线显示出红色线条，而检测线颜色比对照试纸条检测线颜色浅，则判为阳性结果，表明待测样品中黄曲霉毒素 B1 的含量高于或等于 1ng/mL ，并小于 10 ng/mL ，见图 3-2 。
 序列表自由内容
序列表
＜ 110 ＞ 中国农业科学院油料作物研究所
＜ 120 ＞ 杂交瘤细胞株 3G1 及其产生的抗黄曲霉毒素 B1 单克隆抗体
＜ 160 ＞ 4
＜ 210 ＞ 1
＜ 211 ＞ 347bp
＜ 212 ＞ DNA
＜ 213 ＞ 小鼠
＜ 400 ＞ 1
tgaggagacg gtgaccgtgg tcccttggcc ccagtagtcc atagcccagt 50
aggccgatct tgcacagtaa tatgtggctg tgtcctcagt agtcacagaa 100
tttaactgca ggtagtactg gttcttggat gtgtctcgag tgatggagat 150
tcgactcttg agagatggat tgtagtaagg gttaccactg tagctcatga 200
accccatgta ctcaagttta ttcccaggga atttccggat ccagtgccag 250
taatcactgg tgatggagtc gccagtgaca gaacaggtga gggacagagt 300
ctgagaaggt ttcacgaggc taggtcctga ctcctgcagc tgcacct 347
＜ 210 ＞ 2
＜ 211 ＞ 338bp
＜ 212 ＞ DNA
＜ 213 ＞ 小鼠
＜ 400 ＞ 2
gacattgagc tcacccagtc tccaaaattc atgtccacat cagtaggaga 50
cagggtcagc atctcctgca aggccagtca ggatgtgggt actgatgtag 100
cctggtacgg agtccctgat cgcctcacag gcagtggatc tgggacagat 150
ttcactctca ccattagcaa tgtgcagtct gaagacttgg cagattattt 200
ctgtcaacaa tatagcagct atattcacgt tcggctcggg gacaaagttg 250
gaaataaaac gtttttgtgg agaggggcat gtcatagtcc tcactgtgtc 300
tcacgttcgg tgctgggacc aagctggagc tgaaacgg 338
＜ 210 ＞ 3
＜ 211 ＞ 115
＜ 212 ＞ PRT
＜ 213 ＞ 小鼠
＜ 400 ＞ 3
Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
1 5 10 15 20
Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asp Tyr Trp His Trp Ile Arg Lys Phe Pro
25 30 35 40
Gly Asn Lys Leu Glu Tyr Met Gly Phe Met Ser Tyr Ser Gly Asn Pro Tyr Tyr Asn Pro
45 50 55 60
Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu Gln
65 70 75 80
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Ala Tyr Trp
85 90 95 100
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
105 110 115
＜ 210 ＞ 4
＜ 211 ＞ 110
＜ 212 ＞ PRT
＜ 213 ＞ 小鼠
＜ 400 ＞ 4
Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser
1 5 10 15 20
Ile Ser Cys Lys Ala Ser Gln Asp Val Gly Thr Asp Val Ala Trp Tyr Gly Val Pro Asp
25 30 35 40
Arg Leu Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
45 50 55 60
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Ile His Val Arg Leu Gly
65 70 75 80
Asp Lys Val Gly Asn Lys Thr Phe Leu Trp Arg Gly Ala Cys His Ser Pro His Cys Val
85 90 95 100
Ser Arg Ser Val Leu Gly Pro Ser Trp Ser
105 110

Claims (4)

1. 杂交瘤细胞株 3G1 ，其特征在于：它保藏于中国典型培养物保藏中心，保藏编号为 CCTCC NO. C201014 。
2. 抗黄曲霉毒素 B1 单克隆抗体，它由保藏编号为 CCTCC NO. C201014 的杂交瘤细胞株 3G1 分泌产生。
3. 根据权利要求 2 所述的抗黄曲霉毒素 B1 单克隆抗体在黄曲霉毒素 B1 测定中的应用。
4. 根据权利要求 2 所述的抗黄曲霉毒素 B1 单克隆抗体的的制备方法，其特征在于：将获得的杂交瘤细胞株 1C8 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 B1 单克隆抗体。
PCT/CN2013/070614 2012-04-20 2013-01-17 杂交瘤细胞株 3g1 及其产生的抗黄曲霉毒素b1单克隆抗体 WO2013155883A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN102747043B (zh) * 2012-04-20 2013-07-17 中国农业科学院油料作物研究所 杂交瘤细胞株3g1及其产生的抗黄曲霉毒素b1单克隆抗体
CN103116024B (zh) * 2012-11-30 2014-04-09 中国农业科学院油料作物研究所 抗黄曲霉毒素通用单克隆抗体1c11在黄曲霉毒素b1荧光淬灭中的应用及方法
CN103215652B (zh) * 2013-01-25 2014-04-30 中国农业科学院油料作物研究所 黄曲霉毒素阳性抗体基因库、构建方法、用途及黄曲霉毒素重组单链抗体1a7
CN103130892B (zh) * 2013-01-25 2014-04-30 中国农业科学院油料作物研究所 黄曲霉毒素重组单链抗体2g7、编码基因及其应用
CN103215229B (zh) * 2013-04-03 2014-04-30 中国农业科学院油料作物研究所 杂交瘤细胞株afb3g1、其单克隆抗体及半定量化检测黄曲霉毒素b1的多检测线免疫层析试纸条
CN103866401B (zh) * 2014-03-28 2014-11-26 中国农业科学院油料作物研究所 一种黄曲霉毒素纳米抗体基因库、构建方法、用途及黄曲霉毒素b1纳米抗体2014afb-g15
CN104762267B (zh) * 2015-01-15 2018-03-20 江苏衡谱分析检测技术有限公司 杂交瘤细胞afb1‑2a4及其产生的黄曲霉毒素b1单克隆抗体
CN105586317A (zh) * 2016-01-19 2016-05-18 北京龙科方舟生物工程技术有限公司 一种黄曲霉毒素b1的单克隆抗体及其应用
CN106970223B (zh) * 2017-03-07 2018-12-11 中国农业科学院油料作物研究所 净化伏马毒素b1、黄曲霉毒素b1等五种真菌毒素免疫吸附剂及复合亲和柱
CN107012128B (zh) * 2017-04-10 2020-04-28 北京勤邦生物技术有限公司 一种分泌抗黄曲霉毒素b1单克隆抗体的杂交瘤细胞株及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101993855A (zh) * 2010-08-05 2011-03-30 中国农业科学院油料作物研究所 杂交瘤细胞株1c11、其产生的抗黄曲霉毒素通用单克隆抗体及其应用
CN102747043A (zh) * 2012-04-20 2012-10-24 中国农业科学院油料作物研究所 杂交瘤细胞株3g1及其产生的抗黄曲霉毒素b1单克隆抗体
CN102746403A (zh) * 2012-04-20 2012-10-24 中国农业科学院油料作物研究所 用于elisa检测黄曲霉毒素的标准品通用替代物、其制备方法及黄曲霉毒素elisa检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101429250A (zh) * 2008-12-12 2009-05-13 湖南农业大学 霉菌毒素青霉酸单克隆抗体及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101993855A (zh) * 2010-08-05 2011-03-30 中国农业科学院油料作物研究所 杂交瘤细胞株1c11、其产生的抗黄曲霉毒素通用单克隆抗体及其应用
CN102747043A (zh) * 2012-04-20 2012-10-24 中国农业科学院油料作物研究所 杂交瘤细胞株3g1及其产生的抗黄曲霉毒素b1单克隆抗体
CN102746403A (zh) * 2012-04-20 2012-10-24 中国农业科学院油料作物研究所 用于elisa检测黄曲霉毒素的标准品通用替代物、其制备方法及黄曲霉毒素elisa检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
XIAO, ZHI: "Preparation and Characterization of High Speciality Monoclonal Antibodies for Aflatoxin B1", AGRICULTURE, CHINA MASTER' S THESES FULL-TEXT DATABASE, 15 February 2011 (2011-02-15), pages 1 - 51 *
ZHANG, DAOHONG ET AL.: "A high selective immunochromatographic assay for rapid detection of aflatoxin B1", TALANTA, vol. 85, 29 April 2011 (2011-04-29), pages 736 - 742 *

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