WO2022183762A1 - 一种抗非那西丁单克隆抗体杂交瘤细胞株ad及其制备方法与应用 - Google Patents

一种抗非那西丁单克隆抗体杂交瘤细胞株ad及其制备方法与应用 Download PDF

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WO2022183762A1
WO2022183762A1 PCT/CN2021/129509 CN2021129509W WO2022183762A1 WO 2022183762 A1 WO2022183762 A1 WO 2022183762A1 CN 2021129509 W CN2021129509 W CN 2021129509W WO 2022183762 A1 WO2022183762 A1 WO 2022183762A1
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phenacetin
monoclonal antibody
phe
hybridoma cell
cell line
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English (en)
French (fr)
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胥传来
姚静静
匡华
徐丽广
马伟
孙茂忠
刘丽强
吴晓玲
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江南大学
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Priority to US17/779,989 priority Critical patent/US20230167196A1/en
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/9486—Analgesics, e.g. opiates, aspirine
    • 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
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/10—Competitive assay format

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  • the invention belongs to the technical field of food safety immune detection, in particular to an anti-phenacetin monoclonal antibody hybridoma cell line AD and a preparation method and application thereof.
  • Phenacetin belongs to Nonsteroidal Antiinflammatory Drugs (NSAIDs), has anti-inflammatory, analgesic, antipyretic and other effects, and is widely used in clinical for a variety of fever and various pain symptoms
  • NSAIDs are one of the most used drugs in the world. About 30 million people use them every day in the world. With the increase in the use of NSAIDs, the safe use of these drugs has also attracted more and more attention. The FDA believes that NSAIDs have potential cardiovascular and gastrointestinal bleeding risks, which makes the safe use of NSAIDs a hot issue in the global medical community.
  • extensive epidemiological evidence indicates that the abuse of phenacetin is the cause of renal pelvis cancer However, the genetic alterations of the resulting tumors remain largely unknown.
  • An efficient and sensitive detection method for phenacetin is an urgent problem to be solved.
  • Current detection methods include instrumental assays and immunoassays.
  • Instrumental analysis methods such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry, etc., due to the cumbersome sample pretreatment, many interfering substances and limitations of instrument working conditions, and high technical requirements for operators, so the instrument method Not suitable for field testing.
  • immunoassay methods Compared with instrument detection methods, immunoassay methods have the characteristics of low cost, high throughput, high sensitivity, and low requirements for technicians, so they are suitable for rapid screening of large numbers of samples. Therefore, the immunoassay method is of great significance for the detection of phenacetin.
  • Enzyme-linked immunosorbent assay is a low-cost, rapid and portable immunological detection method. It does not require high sample purity and is easy to operate. It is suitable for on-site rapid detection of a large number of samples. The establishment of an efficient immunological detection method and the screening of highly specific monoclonal antibodies are important prerequisites.
  • Paracetamol also known as Tylenol, Panadol, Bufferin, and Paracetamol, is a metabolite of phenacetin in the body, which regulates central prostaglandin synthase by inhibiting hypothalamus body temperature. , reduce the synthesis and release of prostaglandins PGE1, bradykinin and histamine. But so far, there is no relevant report on the use of enzyme-linked immunosorbent assay for the detection of phenacetin.
  • the present invention provides a phenacetin monoclonal antibody hybridoma cell line and its preparation method and application. High detection sensitivity can be used to establish an immunological detection method for phenacetin.
  • One object of the present invention is to provide a phenacetin monoclonal antibody hybridoma cell line AD, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the preservation name is monoclonal cell line AD, and the preservation number is CGMCC No.19681, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preserved on April 23, 2020.
  • Another object of the present invention is to provide a method for preparing a phenacetin monoclonal antibody hybridoma cell line AD, wherein the phenacetin complete antigen-immunized mice are cultured through cell fusion and HAT selective medium to establish The phenacetin monoclonal antibody hybridoma cell line AD; the phenacetin complete antigen-immunized mice are obtained by immunizing mice with Phe-BA-BSA immunogen.
  • the complete phenacetin antigen is obtained by coupling the Phe-BA hapten with a carrier protein, wherein the carrier protein includes bovine serum albumin (BSA), ovalbumin (OVA), and the corresponding phenacetin.
  • BSA bovine serum albumin
  • OVA ovalbumin
  • the butyl immunogen includes Phe-BA-BSA and Phe-BA-OVA; the Phe-BA hapten is obtained by hydrolysis of the product of the reaction between acetaminophen and ethyl 4-bromobutyrate. Ethyl 4-bromobutyrate is a commonly used intermediate in pesticide/pharmaceutical synthesis.
  • Paracetamol has a similar structure to phenacetin and has a high reactive site, and can obtain a hapten with a high reaction rate with ethyl 4-bromobutyrate.
  • the structure of the Phe-BA hapten is shown in the following formula:
  • Phe-BA is obtained after the hydrolysis of the reaction product of acetaminophen and ethyl bromobutyrate, and the Phe-BA is used as a hapten.
  • the carrier proteins including BSA and OVA are coupled to prepare the immunogen and the coating agent.
  • the complete antigen and the unconjugated small molecule hapten are separated by dialysis.
  • the isolated complete antigen is the phenacetin immunogen and the coating.
  • the quilt wherein the immunogen Phe-BA-BSA is used for the preparation of hybridoma cell lines by immunization in mice, and the original Phe-BA-OVA is coated for subsequent detection by the kit;
  • mice Using Freund's adjuvant, incomplete Freund's adjuvant, and Phe-BA-BSA immunogen, albino laboratory mice, namely BALB/c mice, were subcutaneously injected into the back of the neck to conduct mouse immunization. immunized, phenacetin-immunized mice were obtained.
  • For the first immunization 100 ⁇ g/only, an equal volume of mixed emulsion of Freund's complete adjuvant and phenacetin immunogen was used as injection, and for several booster immunizations (50 ⁇ g/only) incomplete Freund's adjuvant and phenacetin were used. An equal volume of mixed emulsion of immunogens was used as an injection.
  • the interval between the first immunization and the first booster immunization was one month, and the interval between multiple booster immunizations was 21 days; the last time was with 50 ⁇ L of Phe diluted with normal saline to 0.5mg/mL -BA-BSA immunogen (25 ⁇ g/only, without adjuvant) sprint immunization; serum titer and inhibition rate were detected by indirect competitive enzyme-linked immunosorbent assay (icELISA); several booster immunizations were 4 to 6 times, preferably 5 Second-rate;
  • the preparation method of the Phe-BA hapten is, in molar ratio, dissolve 1equ. of paracetamol in dimethyl sulfoxide, add 4-6equ.K 2 CO 3 , and add 2-5equ .4-ethyl bromobutyrate, heated to reflux, stirred at about 100°C (80-120°C), after the reaction was complete, lowered to room temperature and concentrated; added NaOH solution, heated to 70-80°C, stirred the reaction, lowered to room temperature, and used The pH was adjusted to 4-5 by HCl, and the hapten Phe-BA was obtained as a white solid after extraction, purification and drying.
  • Another object of the present invention is to provide a phenacetin monoclonal antibody, which is obtained from the phenacetin monoclonal antibody hybridoma cell line with the deposit number CGMCC19681---monoclonal cell line AD secreted.
  • Another object of the present invention is to provide the application of the above-mentioned phenacetin monoclonal antibody for preparing a detection body for the analysis and detection of phenacetin residues in food safety testing, the detection body includes reagents, a detection plate ,Reagent test kit.
  • the detection subject also includes a phenacetin coating original, which is obtained when the Phe-BA hapten is coupled with a carrier protein to prepare a phenacetin immunogen.
  • the carrier protein includes bovine serum albumin BSA and ovalbumin OVA
  • the corresponding phenacetin coating material includes Phe-BA-BSA and Phe-BA-OVA.
  • the phenacetin immunogen is Phe-BA-BSA
  • the corresponding phenacetin coating is preferably Phe-BA-OVA, which has better specificity and detection sensitivity.
  • the monoclonal antibody secreted by the cell line AD provided by the present invention has good specificity and detection sensitivity to phenacetin (IC 50 value is 3.0 ⁇ g/L), and can realize the detection of phenacetin.
  • IC 50 value is 3.0 ⁇ g/L
  • the detection of tin residues provides raw materials for the immunodetection of phenacetin residues in food, and has practical application value.
  • Fig. 1 is the subtype identification of AD monoclonal antibody
  • Fig. 2 is the AD affinity determination of monoclonal antibody
  • Figure 3 is a standard curve for the inhibition of phenacetin by AD monoclonal antibodies.
  • a monoclonal antibody hybridoma with higher sensitivity to phenacetin is finally obtained by immunizing mice with phenacetin complete antigen, culturing in HAT selective medium through cell fusion, and screening cell supernatant through icELISA. cell line.
  • the synthetic route is as follows:
  • hapten Phe-BA prepared in the above step 1 is coupled with bovine serum albumin (BSA) to obtain the complete antigen Phe-BA-BSA.
  • BSA bovine serum albumin
  • the preparation method of the complete antigen Phe-BA-BSA is as follows:
  • Dialysis take a 10cm dialysis bag, boil it in boiling water for 5 minutes, rinse it with deionized water at 60°C for 3 minutes, and store it in deionized water at 4°C for later use; mix the mixture containing Phe-BA-BSA in step (a).
  • the solution was placed in a dialysis bag, and 0.01mol/L PBS was used as the dialysate, dialyzed at 4°C for 3 days, and the dialysate was replaced three times a day to separate the complete antigen from the unconjugated hapten and other small molecular substances, that is, the complete antigen, including The immunogen and the coating original, wherein the immunogen Phe-BA-BSA is used for the next step of mouse immunization, and the coating original is used for the subsequent step 7 when it is used for detection.
  • the hapten Phe-BA is coupled with ovalbumin (OVA) to obtain the complete antigen Phe-BA-OVA, including the immunogen and the coating.
  • OVA ovalbumin
  • the immunogen is used for the next step of mouse immunization, and the coating is Used for subsequent step 7 when applied to detection.
  • the subsequent immunization and detection-related steps are specifically described by using the immunogen Phe-BA-BSA and the coating original Phe-BA-OVA as examples.
  • mice Phe-BA-BSA immunogen was mixed and emulsified with an equal volume of Freund's adjuvant to obtain an injection, and BALB/c mice were subcutaneously injected through the back of the neck.
  • the first immunization (100 ⁇ g/only), an equal volume of mixed emulsion of Freund's complete adjuvant and Phe-BA-BSA immunogen was used as an injection. - Equal volume mixed emulsion of BSA immunogen as injection. The interval between the first immunization and the first booster immunization is one month, and the interval between multiple booster immunizations is 21 days.
  • the last sprint immunization was performed with Phe-BA-BSA immunogen (25 ⁇ g/only, without adjuvant) diluted to a concentration of 0.5 mg/mL with normal saline; serum titers and Inhibition rate.
  • spleen cell suspension was obtained through a 200-mesh cell screen, collected, centrifuged (1200 rpm, 8 min), washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells were diluted to a certain volume, counted, and used for later use;
  • SP2/0 tumor cells Collection of murine myeloma SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were treated with RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO 2 incubator. Before fusion, the number of SP2/0 tumor cells was required to reach 1-4 ⁇ 10 7 to ensure that the SP2/0 tumor cells were in the logarithmic growth phase before fusion. During fusion, tumor cells were collected, suspended in RPMI-1640 basal medium, and counted;
  • FBS fetal bovine serum
  • the fusion process is 7 minutes.
  • 1 mL of PEG 1500 was added dropwise to the cells from slow to fast; for the 2nd minute, let stand.
  • 1mL of RPMI-1640 medium was added dropwise within 1min;
  • 2mL of RPMI-1640 medium was added dropwise within 1min;
  • 1mL of RPMI-1640 medium was added dropwise every 10s .
  • incubate at 37°C for 5min.
  • Centrifuge 800rpm, 8min
  • discard the supernatant resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 ⁇ HAT
  • add 200 ⁇ L/well to a 96-well cell plate, and place at 37 °C, 5% CO 2 incubator.
  • the fused cells were screened with 2% 50 ⁇ HAT in RPMI-1640, and the medium was half-changed on the 5th day with 20% fetal bovine serum and 1% 100 ⁇ HT in RPMI-1640. The transition medium was completely changed, and the cell supernatant was taken for screening on the 7th day.
  • the screening is divided into two steps: in the first step, icELISA is used to screen out the positive cell wells. Cell wells with better inhibition of phenacetin standard were selected, subcloned by limiting dilution method, and detected by the same method. Repeat three times to obtain monoclonal hybridoma cell line AD.
  • mouse monoclonal antibody subtype identification kit to identify the immunoglobulin subtype of the phenacetin monoclonal antibody purified from ascites, its subtype is IgG2b, and it was detected by the mouse monoclonal antibody subtype identification kit.
  • the chain type is the kappa type, as shown in Figure 1.
  • the affinity of the phenacetin monoclonal antibody determined by indirect ELISA was 5.36 ⁇ 10 8 L/mol, as shown in FIG. 2 .
  • the sensitivity to phenacetin was detected by icELISA.
  • the phenacetin monoclonal antibody prepared by the hybridoma cell line AD through in vivo ascites was applied to the addition and recovery test of phenacetin, and the specific steps were as follows:
  • Blocking Add 200 ⁇ L/well blocking solution, and react at 37°C for 2h. Dry after washing.
  • Stop and measure Add 50 ⁇ L of stop solution to each well to stop the reaction, and then measure the OD 450 value of each well with a microplate reader.
  • IC 50 of phenacetin monoclonal antibody determined by icELISA was 3.0 ⁇ g/L, indicating that it has high sensitivity to phenacetin and can be used for phenacetin immunoassay detection.
  • Carbonate buffer solution (CBS): Weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, respectively dissolve in a small amount of double-distilled water, mix, add double-distilled water to about 800mL and mix well, adjust pH to 9.6, add Make up to 1000mL with double-distilled water and store at 4°C for later use;
  • PBS Phosphate buffered saline
  • PBST PBS containing 0.05% Tween-20
  • TMB color developing solution Solution A: Na 2 HPO 4 ⁇ 12H 2 O 18.43 g, citric acid 9.33 g, and purified water to 1000 mL; Solution B: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A and B liquids are mixed at a ratio of 1:5, which is the TMB color developing liquid.

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Abstract

本发明公开了一种抗非那西丁单克隆抗体杂交瘤细胞株AD及其制备方法与应用，涉及食品安全免疫检测技术领域，该单克隆抗体杂交瘤细胞株的保藏名为单克隆细胞株AD，保藏编号为CGMCC19681。以非那西丁代谢物对乙酰氨基酚与4-溴丁酸乙酯的反应产物经水解后得到的Phe-BA为半抗原，将半抗原与载体蛋白偶联制备免疫原Phe-BA-BSA。对小鼠进行免疫原Phe-BA-BSA注射免疫后，再通过PEG方法与骨髓瘤细胞融合，经过间接竞争酶联免疫法的筛选和五次亚克隆，得到杂交瘤细胞株。此细胞株分泌的单克隆抗体可制成非那西丁检测盒，对非那西丁具有较好亲和力和检测灵敏度，可用于食品中非那西丁残留量的免疫检测。

Description

一种抗非那西丁单克隆抗体杂交瘤细胞株AD及其制备方法与应用 技术领域
本发明属于食品安全免疫检测技术领域，具体涉及一种抗非那西丁单克隆抗体杂交瘤细胞株AD及其制备方法与应用。
背景技术
非那西丁(phenacetin，Phe)属于非淄体抗炎药((Nonsteroidal Antiinflammatory Drugs，NSAIDs)，具有抗炎、止痛、退热等作用，在临床上广泛用于多种发热和各种疼痛症状的缓解。目前NSAIDs是全球使用最多的药物种类之一。全世界大约每天有3000万人在使用。随着NSAIDs使用的增多，这类药物的安全使用问题也越来越受到关注，美国食品药品监督管理局(FDA)认为NSAIDs存在潜在的心血管和消化道出血风险，这使NSAIDs的安全用药成为目前全球医药界的热点问题。尽管广泛的流行病学证据表明滥用非那西汀是肾盂癌的危险因素，但由此引起的肿瘤的基因改变在很大程度上仍不清楚。近年来，因为它有危险的副作用在一些国家已经退出了市场。目前，仍然存在非那西丁用于小动物的解热镇痛。因此，建立快速有效的检测非那西丁含量的方法具有重要意义及市场价值。
针对非那西丁的高效灵敏的检测方法是亟待解决的问题。目前的检测方法包括仪器分析法和免疫分析法。仪器分析法如高效液相色谱法、高效液相色谱串联质谱法等，由于对样品前处理繁琐，干扰物较多及仪器工作条件的限制，同时对操作人员的技术要求较高，因此仪器方法并不适用于现场检测。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。因此免疫检方法对非那西丁的检测具有重要意义。
酶联免疫法(ELISA)是一种低成本、快速且便携的免疫学检测方法，检测时对样本的纯度要求不高而且操作简便，适用于大量样本的现场快速检测出结果。建立高效的免疫学检测方法，筛选高特异性的单克隆抗体是其重要前提。
对乙酰氨基酚(Paracetamol)，别称泰诺林(Tylenol)、必理通(Panadol)、百服宁(Bufferin)、扑热息痛，是非那西丁的体内代谢产物，通过抑制下丘脑体温调节中枢前列腺素合成酶，减少前列腺素PGE1、缓激肽和组胺等的合成和释放。但是至今尚未有利用酶联免疫法用于非那西丁检测的相关报道。
发明内容
发明目的：为了克服现有技术中存在的不足，本发明提供一种非那西丁单克隆抗体杂交瘤细胞株及其制备方法与应用，由该细胞株分泌的抗体针对非那西丁具有较高的检测灵敏度，可以用来建立针对非那西丁的免疫学检测方法。
技术方案：为实现上述目的，本发明采用的技术方案为：
本发明的一个目的是，提供一株非那西丁单克隆抗体杂交瘤细胞株AD，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏名为单克隆细胞株AD，保藏编号为 CGMCC No.19681，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期2020年04月23日。
本发明的另一个目的是，提供一种非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，非那西丁完全抗原免疫小鼠通过细胞融合、HAT选择性培养基培养后，建立得到所述非那西丁单克隆抗体杂交瘤细胞株AD；所述非那西丁完全抗原免疫小鼠是由小鼠经Phe-BA-BSA免疫原免疫所得。
进一步的，所述非那西丁完全抗原由Phe-BA半抗原与载体蛋白偶联获得，其中，所述载体蛋白包括牛血清蛋白(BSA)、卵清蛋白(OVA)，对应的非那西丁免疫原包括Phe-BA-BSA、Phe-BA-OVA；所述Phe-BA半抗原由对乙酰氨基酚与4-溴丁酸乙酯反应后的产物经水解后得到。4-溴丁酸乙酯是一种常用的农药/医药合成中间体。对乙酰氨基酚与非那西丁结构类似，且具有活性较高的反应位点，能够与4-溴丁酸乙酯以较高的反应率获得半抗原。所述Phe-BA半抗原的结构如下式所示：
Figure PCTCN2021129509-appb-000001
以Phe-BA-BSA免疫原为例，基本步骤为：
(1)免疫原的制备与鉴定：乙酰氨基酚与溴丁酸乙酯的反应产物水解后得到Phe-BA，以所述Phe-BA为半抗原，采用碳二亚胺法将半抗原分别与包括BSA、OVA在内的载体蛋白偶联，制备得到免疫原与包被原，通过透析分离完全抗原和未偶联的小分子半抗原，分离出的完全抗原就是非那西丁免疫原和包被原，其中，免疫原Phe-BA-BSA用于小鼠免疫制备杂交瘤细胞株，包被原Phe-BA-OVA用于后续的试剂盒检测时使用；
(2)小鼠的免疫：使用弗氏佐剂、弗氏不完全佐剂、Phe-BA-BSA免疫原，通过颈背部皮下注射白变种实验室老鼠即BALB/c小鼠，进行小鼠的免疫，获得非那西丁免疫小鼠。首次免疫(100μg/只)用弗氏完全佐剂与非那西丁免疫原的等体积混合乳化液作为注射剂，数次加强免疫用(50μg/只)弗氏不完全佐剂与非那西丁免疫原的等体积混合乳化液作为注射剂，首次免疫与第一次加强免疫之间间隔一个月，多次加强免疫之间间隔21天；最后一次用50μL经生理盐水稀释至0.5mg/mL的Phe-BA-BSA免疫原(25μg/只，不含佐剂)冲刺免疫；通过间接竞争酶联免疫法(icELISA)检测血清效价和抑制率；数次加强免疫为4～6次，优选为5次；
(3)细胞融合与细胞株建立：通过聚乙二醇(PEG4000)法将非那西丁免疫小鼠的脾细胞和骨髓瘤细胞融合，通过HAT培养基培养，利用间接ELISA检测阳性细胞孔，并进一步利用icELISA法测定阳性细胞孔的抑制效果，通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆，最终筛选获得杂交瘤细胞株AD；
(4)杂交瘤细胞株性质的鉴定：抗体亚型的鉴定采用小鼠单克隆抗体免疫胶体金亚型试剂盒方法，IC 50值、交叉反应率和亲和力常数采用ELISA法测定。
进一步的，所述Phe-BA半抗原的制备方法为，按摩尔比计，将1equ.对乙酰氨基酚溶于二甲亚砜，加入4-6equ.K 2CO 3，搅拌下加入2-5equ.4-溴丁酸乙酯，加热回流，100℃左右 (80～120℃)搅拌，待反应完全，降至室温，浓缩；加入NaOH溶液加热至70～80℃搅拌反应，降至室温，用HCl调节PH至4～5，经萃取、纯化、干燥，得白色固体即为半抗原Phe-BA。
本发明的另一个目的是，提供一种非那西丁单克隆抗体，它由所述保藏编号为CGMCC19681的非那西丁单克隆抗体杂交瘤细胞株------单克隆细胞株AD分泌产生。
本发明的另一个目的是，提供上述非那西丁单克隆抗体的应用，用于制备食品安全检测中非那西丁残留量的分析检测用的检测主体，所述检测主体包括试剂、检测板、试剂盒。
进一步的，所述检测主体中还包括有非那西丁包被原，所述非那西丁包被原是在Phe-BA半抗原与载体蛋白偶联制备非那西丁免疫原时获得的，其中，所述载体蛋白包括牛血清蛋白BSA、卵清蛋白OVA，对应的非那西丁包被原包括Phe-BA-BSA、Phe-BA-OVA。非那西丁免疫原为Phe-BA-BSA时，对应的非那西丁包被原优选为Phe-BA-OVA，具有更好的特异性和检测灵敏度。
本发明的有益效果：本发明提供的细胞株AD分泌的单克隆抗体，对非那西丁具有较好的特异性和检测灵敏度(IC 50值为3.0μg/L)，可实现对非那西丁残留量的检测，为食品中非那西丁残留的免疫检测提供了原料，具有实际应用价值。
附图说明
图1为AD单克隆抗体的亚型鉴定；
图2为单克隆抗体AD亲和力测定；
图3为AD单克隆抗体对非那西丁的抑制标准曲线。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。
实施例
根据下述实施例，可以更好的理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
本发明通过将非那西丁完全抗原免疫小鼠，通过细胞融合，HAT选择性培养基培养，通过icELISA筛选细胞上清，最终得到了对非那西丁有较高灵敏度的单克隆抗体杂交瘤细胞株。
1.半抗原的合成
(1)半抗原Phe-BA的合成：
合成路线如下：
Figure PCTCN2021129509-appb-000002
将0.20g对乙酰氨基酚溶于2mL二甲亚砜，加入1.0g K 2CO 3，搅拌0.5h。然后，搅拌下加入0.77g 4-溴丁酸乙酯，加热回流，110℃搅拌反应24h，TLC监测。待反应完全，停止加热，直至反应液温度降至室温，浓缩；加入2mL 1mol/L NaOH溶液加热至80℃搅拌反应1h；待反应液温度降至室温，用1mol/L HCl调节PH至4，用乙酸乙酯萃取，无水Na 2SO 4干燥，浓缩；经硅胶柱纯化，浓缩，干燥，得到白色固体，即半抗原Phe-BA。
2.完全抗原的制备：上述步骤1制备的半抗原Phe-BA与牛血清蛋白(BSA)偶联得到完全抗原Phe-BA-BSA。
所述完全抗原Phe-BA-BSA制备方法如下：
a、称取步骤(1)制得的半抗原Phe-BA 2.85mg溶于200μL无水N，N-二甲基甲酰胺，依次加入1.6mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI)，N-羟基琥珀酰亚胺(NHS)1.0mg，室温搅拌反应0.5h，称为A液；称取牛血清蛋白BSA 10mg，加入2mL硼酸缓冲溶液，称为B液；搅拌下，缓慢将A液逐滴加至B液中，室温反应2h，得到含有Phe-BA-BSA的混合液；
b、透析：取10cm的透析袋，于沸水中煮沸5min，再用60℃的去离子水冲洗3min，保存在4℃去离子水中备用；将步骤(a)中含有Phe-BA-BSA的混合液置于透析袋，用0.01mol/L PBS作为透析液，4℃透析3d，每天更换三次透析液，以分离完全抗原与未偶联的半抗原及其他小分子物质，即得完全抗原，包括免疫原和包被原，其中免疫原Phe-BA-BSA用作下一步的小鼠免疫，包被原用于后续步骤7应用于检测时使用。
同理，采用半抗原Phe-BA与卵清蛋白(OVA)偶联得到完全抗原Phe-BA-OVA，包括免疫原和包被原，其中免疫原用作下一步的小鼠免疫，包被原用于后续步骤7应用于检测时使用。
本实施例中，后续的免疫及检测相关步骤具体采用免疫原Phe-BA-BSA、包被原Phe-BA-OVA作为示例说明。
3.小鼠的免疫：Phe-BA-BSA免疫原与等体积弗氏佐剂混合乳化后，获得注射剂，通过颈背部皮下注射BALB/c小鼠。
首次免疫(100μg/只)用弗氏完全佐剂与Phe-BA-BSA免疫原的等体积混合乳化液作为注射剂，5次加强免疫(50μg/只)用弗氏不完全佐剂与Phe-BA-BSA免疫原的等体积混合乳化液作为注射剂。首次免疫与第一次加强免疫之间间隔一个月，多次加强免疫之间间隔 21天。最后一次用经生理盐水稀释至浓度为0.5mg/mL的Phe-BA-BSA免疫原(25μg/只，不含佐剂)冲刺免疫；通过间接竞争酶联免疫法(icELISA)检测血清效价和抑制率。
4.细胞融合
在冲刺免疫三天后，按照常规PEG法进行细胞融合，具体步骤如下：
a、摘眼球取血，颈椎脱臼法处死非那西丁免疫小鼠后，立即放入75％酒精中消毒，浸泡5min左右，无菌操作取出该小鼠的脾脏，用注射器的胶头适度研磨并通过200目细胞筛网得到脾细胞悬液，收集，离心(1200rpm，8min)，用RPMI-1640培养基洗涤脾细胞三次，最后一次离心后，将脾细胞稀释到一定体积，计数，备用；
b、收集鼠源骨髓瘤SP2/0细胞：融合前7-10天，将SP2/0瘤细胞用含10％FBS(胎牛血清)RPMI-1640培养基在5％CO 2培养箱中。融合前要求SP2/0瘤细胞数量达到1～4×10 7，保证融合前SP2/0瘤细胞处于对数生长期。融合时，收集瘤细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
c、融合过程7min。第1min，将1mL的PEG 1500由慢到快滴加到细胞中；第2min，静置。第3min和第4min，在1min内滴加1mL RPMI-1640培养基；第5min和第6min，在1min内滴加2mL RPMI-1640培养基；第7min，每10s滴加1mL的RPMI-1640培养基。然后37℃温浴5min。离心(800rpm，8min)，弃上清，重悬入含20％胎牛血清，2％的50×HAT的RPMI-1640筛选培养液中，按照200μL/孔加到96孔细胞板，置于37℃，5％CO 2培养箱中培养。
5.细胞筛选与细胞株建立
在细胞融合的第3天对融合细胞进行2％的50×HAT的RPMI-1640筛选培养液半换液，第5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7天取细胞上清进行筛选。筛选分两步：第一步先用icELISA筛选出阳性细胞孔，第二步选用对非那西丁为标准品，用icELISA对阳性细胞进行抑制效果测定。选择对非那西丁标准品均有较好抑制的细胞孔，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，获得单克隆杂交瘤细胞株AD。
6.非那西丁单克隆抗体的制备与鉴定
取8-10周龄非那西丁免疫BALB/c小鼠，每只小鼠腹腔注射无菌石蜡油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。最终得到纯化后的非那西丁单克隆抗体，-20℃贮存。
使用小鼠单抗亚型鉴定试剂盒对腹水纯化获得的非那西丁单克隆抗体进行免疫球蛋白亚型鉴定，其亚型为IgG2b型，经小鼠单抗亚型鉴定试剂盒检测得轻链类型为κ型，如图1所示。
采用间接ELISA测定非那西丁单克隆抗体的亲和力为5.36×10 8L/mol，如图2所示。采用icELISA检测对非那西丁的灵敏度，如图3所示，根据标准方程y＝-0.3479x+0.6733(R 2＝0.9863)计算出IC 50为3.0μg/L。
以上结果说明所制备的非那西丁单克隆抗体具有较高的亲和力和灵敏度，可用于非那西丁免疫分析检测和亲和柱的制备。
7.抗体应用
将杂交瘤细胞株AD通过体内腹水制备的非那西丁单克隆抗体应用于非那西丁的添加回收试验，具体步骤如下：
7.1包被：将前述步骤2中得到的包被原Phe-BA-OVA用0.05mol L -1pH 9.6碳酸盐缓冲液稀释至0.1μg/mL，100μL/孔，37℃反应2h。
7.2洗涤：将板内溶液倾去，并用洗涤液洗涤3次，每次3min.
7.3封闭：加入200μL/孔封闭液，37℃反应2h。洗涤后烘干备用。
7.4加样：在阳性对照孔加入100μL PBS；检测孔加入100μL浓度为0.3～50μg/L非那西丁标准溶液。然后，将非那西丁单克隆抗体用0.01mol L -1PBS稀释至0.10.1μg/mL，并加入到各稀释度的包被孔中，100μL/孔，37℃反应30min；充分洗涤后，加入1∶3000稀释的HRP-羊抗鼠IgG，100μL/孔，37℃反应30min。
7.5显色：将酶标板取出，充分洗涤后，每孔加入100μL的TMB显色液，37℃避光反应15min.
7.6终止和测定：每孔加入50μL终止液以终止反应，然后用酶标仪测定各孔的OD 450值。
用icELISA测定非那西丁单克隆抗体的IC 50为3.0μg/L，说明对非那西丁有很高的灵敏度，可用于非那西丁免疫分析检测。
以上步骤中，各个溶液的配置为：
碳酸盐缓冲液(CBS)：称取Na 2CO 3 1.59g，NaHCO 3 2.93g，分别溶于少量双蒸水后混合，加双蒸水至约800mL混匀，调pH值至9.6，加双蒸水定容至1000mL，4℃贮存备用；
磷酸盐缓冲液(PBS)：8.0g NaCl，0.2g KCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，溶于800mL纯水中，用NaOH或HCl调pH到7.2～7.4，定容至1000mL；
PBST：含0.05％吐温-20的PBS；
TMB显色液：A液：Na 2HPO 4·12H 2O 18.43g，柠檬酸9.33g，纯水定容至1000mL；B液：60mg TMB溶于100mL乙二醇中。A、B液按1∶5混合即为TMB显色液，现用现混。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种非那西丁单克隆抗体杂交瘤细胞株AD，其特征在于：已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏名为单克隆细胞株AD，保藏编号为CGMCC19681。
  2. 根据权利要求1所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：非那西丁免疫小鼠通过细胞融合、HAT选择性培养基培养后，建立得到所述非那西丁单克隆抗体杂交瘤细胞株AD；所述非那西丁免疫小鼠是由小鼠经非那西丁免疫原免疫所得。
  3. 根据权利要求2所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：所述非那西丁免疫原由Phe-BA半抗原与载体蛋白偶联获得，其中，所述载体蛋白包括牛血清蛋白BSA、卵清蛋白OVA，对应的非那西丁免疫原包括Phe-BA-BSA、Phe-BA-OVA；所述Phe-BA半抗原由对乙酰氨基酚与4-溴丁酸乙酯反应后的产物经水解后得到，结构如下式所示：
    Figure PCTCN2021129509-appb-100001
  4. 根据权利要求3所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：所述Phe-BA半抗原的制备方法为，按摩尔比计，将1equ.对乙酰氨基酚溶于二甲亚砜，加入4-6equ.K 2CO 3，搅拌下加入2-5equ.4-溴丁酸乙酯，加热回流，80～120℃搅拌，待反应完全，降至室温，浓缩；加入NaOH溶液加热至70～80℃搅拌反应，降至室温，用HCl调节PH至4～5，经萃取、纯化、干燥，得白色固体即为半抗原Phe-BA。
  5. 根据权利要求2所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：所述非那西丁免疫小鼠的免疫方法为，通过颈背部皮下注射BALB/c小鼠，先用弗氏完全佐剂与非那西丁免疫原的等体积混合乳化液作为注射剂进行首次免疫，再用弗氏不完全佐剂与非那西丁免疫原的等体积混合乳化液作为注射剂进行数次加强免疫，最后一次用50μL经生理盐水稀释至0.5mg/mL的非那西丁免疫原进行冲刺免疫。
  6. 根据权利要求5所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：首次免疫的注射量为100μg/只，每次加强免疫的注射量为50μg/只，冲刺免疫的注射量为25μg/只；
    首次免疫与第一次加强免疫之间间隔一个月，数次加强免疫之间间隔21天，所述加强免疫进行4～6次，优选5次。
  7. 根据权利要求2所述的非那西丁单克隆抗体杂交瘤细胞株AD的制备方法，其特征在于：所述细胞融合的具体方法为：通过聚乙二醇法将非那西丁完全抗原免疫小鼠的脾细胞和骨髓瘤细胞融合，通过HAT培养基培养，利用间接ELISA检测阳性细胞孔，并进一步利用icELISA法测定阳性细胞孔的抑制效果，通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆，最终筛选获得杂交瘤细胞株AD。
  8. 一种非那西丁单克隆抗体，其特征在于：由权利要求1所述的或由权利要求2-7任一所述方法制备的单克隆杂交瘤细胞株AD分泌产生。
  9. 根据权利要求8所述的非那西丁单克隆抗体的应用，其特征在于：用于制备食品安全检测中非那西丁残留量的分析检测用的检测主体，所述检测主体包括试剂、检测板、试剂 盒。
  10. 根据权利要求9所述的非那西丁单克隆抗体的应用，其特征在于：所述检测主体中还包括有非那西丁包被原，所述非那西丁包被原是在Phe-BA半抗原与载体蛋白偶联制备非那西丁免疫原时获得的，其中，所述载体蛋白包括牛血清蛋白BSA、卵清蛋白OVA，对应的非那西丁包被原包括Phe-BA-BSA、Phe-BA-OVA。
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