WO2019129082A1 - 一株林可胺类通用性单克隆抗体杂交瘤细胞株及其应用 - Google Patents

一株林可胺类通用性单克隆抗体杂交瘤细胞株及其应用 Download PDF

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WO2019129082A1
WO2019129082A1 PCT/CN2018/123952 CN2018123952W WO2019129082A1 WO 2019129082 A1 WO2019129082 A1 WO 2019129082A1 CN 2018123952 W CN2018123952 W CN 2018123952W WO 2019129082 A1 WO2019129082 A1 WO 2019129082A1
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lincosamide
monoclonal antibody
clindamycin
cell strain
lincomycin
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匡华
郭玲玲
胥传来
徐丽广
马伟
刘丽强
宋珊珊
吴晓玲
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Jiangnan University
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], 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
    • 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
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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

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  • the invention relates to a lincosamide universal monoclonal antibody hybridoma cell strain and an application thereof, and belongs to the technical field of food safety immunological detection.
  • Lincosamides have anti-gram-positive aerobic bacteria and Gram-positive or negative anaerobic bacteria activities, and lincomycin, clindamycin and pyrimycin are commonly used in veterinary clinics.
  • Residues of lincoamines in animal-derived foods can cause renal dysfunction and increased drug resistance of Gram-positive bacteria. Both China and the European Union have made relevant regulations on the maximum residue limits in animal-derived foods. Therefore, it is urgent to establish a Fast, sensitive and accurate method for detecting lincosamide residues to ensure the safety of animal foods.
  • the methods for detecting lincosamide residues mainly include high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS), and the immunoassay methods are relatively few.
  • HPLC high performance liquid chromatography
  • LC/MS liquid chromatography-mass spectrometry
  • the immunoassay method is relatively few.
  • the instrument detection method is accurate, but the instrument is expensive, the operation is complicated, the time is long, and the sample preparation is cumbersome.
  • the immunoassay method has the characteristics of low cost, high throughput, high sensitivity, low requirements for technicians, and is therefore suitable for rapid screening of a large number of samples.
  • the invention provides a preparation method of a lincosamide universal monoclonal antibody, which is prepared by separating a hybridoma cell line and extracting monoclonal antibodies of clindamycin, lincomycin and pyrimycin from the hybridoma cell line.
  • the antibody which is used for the detection of clindamycin, lincomycin and pyrimycin, has good detection sensitivity and can be used to establish an immunological detection method of lincosamide.
  • the first object of the present invention is to provide a monoclonal cell strain which has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 5, 2017, and the deposit number is CGMCC No. 14691, and the deposit address is Beijing. No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District.
  • a second object of the present invention is to provide a method for detecting lincosamides by using the lincosamide-specific monoclonal antibody secreted by the above monoclonal cell strain CGMCC No. 14691, through an enzyme Linkage immunoassay for the detection of lincosamides.
  • the lincosamides include clindamycin, lincomycin or pyrimycin.
  • a third object of the present invention is to provide a lincosamide-specific monoclonal antibody which is secreted by the above monoclonal cell strain CGMCC No. 14691.
  • the lincosamide-specific monoclonal antibody can be used for the analysis of the residual amount of lincosamides in foods.
  • a fourth object of the present invention is to provide a kit comprising the monoclonal cell strain CGMCC No. 14691 or the lincosamide-specific monoclonal antibody.
  • the kit is for analyzing the residual amount of lincosamide in a food.
  • the lincosamides include clindamycin, lincomycin or pyrimycin.
  • a fifth object of the present invention is to provide a method for preparing a lincosamide-based versatile monoclonal antibody, the method steps being as follows:
  • hapten It is prepared by condensation reaction of ⁇ -mercaptopropionic acid with clindamycin. The specific synthetic route is shown in Fig. 1. Dissolve 9.6 g of clindamycin in 20 mL of dimethyl sulfoxide (DMSO), add 2.3 g of KOH, and slowly add ⁇ -mercaptopropionic acid solution (3.2 g of ⁇ -mercaptopropionic acid dissolved in 10 mL of DMSO) while stirring. The temperature of the bath was raised to 100 ° C, and the reaction was carried out for 2 h.
  • DMSO dimethyl sulfoxide
  • the screening is divided into two steps: the first step is to select positive cells by indirect ELISA, the second step is to use clindamycin, lincomycin and pyrimycin as standard, and the inhibition effect of positive cells by indirect competition ELISA is determined. Positive cells with good inhibition of the four standards were selected, subcloned by the limiting dilution method, and detected by the same method. Repeat three times to obtain a cell line capable of stably secreting a ubiquitin-like monoclonal antibody;
  • the invention designs a chlorine substitute of clindamycin as a hapten, and couples it with BSA by an activated ester method to obtain an immunogen; and immunizes BALB/c mice, and uses the clindamycin original drug.
  • the coating was obtained by coupling with OVA by CDI method and used for screening of mouse serum and cell supernatant. Through cell fusion and screening, a cell strain capable of stably secreting monoclonal antibodies having good affinity and detection sensitivity to clindamycin, lincomycin, and pyrimycin is finally obtained, and monoclonal antibody is determined.
  • the half-inhibition rate (IC 50 ) of lincomycin, lincomycin and pyrimycin is: 0.379 ng/mL, 4.339 ng/mL, 19.314 ng/mL, respectively; It has become possible.
  • a monoclonal cell line was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 5, 2017. The deposit number is CGMCC No.14691, and the deposit address is No. 1 Courtyard of Beichen West Road, Chaoyang District, Beijing. number.
  • Figure 1 shows the design of a hapten
  • Figure 2 is a mass spectrum of clindamycin chloride substituted derivative
  • Figure 3 is a standard curve of antibodies against clindamycin, lincomycin and pyrimycin.
  • the invention immunizes mice by complete antigen, cultures through cell fusion, HAT selective medium, screens cell supernatant by indirect ELISA and indirect competition ELISA, and finally obtains clindamycin, lincomycin and pyrioxazole.
  • hapten It is prepared by condensation reaction of ⁇ -mercaptopropionic acid with clindamycin. The specific synthetic route is shown in Fig. 1. Dissolve 9.6 g of clindamycin in 20 mL of dimethyl sulfoxide (DMSO), add 2.3 g of KOH, and slowly add ⁇ -mercaptopropionic acid solution (3.2 g of ⁇ -mercaptopropionic acid in 10 mL of DMSO) while stirring. The oil bath was heated to 100 ° C, and reacted for 2 h.
  • DMSO dimethyl sulfoxide
  • Lin-BSA Weigh 10 mg of hapten and dissolve it in 3 ml of 0.1M pH 6.0 2-(N-morpholine)ethanesulfonic acid (MES) solution; stir constantly, add 24mg N- Hydroxysuccinimide, after 15 min, add 31 mg of 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide); stir at room temperature for 2 h to obtain an activation solution; weigh 20 mg BSA Dissolve in 1ml 0.1M pH 9.0 carbonate buffer solution; add the activation solution dropwise under constant stirring, and react at room temperature for 4h, then dialyze with 0.01M pH 7.4 phosphate buffer solution. Get Lin-BSA.
  • MES 2-(N-morpholine)ethanesulfonic acid
  • Lin-OVA Weigh 10 mg of clindamycin and 29.89 mg of CDI completely dissolved in 2 ml of anhydrous N,N-dimethylformamide (DMF), and activate at room temperature for 5 h; The activation solution was added dropwise to the OVA solution (20 mg/ml, 0.1 M pH 9.0 carbonate buffer solution), and reacted at room temperature overnight. The reaction solution was dialyzed against 0.01 M phosphate buffer pH 7.4 to obtain Lin. -OVA.
  • DMF N,N-dimethylformamide
  • Coat The coated antigen was serially diluted with 0.05 M pH 9.6 carbonate buffer, 100 ⁇ L/well, and incubated at 37 °C for 2 h.
  • D. Loading PBS was added to the half of the plate, 50 ⁇ L/well (the upper part is called 0), and the lower part was added with different concentrations of lincosamide standard. The antiserum was started from 1:1000. Dilution, 50 ⁇ L/well (the lower half becomes spiked), the upper and lower portions are correspondingly added to the wells of different dilution gradient coated antigen, and incubated at 37 ° C for 30 min; after thorough washing, add 1:3000 diluted mouse secondary antibody, 100 ⁇ L/well Incubate at 37 ° C for 30 min, wash and pat dry.
  • Termination and determination The plate was taken out, and 50 ⁇ L of stop solution (2 mol/L of sulfuric acid) was added to each well to terminate the reaction, and then the absorbance A450 of each well was measured by a microplate reader.
  • the highest dilution factor of serum corresponding to 2.1 times (or P/N ⁇ 2.1) of the OD value greater than or equal to the negative serum control well is the serum ELISA titer.
  • the standard OD concentration of the added standard is the half of the OD value.
  • the screening is divided into two steps: the first step is to select positive cells by indirect ELISA, the second step is to use clindamycin, lincomycin and pyrimycin as standard, and the inhibition effect of positive cells by indirect competition ELISA is determined. . Positive cells with good inhibition of the four standards were selected, subcloned by limiting dilution method, and detected by the same method. After repeating three times, a cell strain capable of stably secreting a versatile monoclonal antibody of lincosamide can be obtained.
  • the IC 50 of the monoclonal antibody against clindamycin, lincomycin, and pirimimycin was determined to be: 0.379 ng/mL, 4.339 ng/mL, and 19.314 ng/mL, respectively; To meet the current demand for lincoamine immunoassay products on the market.

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Abstract

本发明公开了一株林可胺类通用性单克隆抗体杂交瘤细胞株及其应用，属于食品安全免疫学检测技术领域。本发明采用克林霉素氯取代衍生物作为半抗原，将半抗原通过活化酯法偶联牛血清蛋白(BSA)，得到免疫抗原，与弗氏佐剂混合均匀后，通过皮下注射免疫BALB/c小鼠；将克林霉素采用羰基二咪唑(CDI)法与鸡蛋白蛋白(OVA)偶联，作为包被抗原，用于小鼠血清以及细胞上清检测。将免疫小鼠的脾细胞通过PEG方法与小鼠骨髓瘤细胞融合，经过间接ELISA和间接竞争ELISA筛选和三次亚克隆，得到一株群选性的杂交瘤细胞株。本发明提供的细胞株对克林霉素、林可霉素和吡利霉素均具有较好的抑制，可以满足目前市场上对林可胺类多残免疫检测产品的需求。

Description

一株林可胺类通用性单克隆抗体杂交瘤细胞株及其应用 技术领域
本发明涉及一株林可胺类通用性单克隆抗体杂交瘤细胞株及其应用，属于食品安全免疫学检测技术领域。
背景技术
林可胺类药物(lincosamides)具有抗革兰氏阳性需氧菌和革兰氏阳性或阴性厌氧菌活性，兽医临床常用的有林可霉素、克林霉素和吡利霉素。
动物源食品中林可胺类药物残留可引起肾功能障碍和革兰氏阳性菌耐药性增加，我国和欧盟都对动物源食品中的最大残留限量做了相关规定，因此，亟待建立一种快速、灵敏、准确的林可胺类药物残留检测方法，确保动物性食品的安全。
目前检测林可胺类药物残留的方法主要有高效液相色谱法(HPLC)、液质联用法(LC/MS)等仪器检测方法，而免疫分析方法相对较少。其中仪器检测方法检测准确，但仪器昂贵，操作复杂，时间长，样品前处理繁琐。免疫分析方法相对于仪器检测方法，具有低成本、高通量、高灵敏、对技术人员要求低等特点，因此适用于大量样品的快速筛查。
发明内容
本发明提供了一种林可胺类通用性单克隆抗体的制备方法，通过制备杂交瘤细胞株，从杂交瘤细胞株中分离得到克林霉素、林可霉素和吡利霉素单克隆抗体，该单克隆抗体用于检测克林霉素、林可霉素和吡利霉素时，都具有较好检测灵敏度，可以用来建立林可胺类药物的免疫学检测方法。
本发明的第一个目的是提供一株单克隆细胞株，已于2017年9月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14691，保藏地址为北京市朝阳区北辰西路1号院3号。
本发明的第二个目的是提供一种检测林可胺类药物的方法，所述方法是利用上述的单克隆细胞株CGMCC No.14691分泌产生的林可胺类特异性单克隆抗体，通过酶联免疫法检测林可胺类药物。所述林可胺类药物包括克林霉素、林可霉素或吡利霉素。
本发明的第三个目的是提供一种林可胺类特异性单克隆抗体，由上述的单克隆细胞株CGMCC No.14691所分泌产生。
所述的林可胺类特异性单克隆抗体可用于分析检测食品中林可胺类药物的残留量。
本发明的第四个目的是提供一种试剂盒，所述试剂盒含有所述的单克隆细胞株CGMCC No.14691或者所述的林可胺类特异性单克隆抗体。
所述试剂盒用于分析检测食品中林可胺类药物的残留量。所述林可胺类药物包括克林霉素、林可霉素或吡利霉素。
本发明的第五个目的是提供一种林可胺类通用性单克隆抗体的制备方法，所述方法步骤为：
(1)半抗原的设计：利用β-巯基丙酸与克林霉素的缩合反应制备得到，具体合成路线见图1。用20mL二甲基亚砜(DMSO)溶解9.6g克林霉素原药，加入2.3g KOH，边搅拌边缓慢滴加β-巯基丙酸溶液(3.2gβ-巯基丙酸溶解于10mLDMSO)，油浴升温至100℃，反应2h，待产物自然冷却至室温后，加入50mL水，以6mol/LHCl调反应液至pH 3，然后以50mL二氯甲烷萃取，提取液经无水Na 2SO 4干燥、浓缩、结晶，得黄色晶体，即目标化合物，通过质谱鉴定。
(2)完全抗原的制备：采用步骤(1)制备得到的克林霉素氯取代衍生物，通过活化酯法偶联牛血清蛋白(BSA)，得到免疫抗原Lin-BSA；将克林霉素采用羰基二咪唑(CDI)法与鸡蛋白蛋白(OVA)偶联，作为包被抗原Lin-OVA。
(3)动物免疫与效价测定：采用小剂量短周期方案免疫健康BALB/c雌性小鼠，首次免疫用100μg偶联抗原与等量福氏完全佐剂混匀后进行皮下注射，间隔3周后，再用100μg偶联抗原与等量福氏不完全佐剂加强免疫，此后每隔3周用半量偶联抗原加强免疫一次；冲刺免疫剂量减半，与等体积的生理盐水混合后采用腹腔免疫，通过间接竞争ELISA检测血清效价和抑制；
(4)细胞融合与筛选：在冲刺免疫三天后，按照常规PEG(聚乙二醇，分子量为1450)方法进行细胞融合，具体步骤如下：
A.无菌取小鼠脾脏，研磨并通过200目细胞筛网得到脾细胞悬液，并进行细胞计数；
B.收集SP2/0细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
C.将脾细胞和SP2/0细胞按照10:1(数量比)的比例混合，离心后用50％PEG融合，时间1min，之后按照从慢到快，加入RPMI-1640基础培养液，离心后悬浮于含20％胎牛血清、2％的50×HAT的RPMI-1640筛选培养液中，加到96孔细胞培养板，置于37℃、5％CO 2的培养箱中培养。在细胞融合的第三天对融合细胞进行RPMI-1640筛选培养液半换液，第6天进行用含20％胎牛血清、1％的100×HT的RPMI-1640过渡培养液进行全换液，在第9天取细胞上清进行筛选；
筛选分两步：第一步先用间接ELISA筛选出阳性细胞，第二步选用克林霉素、林可霉素和吡利霉素为标准品，用间接竞争ELISA对阳性细胞进行抑制效果测定，选出对四个标准品均具有较好抑制的阳性细胞，采用有限稀释法进行亚克隆，用同样的方法进行检测。重复三 次，即可得到能稳定分泌林可胺类通用性单克隆抗体的细胞株；
(5)单克隆抗体的制备与鉴定：取8-10周龄BALB/c小鼠，每只小鼠腹腔注射石蜡油1mL；7天后每只小鼠腹腔注射1×10 6杂交瘤细胞，从第七天开始收集腹水，将腹水通过辛酸-硫酸铵法纯化，获得的单抗置于-20℃保存。
本发明的有益效果：本发明设计克林霉素的氯取代物作为半抗原，将其与BSA采用活化酯法进行偶联得到免疫原；免疫BALB/c小鼠，将克林霉素原药采用CDI法与OVA偶联得到包被原，用于小鼠血清和细胞上清的筛选。通过细胞融合和筛选，最终获得了能够稳定分泌对克林霉素、林可霉素、吡利霉素等均具有较好亲和力和检测灵敏度的单克隆抗体的细胞株，测定单克隆抗体对克林霉素、林可霉素、吡利霉素的半数抑制率(IC 50)分别为：0.379ng/mL、4.339ng/mL、19.314ng/mL；使得氯霉素类的多残免疫检测方法成为了可能。
生物材料保藏
一株单克隆细胞株，已于2017年9月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14691，保藏地址为北京市朝阳区北辰西路1号院3号。
附图说明
图1为半抗原的设计；
图2为克林霉素氯取代衍生物质谱图；
图3为抗体对克林霉素、林可霉素和吡利霉素的标准曲线。
具体实施方式
本发明通过将完全抗原免疫小鼠，通过细胞融合，HAT选择性培养基培养，通过间接ELISA和间接竞争ELISA筛选细胞上清，最终得到了对克林霉素、林可霉素、吡利霉素均有较好灵敏度的通用性单克隆抗体。
实施例1：林可胺类通用性单克隆抗体的制备
(1)半抗原的设计：利用β-巯基丙酸与克林霉素的缩合反应制备得到，具体合成路线见图1。用20mL二甲基亚砜(DMSO)溶解9.6g克林霉素原药，加入2.3g KOH，边搅拌边缓慢滴加β-巯基丙酸溶液(3.2gβ-巯基丙酸溶解于10mL DMSO)，油浴升温至100℃，反应2h，待产物自然冷却至室温后，加入50mL水，以6mol/LHCl调反应液至pH3，然后以50mL二氯甲烷萃取，提取液经无水Na2SO4干燥、浓缩、结晶，得黄色晶体，即目标化合物，通过质谱鉴定。
(2)完全抗原的制备：采用克林霉素氯取代衍生物，通过活化酯法偶联牛血清蛋白(BSA)，得到免疫抗原Lin-BSA；将克林霉素采用羰基二咪唑(CDI)法与鸡蛋白蛋白(OVA)偶联，作为包被抗原Lin-OVA。具体合成方法如下：
A.Lin-BSA的合成：称取10mg半抗原，溶于3ml 0.1M pH 6.0的一水合2-(N-吗啡啉)乙磺酸(MES)溶液中；不断搅拌下，先加入24mg N-羟基琥珀酰亚胺，15min后，再加入31mg 1-乙基-3-(3-二甲基氨基丙基-碳二亚胺)；在室温下，搅拌2h，得到活化液；称取20mg BSA，溶于1ml 0.1M pH 9.0碳酸盐缓冲溶液中；在不断搅拌下，逐滴加入活化液，室温下，反应4h后，将反应液用0.01M pH 7.4的磷酸盐缓冲液透析，即可得到Lin-BSA.
B.Lin-OVA的合成：分别称取10mg克林霉素原药和29.89mg CDI完全溶于2ml无水N,N-二甲基甲酰胺(DMF)中，于室温条件下，活化5h；将活化液逐滴滴加到OVA溶液(20mg/ml,0.1M pH 9.0碳酸盐缓冲溶液)中，室温反应过夜，将反应液用0.01M pH 7.4的磷酸盐缓冲液透析，即可得到Lin-OVA.
(3)动物免疫与效价测定：采用小剂量短周期方案免疫健康BALB/c雌性小鼠，首次免疫用100μg偶联抗原与等量福氏完全佐剂混匀后进行皮下注射，间隔3周后，再用100μg偶联抗原与等量福氏不完全佐剂加强免疫，此后每隔3周用半量偶联抗原加强免疫一次；冲刺免疫剂量减半，与等体积的生理盐水混合后采用腹腔免疫，通过间接竞争ELISA检测血清效价和抑制；
其具体ELISA程序如下：
A.包被：将包被抗原用0.05M pH9.6碳酸盐缓冲液梯度稀释，100μL/孔，37℃孵育2h。
B.洗涤：将板内溶液倾去，每孔注入200μLPBST溶液，置于摇床上振荡3min，甩干，洗涤3次。以下洗涤方法相同。
C.封闭：拍干后，加入200μL/孔封闭液，37℃孵育2h。洗涤后烘干备用。
D.加样：酶标板上半部分加入PBS，50μL/孔(上半部分称为0标)，下半部分加入不同浓度的林可胺类标准品，将抗血清从1:1000开始梯度稀释，50μL/孔(下半部分成为加标)，上下部分对应加入不同稀释梯度包被抗原的孔中，37℃孵育30min；充分洗涤后，加入1:3000稀释的鼠二抗，100μL/孔，37℃孵育30min，洗涤后拍干。
E.显色：将酶标板取出，充分洗涤后，每孔加入100μL的显色液(TMB与底物液比例1:5)，37℃避光反应15min。
F.终止和测定：取出酶标板，每孔加入50μL终止液(2mol/L的硫酸)终止反应，然后用酶标仪测定各孔的吸光值A450。
G.结果判读：以OD值大于或等于阴性血清对照孔的2.1倍(即P/N≥2.1)所对应的血清最高稀释倍数即为血清的ELISA效价。上下部分对照，加标OD值为0标一半的即是所加的标准品浓度。
(4)细胞融合与筛选：在冲击免疫三天后，按照常规PEG(聚乙二醇，分子量为1450) 方法进行细胞融合，具体步骤如下：
A.无菌取小鼠脾脏，研磨并通过200目细胞筛网得到脾细胞悬液，并进行细胞计数；
B.收集SP2/0细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
C.将脾细胞和SP2/0细胞按照10:1(数量比)的比例混合，离心后用50％PEG融合，时间1min，之后按照从慢到快，加入RPMI-1640基础培养液，离心后悬浮于含20％胎牛血清、2％的50×HAT的RPMI-1640筛选培养液中，加到96孔细胞培养板，置于37℃、5％CO2的培养箱中培养。在细胞融合的第三天对融合细胞进行RPMI-1640筛选培养液半换液，第6天进行用含20％胎牛血清、1％的100×HT的RPMI-1640过渡培养液进行全换液，在第9天取细胞上清进行筛选。
筛选分两步：第一步先用间接ELISA筛选出阳性细胞，第二步选用克林霉素、林可霉素和吡利霉素为标准品，用间接竞争ELISA对阳性细胞进行抑制效果测定。选出对四个标准品均具有较好抑制的阳性细胞，采用有限稀释法进行亚克隆，用同样的方法进行检测。重复三次，即可得到能稳定分泌林可胺类通用性单克隆抗体的细胞株。
(5)单克隆抗体的制备与鉴定：取8-10周龄BALB/c小鼠，每只小鼠腹腔注射石蜡油1mL；7天后每只小鼠腹腔注射1×106杂交瘤细胞，从第七天开始收集腹水，将腹水通过辛酸-硫酸铵法纯化，获得的单抗置于-20℃保存。
使用间接竞争ELISA和间接ELISA，测定得到单克隆抗体对克林霉素、林可霉素、吡利霉素的IC 50分别为：0.379ng/mL、4.339ng/mL、19.314ng/mL；可以满足目前市场上对林可胺类免疫检测产品的需求。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一株单克隆细胞株，已于2017年9月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14691，保藏地址为北京市朝阳区北辰西路1号院3号。
  2. 一种检测林可胺类药物的方法，其特征在于，所述方法是利用权利要求1所述的单克隆细胞株分泌产生的林可胺类特异性单克隆抗体，通过酶联免疫法检测林可胺类药物。
  3. 根据权利要求2所述的一种检测林可胺类药物的方法，其特征在于，所述林可胺类药物包括克林霉素、林可霉素或吡利霉素。
  4. 一种林可胺类特异性单克隆抗体，其特征在于，由权利要求1所述的杂交瘤细胞所分泌产生。
  5. 权利要求4所述的林可胺类特异性单克隆抗体的应用，其特征在于，用于食品安全检测中林可胺类药物残留的分析检测。
  6. 根据权利要求5所述的林可胺类特异性单克隆抗体的应用，其特征在于，所述林可胺类药物包括克林霉素、林可霉素或吡利霉素。
  7. 一种试剂盒，其特征在于，含有权利要求1所述的单克隆细胞株或者权利要求4所述的林可胺类特异性单克隆抗体。
  8. 根据权利要求7所述的一种试剂盒，其特征在于，所述试剂盒用于检测林可胺类药物。
  9. 权利要求7或8所述的一种试剂盒，其特征在于，所述林可胺类药物包括克林霉素、林可霉素或吡利霉素。
  10. 权利要求7～9任一所述的试剂盒在检测林可胺类药物中的应用。
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