WO2013155882A1 - 杂交瘤细胞株10g4及其产生的抗黄曲霉毒素b1、b2、g1、g2总量单克隆抗体 - Google Patents

杂交瘤细胞株10g4及其产生的抗黄曲霉毒素b1、b2、g1、g2总量单克隆抗体 Download PDF

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WO2013155882A1
WO2013155882A1 PCT/CN2013/070612 CN2013070612W WO2013155882A1 WO 2013155882 A1 WO2013155882 A1 WO 2013155882A1 CN 2013070612 W CN2013070612 W CN 2013070612W WO 2013155882 A1 WO2013155882 A1 WO 2013155882A1
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aflatoxin
aflatoxins
monoclonal antibody
hybridoma cell
cell line
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PCT/CN2013/070612
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English (en)
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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Priority to US13/984,231 priority Critical patent/US8841419B2/en
Priority to KR1020137019060A priority patent/KR101491835B1/ko
Priority to NZ613306A priority patent/NZ613306A/en
Publication of WO2013155882A1 publication Critical patent/WO2013155882A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • 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

Definitions

  • the present invention relates to a hybridoma cell line 10G4 and a monoclonal antibody against the total amount of aflatoxin 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 the characteristics of wide pollution, strong toxicity and serious harm. Therefore, the world has at least 70 More than one country has established aflatoxin limits in agricultural products and foods, and many countries have developed major aflatoxins B1, B2, G1, G2. The limit of the total amount. Therefore, the total aflatoxin analysis method is particularly important.
  • Existing methods for detecting aflatoxins include thin layer chromatography, precision instrumental analysis, and immunological analysis.
  • thin layer chromatography is the most commonly used detection method for detecting aflatoxin. It does not require special equipment and equipment. It can be carried out in general laboratories, but the amount of reagents is large, the operation is cumbersome, the interference of other components is serious, and the accuracy is poor. It is accurate and quantitative, and it is harmful to the experimental personnel and the surrounding environment. It is not suitable for rapid detection on site.
  • the precision instrumental analysis method includes fluorescence spectrophotometry and high performance liquid chromatography. Its sensitivity and accuracy are good, but the instrument is expensive. It requires high degree of purification of aflatoxin samples.
  • the sample preparation process is cumbersome and time consuming, and it requires the experimental environment. High, it is difficult to achieve fast detection.
  • the immunoassay technology developed in recent years has overcome the shortcomings of the former two, with strong specificity, high sensitivity, simple sample preparation, low cost, low pollution hazard to the experimenter and the surrounding environment, suitable for on-site batch inspection, etc. advantage.
  • Immunoassay is the qualitative and quantitative detection of ultra-micro residues by the specific reaction of antigen and antibody and the biological, physical or chemical amplification of the label on the antibody (or antigen), so it is necessary to study the total amount of aflatoxin. Any of the immunological detection techniques of the analytical method must first produce antibodies against the total amount of aflatoxin B1, B2, G1, G2. In fact, there have been many reports on the development of aflatoxin antibodies both domestically and internationally. Aflatoxin universal antibodies have also been reported, and some researchers have even used aflatoxin universal antibodies to establish a total aflatoxin analysis method.
  • the aflatoxin universal antibody mainly emphasizes that the antibody can have a strong specific binding reaction to various aflatoxins, and can be used to establish an immunoassay method for each aflatoxin separately; and aflatoxins B1, B2, G1
  • the G2 total antibody not only emphasizes that the antibody has a strong specific binding reaction to various aflatoxins, but also emphasizes that the sensitivity of the immunoassay method for each aflatoxin established by the antibody is strong. .
  • the versatility of aflatoxin universal antibodies reported at home and abroad is relatively strong, but the analytical sensitivity uniformity for each aflatoxin is generally poor. Therefore, these reported aflatoxin universal antibodies are not suitable.
  • the problem to be solved by the present invention is to provide a hybridoma cell line 10G4 and the aflatoxin-resistant B1, B2, G1 produced thereby. , G2 total monoclonal antibody.
  • the present invention provides a hybridoma cell line 10G4 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. C201016. The classification is mouse hybridoma cells. 10G4. It has the anti-aflatoxin B1, B2, G1, G2 total monoclonal antibody heavy chain variable region coding gene sequence and sequence listing in SEQ ID NO. The anti-aflatoxin B1, B2, G1, G2 total monoclonal antibody light chain variable region coding gene sequence shown in SEQ ID NO.
  • Monoclonal antibody against aflatoxin B1, B2, G1, G2, which is deposited under the accession number CCTCC The hybridoma cell line 10G4 of NO. C201016 was secreted. Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 3 in the sequence listing; the light chain variable region has the SEQ in the sequence listing The amino acid sequence shown in ID NO.
  • the hybridoma cell line 10G4 provided by the present invention is obtained by a two-step screening method, and the specific steps are as follows: BALB/c After immunization of mice with aflatoxin complete antigen AFB1-BSA 4-6 times, the last booster immunization with aflatoxin complete antigen AFB1-BSA twice the previous immunization dose, 3 Cell fusion was performed in the day after, and the fusion cells were screened in two steps by ELISA. The first step was to screen for anti-aflatoxin B1 but not carrier protein BSA by indirect ELISA.
  • the second step uses indirect competitive ELISA to detect the positive well cultured in the first step, using aflatoxin G2 As a competitive source - because the aflatoxin universal antibody reported at home and abroad tends to have a minimal cross-reaction rate to aflatoxin G2 ( ⁇ 50%) ), select the wells with higher absorbance and sensitivity, clone by limiting dilution method, and use the same two-step screening method for detection about 10 days after cloning, so repeat clone 2-3 After the second screening, the hybridoma cell line 10G4 was obtained.
  • Anti-aflatoxin B1, B2, G1, G2 total monoclonal antibodies in aflatoxins B1, B2, Application in G1 and G2 total determination.
  • the invention provides anti-aflatoxin B1, B2, G1, G2
  • the preparation method of the total amount of monoclonal antibody is as follows: the obtained hybridoma cell line 10G4 is injected with BALB/c previously treated with Freund's incomplete adjuvant. In mice, the ascites of the mice was collected and purified to obtain monoclonal antibodies against the total amount of aflatoxins B1, B2, G1 and G2.
  • 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, 4 ° C, Centrifuge at 12000r/min for more than 15min, aspirate the supernatant, mix the obtained abdomen water with 4 volumes of acetate buffer, slowly add n-octanoic acid with 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 a pH of 7.4 was added, and
  • the acetate buffer solution is 0.29 g sodium acetate, and 0.141 mL acetic acid is added with water to a volume of 100 mL.
  • the obtained 0.1 mol/L phosphate buffer solution is 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, potassium dihydrogen phosphate 0.02 g, and the solution is adjusted to a volume of water. 100 mL of the obtained.
  • the hybridoma cell line 10G4 provided by the present invention can be used for preparing a monoclonal antibody against aflatoxin B1, B2, G1, G2.
  • the titer of 10G4 rat ascites antibody was 5.12 ⁇ 10 5 by conventional non-competitive enzyme-linked immunosorbent assay (ELISA).
  • the aflatoxin-resistant B1, B2, G1, G2 provided by the present invention
  • the total amount of monoclonal antibodies can consistently identify aflatoxins B1, B2, G1, G2, 50% inhibitory concentration of aflatoxins B1, B2, G1, G2 IC 50 2.09 ng/mL, 2.23 ng/mL, 2.19 ng/mL, 3.21 ng/mL, for aflatoxin B1,
  • the cross-reaction rate of B2, G1 and G2 ranges from 65.2% to 100%.
  • the aflatoxin-resistant B1, B2, G1, G2 provided by the present invention
  • the total amount of monoclonal antibodies can be used to quantitatively determine the total amount of aflatoxins B1, B2, G1, and G2.
  • the first immunization was performed by emulsifying the aflatoxin B1 complete antigen with an equal amount of complete Freund's adjuvant and then subcutaneously injecting multiple injections into the neck of the mouse.
  • the second immunization was performed 4 weeks later, using Freund's incomplete adjuvant and an equal amount of aflatoxin B1 is fully antigen emulsified and injected intraperitoneally in mice.
  • the third immunization and the second immunization interval are 4 weeks, the immunization method is the same, and the fourth immunization is the third immunization.
  • the immunization method is the same as the second immunization, and the same is intraperitoneal injection.
  • the 4 immunization doses were the same, all being 50 ⁇ g per mouse.
  • 8 days after the fourth immunization blood was collected from the tail vein, serum was separated, and indirect ELISA was used.
  • the serum titer of the mice was monitored, and the serum sensitivity of the mice was determined by indirect competitive ELISA.
  • the mice corresponding to the serum with relatively high titer and sensitivity were selected for the last boost, and the immune dose was the previous 2 Times.
  • Aflatoxin B1 Complete antigen AFB1-BSA was purchased from Sigma-Aldrich.
  • PEG polyethylene glycol
  • cell fusion is carried out according to a conventional method. Specific steps: the immunized mice are killed under aseptic conditions, and the splenocytes are isolated and mixed with the murine myeloma cells SP2/0 at a ratio of 5:1. Wash the mixed cells with RPMI-1640 basal medium, fuse with 50% PEG, mix for 1 minute, then top up RPMI-1640 The basal medium, centrifugation, removal of the supernatant, mouse spleen cells and murine myeloma cells.
  • PEG polyethylene glycol
  • SP2/0 fused cells were resuspended in 72 mL of RPMI-1640 basal medium, and the resuspended cells were added to 96.
  • the RPMI-1640 base medium containing 20% (by volume) fetal bovine serum, 2% (% by weight) growth factor and 1% (by weight) hypoxanthine - aminopterin - thymidine HAT.
  • the above SP2/0 was purchased from Shanghai Panke Biotechnology Co., Ltd.; RPMI-1640 base medium was purchased from Hyclone; 1% hypoxanthine - aminopterin - thymidine HAT was purchased from Sigma-Aldrich the company.
  • 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, and the aflatoxin G2 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 original of 0, ie, the positive control wells.
  • 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 10G4 was obtained.
  • Hybridoma cell lines can be produced by using Tiangen's total RNA extraction kit and extracting according to the instructions. 10G4 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 356 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 118 amino acids, and the sequence is shown in SEQ ID NO: 3.
  • Light chain variable region coding gene sequence length 332 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.
  • the anti-aflatoxin B1, B2, G1, G2 total monoclonal antibody hybridoma cell lines obtained in Example 2 were obtained. 10G4 was injected into BALB/c mice pretreated with Freund's incomplete adjuvant. The ascites of the mice was collected and the antibody was purified by caprylic acid-ammonium sulfate method.
  • the specific operation was as follows: filtration of mouse ascites with double-layer filter paper, 4 °C, 12000r/min centrifugation for 15min, aspirate the supernatant, mix the obtained abdomen water with 4 times volume of acetate buffer, slowly add n-octanoic acid under stirring, and the volume of n-octanoic acid required per milliliter of ascites is 33 ⁇ L, mixed at room temperature for 30 min, allowed to stand at 4 °C for 2 h, then centrifuged at 4 °C, 12000 r/min for 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 thereto, and 2 mol/L was used.
  • 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 2 h, then 4 ° C, Centrifuge at 12000r/min for 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, and then lyophilized with a freeze dryer to collect the lyophilized powder.
  • Aflatoxin B1, B2, G1, G2 total monoclonal antibody the antibody was placed in the refrigerator at -20 °C; the acetate buffer was 0.29g sodium acetate, 0.141mL acetic acid was added to the water to a volume of water 100mL obtained; the 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 Add water to a volume of 100mL.
  • the titer of 10G4 rat ascites antibody was determined by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) 5.12 ⁇ 10 5 , ie rat ascites antibody dilution 5.12 ⁇ 10 5
  • ELISA enzyme-linked immunosorbent assay
  • the cross-reaction rate ranged from 65.2% to 100%.
  • Example 4 Aflatoxin B1, B2, G1, G2 Total monoclonal antibody 10G4 Applications.
  • the aflatoxins B1, B2, G1, and G2 obtained in Example 3 were collectively 4
  • the average curve of the standard curve obtained a new quantitative analysis curve as a total analysis curve of aflatoxin B1, B2, G1, G2, and used to detect 5 parts of peanut actual sample aflatoxin B1 , B2, G1, G2 total, and adopt GB/T 18979-2003 High performance liquid chromatography standard method for detection and comparison, the results show that the consistency of the two methods is ideal, detailed results:
  • Sample 1 Based on the total amount of antibody 10G4 ELISA method test result is 0.23ng/mL , GB/T 18979-2003 high performance liquid chromatography standard method detection result is 0.2 ng / mL ;
  • Sample 2 Based on the total amount of antibody 10G4, the ELISA method showed 1.47 ng/mL, GB/T 18979-2003 high performance liquid chromatography standard method detection results were 1.2 ng / mL ;
  • Sample 3 Based on the total amount of antibody 10G4, the ELISA method showed a result of 0.88 ng/mL, GB/T.
  • the standard test method of high performance liquid chromatography was 18 ng/mL .
  • Sample 5 Based on the total amount of antibody 10G4 ELISA method detection result is 0.92ng/mL, GB/T 18979-2003 The standard method for HPLC is 0.8 ng/mL.
  • Hybridoma cell line 10G4 and its anti-aflatoxin B1, B2, G1, G2 Total monoclonal antibody

Abstract

提供杂交瘤细胞株10G4及其分泌产生的抗黄曲霉毒素总量单克隆抗体。杂交瘤细胞株10G4（CCTCC NO.C201016）可以用于制备抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体，并用常规非竞争酶联免疫吸附分析（ELISA）法测得10G4的鼠腹水抗体的效价可达5.12× 105 。抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体可以一致性较好地识别黄曲霉毒素B1、B2、G1、G2，对黄曲霉毒素B1、B2、G1、G2的50%抑制浓度 IC50依次为2.09ng/ml、2.23ng/ml、2.19ng/ml、3.21ng/ml，对黄曲霉毒素B1、B2、G1、G2的交叉反应率范围为62.5%—100%，可应用于定量测定黄曲霉毒素B1、B2、G1、G2总量。

Description

杂交瘤细胞株 10G4 及其产生的抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体 技术领域
本发明涉及杂交瘤细胞株 10G4 及其产生的抗黄曲霉毒素总量单克隆抗体。
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。黄曲霉毒素品种较多，目前被已发现 20 余种。黄曲霉毒素具有污染广、毒性强、危害重等特点。因此，全球至今至少有 70 个以上的国家制定了农产品及食品中黄曲霉毒素限量标准，很多国家更是制定了主要黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量的限量标准。因此黄曲霉毒素总量分析方法显得尤为重要。
现有黄曲霉毒素的检测方法包括薄层层析法、精密仪器分析法和免疫学分析法。其中薄层层析法是检测黄曲霉毒素最常用的检测方法，其不需要特殊的仪器设备，一般实验室都可进行，但试剂用量大、操作繁琐、其它组分干扰严重、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，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，其灵敏度高，准确性好，但仪器昂贵，要求黄曲霉毒素样品纯化程度高，样品前处理过程繁琐，耗时长，对实验环境要求高，难以实现快速检测。近些年发展起来的免疫分析技术克服了前两者的缺点，具有特异性强、灵敏度高、样品前处理简单、成本低、对实验人员和周围环境的污染危害小、适于现场批量检测等优点。
免疫分析是利用抗原和抗体的特异性反应和抗体（或抗原）上标记物的生物、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总量分析方法的任何一种免疫学检测技术，都必须先制得抗黄曲霉毒素B1、B2、G1、G2总量的抗体。事实上无论国内还是国际上研制黄曲霉毒素抗体的报道很多，黄曲霉毒素通用抗体也有报道，甚至也有个别学者将黄曲霉通用抗体用来建立黄曲霉毒素总量分析方法。黄曲霉毒素通用抗体主要强调抗体对各种黄曲霉毒素均可发生较强的特异性结合反应，可被用来分别建立每种黄曲霉毒素的免疫分析方法；而黄曲霉毒素B1、B2、G1、G2总量抗体则不仅强调抗体对各种黄曲霉毒素均可发生较强的特异性结合反应，而且还特别强调应用该抗体建立的针对每种黄曲霉毒素的免疫分析方法的灵敏度一致性要强。国内外现有报道的黄曲霉毒素通用抗体通用性都是比较强的，但针对每种黄曲霉毒素的分析灵敏度一致性却通常都较差，因此，这些报道的黄曲霉毒素通用抗体并不适合用来建立黄曲霉毒素B1、B2、G1、G2总量分析方法，即使建立了黄曲霉毒素总量分析方法，其定量分析的准确性也会大打折扣。因此，研制黄曲霉毒素B1、B2、G1、G2总量抗体对实现黄曲霉毒素B1、B2、G1、G2总量免疫快速定量检测具有重要意义。
 技术问题
本发明所要解决的问题是提供杂交瘤细胞株 10G4 及其产生的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体。
技术解决方案
本发明提供了杂交瘤细胞株 10G4 ，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. C201016 ，分类命名为小鼠杂交瘤细胞 10G4 。其具有序列表中 SEQ ID NO.1 所示的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量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 、 B2 、 G1 、 G2 总量单克隆抗体轻链可变区编码基因序列。
抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体，它由保藏编号为 CCTCC NO. C201016 的杂交瘤细胞株 10G4 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。
本发明提供的杂交瘤细胞株 10G4 是采用两步筛选法获得的，其具体步骤为：将 BALB/c 小鼠经黄曲霉毒素完全抗原 AFB1-BSA 免疫 4-6 次后，用 2 倍于前一次免疫剂量的黄曲霉毒素完全抗原 AFB1-BSA 作最后一次加强免疫， 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培养液进行检测，用黄曲霉毒素 G2 作为竞争原--因为国内外现有报道的黄曲霉毒素通用抗体对黄曲霉毒素 G2 的交叉反应率往往是最小的（＜ 50% ），选择吸光值和灵敏度均较高的孔，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筛选法进行检测，如此重复克隆 2-3 次后，最终筛选获得杂交瘤细胞株 10G4 。
抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体在黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量测定中的应用。
本发明提供的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体的制备方法，步骤如下：将获得的杂交瘤细胞株 10G4 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，纯化即得抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体。
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， 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 ～ 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 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 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，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 、 B2 、 G1 、 G2 总量单克隆抗体，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。
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 10G4 可以用于制备抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体。用常规非竞争酶联免疫吸附分析（ ELISA ）法测得 10G4 的鼠腹水抗体的效价可达 5.12 × 105 。
(2) 本发明提供的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体可以一致性较好地识别黄曲霉毒素 B1 、 B2 、 G1 、 G2 ，对黄曲霉毒素 B1 、 B2 、 G1 、 G2 的 50% 抑制浓度 IC50 依次为 2.09ng/mL 、 2.23ng/mL 、 2.19ng/mL 、 3.21ng/mL ，对黄曲霉毒素 B1 、 B2 、 G1 、 G2 的交叉反应率范围为 65.2%-100% 。
(3) 本发明提供的抗黄曲霉毒素B1、 B2 、 G1 、 G2 总量单克隆抗体可应用于定量测定黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量。
 本发明的实施方式
实施例 1 ：杂交瘤细胞株 10G4 的制备
1. 动物免疫
购买 6 周龄 BALB/c 小鼠 6 只，免疫市售的黄曲霉毒素 B1 完全抗原 AFB1-BSA 。第一次免疫将黄曲霉毒素 B1 完全抗原与等量福氏完全佐剂乳化后，于小鼠颈背部皮下多点注射。第二次免疫于 4 周后进行，采用福氏不完全佐剂与等量黄曲霉毒素 B1 完全抗原乳化，于小鼠腹腔注射。第三次免疫与第二次免疫间隔 4 周，免疫方式与其相同，第四次免疫于第三次免疫 3 周后进行，免疫方式与第二次免疫相同，同样为腹腔注射。 4 次免疫剂量相同，均为每鼠 50 μ g 。前 3 次每次免疫后 8 天，尾静脉采血，分离血清，采用间接 ELISA 法监测小鼠血清效价。第 4 次免疫后 8 天，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，免疫剂量为之前的 2 倍。黄曲霉毒素 B1 完全抗原 AFB1-BSA 购于 Sigma-Aldrich 公司。
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：无菌条件下杀死免疫小鼠，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，用 50%PEG 融合，融合 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 72mLRPMI-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 - 胸腺嘧啶核苷即 HAT 。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 - 胸腺嘧啶核苷即 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 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 G2 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 10G4 。
实施例 2 ：抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体杂交瘤细胞株系 10G4 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 10G4 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 356bp ，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 118 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 332bp ，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 、 B2 、 G1 、 G2 总量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 、 B2 、 G1 、 G2 总量单克隆抗体杂交瘤细胞株系 10G4 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 、 B2 、 G1 、 G2 总量单克隆抗体，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 10G4 分泌的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体的亚型为 IgG1 。
用常规非竞争酶联免疫吸附分析（ ELISA ）法测得 10G4 的鼠腹水抗体的效价可达 5.12 × 105 ，即鼠腹水抗体稀释 5.12 × 105 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 、 B2 、 G1 、 G2 总量的灵敏度 50% 抑制浓度 IC50 依次为 2.09ng/mL 、 2.23ng/mL 、 2.19ng/mL 、 3.21ng/mL ，对黄曲霉毒素 B1 、 B2 、 G1 、 G2 的交叉反应率范围为 65.2%-100% 。
实施例 4 ：黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体 10G4 的应用。
将实施例 3 中获得的黄曲霉毒素 B1 、 B2 、 G1 、 G2 共 4 条标准曲线平均化处理获得一条新的定量分析曲线作为黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量分析曲线，并用于检测 5 份花生实际样品黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量，同时采用 GB/T 18979-2003 高效液相色谱标准方法进行检测比对，结果显示两种方法一致性比较理想，详细结果：
样品 1 ：基于抗体 10G4 的总量 ELISA 方法检测结果为 0.23ng/mL ， GB/T 18979-2003 高效液相色谱标准方法检测结果为 0.2ng/mL ；
样品 2 ：基于抗体 10G4 的总量 ELISA 方法检测结果为 1.47ng/mL ， GB/T 18979-2003 高效液相色谱标准方法检测结果为 1.2ng/mL ；
样品 3 ：基于抗体 10G4 的总量 ELISA 方法检测结果为 0.88ng/mL ， GB/T 18979-2003 高效液相色谱标准方法检测结果为 1.1 ng/mL ；
样品 4 ：基于抗体 10G4 的总量 ELISA 方法检测结果为阴性， GB/T 18979-2003 高效液相色谱标准方法检测结果为阴性；
样品 5 ：基于抗体 10G4 的总量 ELISA 方法检测结果为 0.92ng/mL ， GB/T 18979-2003 高效液相色谱标准方法检测结果为 0.8ng/mL 。
 序列表自由内容
＜ 110 ＞ 中国农业科学院油料作物研究所
＜ 120 ＞ 杂交瘤细胞株 10G4 及其产生的抗黄曲霉毒素 B1 、 B2 、 G1 、 G2 总量单克隆抗体
＜ 160 ＞ 4
＜ 210 ＞ 1
＜ 211 ＞ 356bp
＜ 212 ＞ DNA
＜ 213 ＞ 小鼠
＜ 400 ＞ 1
aggtgcagct gcaggagtca gggggaggct tagtgaagcc tggagggtcc 50
ctgaaactct cctgtgcagc ctcaggattc actctcagta gtaatgacat 100
gtcttgggtt cgccagacac cggagaagag gctggagtgg gtcgcaagta 150
ttagtagagg tggtaggtac acctactatc cagacagtgt gaaggggcga 200
ttcaccatct ccagagacaa agccaagaac accctgtatc tgcaaatgaa 250
cagtctgagg tctgaggata cggccatgta ttattgtgca agacactatg 300
gtagctactg gtacttcgat gtctggggcc aagggaccac ggtcaccgtc 350
tcctca 356
＜ 210 ＞ 2
＜ 211 ＞ 332bp
＜ 212 ＞ DNA
＜ 213 ＞ 小鼠
＜ 400 ＞ 2
ccgtttgatt tccagcttgg tgccccctcc gaacgtgtaa gctccctaat 50
gtgctgacag taataggttg cagcatcctc ctcctccaca ggatggatgt 100
tgagggtgaa gtctgtccca gacccactgc cactgaacct ggcagggacc 150
ccagattcta ggttggatac aagatagatg aggagtctgg gtggctgtcc 200
tggtttctgt tggttccagt gcatataact atagccagat gtactgacac 250
ttttgctggc cctgtatgag atggtggccc tctgccccag agatacagct 300
aaggaagctg gagactgggt gagctcaatg tc 332
＜ 210 ＞ 3
＜ 211 ＞ 118
＜ 212 ＞ PRT
＜ 213 ＞ 小鼠
＜ 400 ＞ 3
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser
1 5 10 15 20
Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Asn Asp Met Ser Trp Val Arg Gln Thr Pro
25 30 35 40
Glu Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Arg Gly Gly Arg Tyr Thr Tyr Tyr Pro
45 50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Tyr Gly
85 90 95 100
Ser Tyr Trp Tyr Phe Asp Val 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 Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr
1 5 10 15 20
Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn
25 30 35 40
Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
45 50 55 60
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg
85 90 95 100
Ser Glu Gly Ala Pro Ser Trp Lys Ser Asn
105 110

Claims (4)

1. 杂交瘤细胞株10G4，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. C201016。
2. 抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体，其特征在于：它由保藏编号为CCTCC NO. C201016的杂交瘤细胞株10G4分泌产生。
3. 根据权利要求2所述的抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体在黄曲霉毒素B1、B2、G1、G2总量测定中的应用。
4. 根据权利要求2所述抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体的制备方法，步骤如下：将获得的杂交瘤细胞株10G4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，纯化即得抗黄曲霉毒素B1、B2、G1、G2总量单克隆抗体。
PCT/CN2013/070612 2012-04-20 2013-01-17 杂交瘤细胞株10g4及其产生的抗黄曲霉毒素b1、b2、g1、g2总量单克隆抗体 WO2013155882A1 (zh)

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