WO2021189522A1 - 基于荧光传感器的单克隆抗体分泌细胞筛选方法 - Google Patents

基于荧光传感器的单克隆抗体分泌细胞筛选方法 Download PDF

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WO2021189522A1
WO2021189522A1 PCT/CN2020/082962 CN2020082962W WO2021189522A1 WO 2021189522 A1 WO2021189522 A1 WO 2021189522A1 CN 2020082962 W CN2020082962 W CN 2020082962W WO 2021189522 A1 WO2021189522 A1 WO 2021189522A1
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monoclonal antibody
fluorescence sensor
fag
ngo
fluorescence
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PCT/CN2020/082962
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French (fr)
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黄峙
邢曦雯
段丽青
凌钦婕
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暨南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

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  • the invention belongs to the field of biotechnology and immunoassay, and particularly relates to a method for screening monoclonal antibody secreting cells based on a fluorescence sensor.
  • Nanographene oxide is obtained by decomposing nanographene material after strong oxidation. Its surface contains oxygen-containing groups such as hydroxyl, alkoxy, and carboxyl groups. The existence of these active groups makes NGO in The dispersion stability in the aqueous solution is significantly improved, and it has hydrophilicity and biocompatibility, and can interact with a variety of biomolecules. In addition, the most interesting thing is the efficient energy absorption and storage functions of nano-graphene oxide.
  • Fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET) is a mature and sensitive molecular interaction monitoring technology, and it is a powerful tool for detecting changes in the nanometer and nanometer distances of biological macromolecules in living bodies.
  • FRET Fluorescence resonance energy transfer
  • donor fluorescent group
  • absorption spectrum of another group acceptor
  • Nano-graphene oxide has an efficient transfer and absorption effect on energy.
  • NGO and fluorescent molecules are close to each other to a nanometer distance, they will hijack the fluorescent energy and cause rapid fluorescence extraction.
  • NGO and fluorescent molecules dissociate, then Without the effect of fluorescence energy transfer, fluorescence can be reproduced. Therefore, NGO can be used as a sensitive fluorescent sensor material for designing and constructing a detection technology system based on FRET.
  • mAb monoclonal antibodies
  • the basic steps include: 1) immunizing animals with specific antigens; 2) fusion of animal B lymphocytes with myeloma cells; 3) screening of hybridoma cells that secrete monoclonal antibodies; 4 ) Cloning of antibody-secreting cells; 5) Characterization of mAb.
  • cell screening and cloning are the key steps for preparing ideal mAbs. That is to say, the traditional monoclonal antibody cell screening method is relatively cumbersome. There are two screening processes.
  • the first is to screen out hybridoma cells with selective medium; the second is to further screen out the antigen-specific antibodies that we need.
  • Hybridoma cells a large number of hybridoma cell samples need to be confirmed in a short time whether they are antibody-secreting cells, and high-affinity antibody-secreting cells must be identified to determine the choice of hybridoma cells. Therefore, it is necessary to establish a convenient, rapid and inexpensive method for screening high-affinity monoclonal antibody secreting cells. At present, methods such as enzyme-linked immunoassay (ELISA), single cell microanalysis and fluorescent cell sorting (FACS) have been established one after another, but they are still not satisfactory. The high-affinity monoclonal antibody secreting cell screening method has always been high-affinity The technical bottleneck of mAb.
  • ELISA enzyme-linked immunoassay
  • FACS fluorescent cell sorting
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for screening monoclonal antibody secreting cells based on a fluorescent sensor.
  • a method for screening monoclonal antibody secreting cells based on a fluorescent sensor including the following steps:
  • NGO nanographene oxide
  • FAg fluorescently labeled antigen
  • step (2) Mix the monoclonal antibody hybridoma cells with the NGO-FAg fluorescence sensor of step (2), and sort the strong fluorescent cells, that is, screen out the high-affinity monoclonal antibody secreting cells.
  • the fluorescently labeled antigen described in step (1) is obtained by labeling fluorescein isothiocyanate (FITC) on the N-terminus of the polypeptide of the immune antigen (Ag).
  • FITC fluorescein isothiocyanate
  • the immune antigen is preferably a specific epitope peptide of selenoprotein K, and its amino acid sequence is: MVYISNGQVLDSRNQSPWRV, and the molecular weight is 2852Da.
  • the excitation light wavelength of the fluorescein isothiocyanate is 488 nm, and the emission light wavelength is 520 nm.
  • the NGO described in step (1) has a sheet diameter of 50-200 nm and a specific surface area of 50-100 m 2 /g; preferably, the sheet diameter is 86 nm and a specific surface area of 90 m 2 /g.
  • the concentration of NGO in the system described in step (1) is 1.0-1.5 ⁇ g/mL; preferably 1.25 ⁇ g/mL.
  • the concentration of FAg in the system in step (1) is 80-120 nM; preferably 100 nM.
  • the ratio of the abundance of FAg to the surface area of the NGO in step (1) is 1 ⁇ 10 7 to 8 ⁇ 10 7 / ⁇ m 2 ; preferably 2.3 ⁇ 10 7 / ⁇ m 2 .
  • the ultrafiltration centrifuge tube used in the ultrafiltration described in step (1) is a 4 mL millipore ultrafiltration centrifuge tube with a molecular retention of 5-10KD.
  • the centrifugation is 4000 rpm centrifugation for 10 minutes at 4°C.
  • the monoclonal antibody hybridoma cells described in step (2) are preferably SelK monoclonal antibody hybridoma cells obtained by animal immunization with a specific epitope peptide of selenoprotein K.
  • the mixing time of the monoclonal antibody hybridoma cells and the NGO-FAg fluorescence sensor in step (2) is 5-20 min; preferably 10 min.
  • the monoclonal antibody hybridoma cells described in step (2) are resuscitated, cultured, and expanded to 10 6 cells, and then mixed with the NGO-FAg fluorescence sensor.
  • the sorting of strong fluorescent cells in step (2) is to use a sorting flow cytometer for cell sorting.
  • the present invention has the following advantages and effects:
  • the present invention uses nanographene oxide and fluorescently labeled antigen peptides to prepare a FRET-based fluorescent sensor, which is added to hybridoma cells, and the high-affinity mAb secreted by the cells snatches Ag, resulting in steric hindrance or dissociation between FAg and NGO , So that the fluorescence reappears ( Figure 1). Therefore, the high-affinity antibody secreting cells can be distinguished and screened according to the strength of the cell fluorescence signal.
  • the method of the present invention can screen high-affinity monoclonal antibody secreting cells in only one step, can simplify the complicated process of traditional screening methods, and has the advantages of convenience and speed, saving manpower, material resources and financial resources.
  • Fig. 1 is a schematic diagram of the principle of the present invention.
  • Figure 2 is a graph showing the detection results of the fluorescence quenching effect of FAg with different concentrations of NGO in Example 1.
  • A is the fluorescence spectrum;
  • B is the fluorescence intensity curve;
  • C is the fluorescence recovery result graph after adding SelK mAb;
  • D is the NGO The kinetic curve of the fluorescence quenching of FAg.
  • Figure 3 is a graph showing the effect of pH and temperature on NGO-FAg in Example 1.
  • A is the graph of the effect of pH on NGO-FAg;
  • B is the graph of the effect of temperature on NGO-FAg.
  • Figure 4 is a graph showing the detection results of fluorescence recovery intensity of NGO-FAg under different titers of SelK mAb in Example 2.
  • A is the fluorescence spectrum;
  • B is the linear fitting graph of relative fluorescence intensity to SelK mAb titers ;
  • C is the standard curve diagram for detecting SelK mAb.
  • Fig. 5 is a graph showing the detection results of changes in fluorescence intensity of the copolymer of SelK mAb and NGO-FAg with different titers added in Example 2.
  • Fig. 6 is a graph showing the specific selectivity analysis results of SelK mAb in Example 2.
  • Figure 7 is the confocal laser fluorescence image of Example 3; where A is HAW-1 cells + FAg; B is RPMI-8226 cells + FAg; C is HAW-1 cells + FAg + NGO; D is RPMI-8226 cells +FAg+NGO; E is HAW-1 cell+Free-Ag+Fag.
  • FIG. 8 is a flow cytometry chart of Example 3.
  • Figure 9 is a flow cytometric sorting and antibody titer affinity test result chart of Example 4; wherein, A is a flow cytometric sorting chart, a and b are strong fluorescent cells, c and d are weak fluorescent cells, e and f is non-fluorescent cells; B is the results of indirect ELISA method to detect antibody titer at different times; C is the results of Western Blot detection.
  • the specific epitope peptide of selenoprotein K (Ag, amino acid sequence: MVYISNGQVLDSRNQSPWRV, molecular weight: 2852Da) was customized by chemical synthesis (Shanghai Xinhao Biotechnology Co., Ltd.). In the process of solid-phase peptide synthesis, fluorescein isothiocyanate (FITC, excitation light wavelength: 488nm; emission light wavelength: 520nm) was labeled on the N-terminal of the peptide, and 5 mg of fluorescently labeled antigen (FAg) was obtained as a custom.
  • FITC fluorescein isothiocyanate
  • Fg fluorescently labeled antigen
  • step (1) Add a series of NGO (purchased from Nanjing Xianfeng Nano Material Technology Co., Ltd.) into the FAg obtained in step (1).
  • the chip diameter is in the range of 50 to 200 nm, the average chip diameter is 86 nm, and the thickness is 0.8 to 1.6 nm.
  • the single layer ratio is 96%, the purity is 98%, the specific surface area (BET): 90 square meters/g.
  • the concentration of NGO in the system is respectively 0 ⁇ g/mL, 0.25 ⁇ g/mL, 0.5 ⁇ g/mL, 0.75 ⁇ g/mL, 1.0 ⁇ g/mL, 1.25 ⁇ g/mL, 1.5 ⁇ g/mL, 2.0 ⁇ g/mL
  • the concentration of FAg in the system is 100nM, mix 1 minute, ultrafiltration to remove free FAg (using a 4mL millipore ultrafiltration centrifuge tube with a retention molecule of 5-10KD, repeated centrifugation with 1 ⁇ PBS buffer (4000rpm at 4°C for 10min) 3 times, each 3mL, collect and Adjust the final volume to 1 mL).
  • the inventors observed the optimal concentration of NGO by adding a selenoprotein K monoclonal antibody (SelK mAb) with a titer of 160 U/mL to the NGO-FAg system constructed under different concentrations of NGO.
  • the fluorescence intensity was measured with a fluorescence spectrophotometer and the fluorescence spectrum was scanned ( Figure 2).
  • the results showed that the fluorescence intensity decreased with the increase of the NGO concentration.
  • the NGO concentration reached 1.25 ⁇ g/mL, the NGO concentration was increased, and the fluorescence intensity was basically not There is a great reduction; when the NGO concentration is 1.25 ⁇ g/mL, the fluorescence recovery is best after adding the SelK mAb to be detected. Therefore, 1.25 ⁇ g/mL is the optimal concentration of NGO, and the fluorescence quenching efficiency of 100nM FAg for 1 minute is more than 90%.
  • Hybridoma cells (specified epitope peptides of selenoprotein K were obtained by animal immunization, purchased from Epigenomics, a German molecular diagnostic company, and we numbered them as HAW-1) cultured at 37°C for one week, and collected and combined hybridoma cells were cultured on Serum (secrete SelK antibody), refrigerate at 4°C (no more than 2 weeks).
  • SSAS half-saturated ammonium sulfate aqueous solution
  • KCl (1mM), CaCl 2 (0.5mM), glycine (0.2mM), glutamine (1mM), bovine serum albumin BSA (2.5 ⁇ g/mL), Glucose glucose (10mM), fetal bovine serum FBS (2.5 ⁇ g/mL), ⁇ -actin mAb (2.5 ⁇ g/mL), etc. are added as interfering substances and SelK mAb with a titer of 20U/mL
  • the NGO-FAg prepared in Example 1.
  • HAW-1 SelK monoclonal antibody hybridoma cells
  • RPMI-8226 cells human multiple myeloma cells
  • the hybridoma cell SelK and RPMI-8226 medium were cultured in DMEM medium 1640 to 10 5 cells / well and incubated for 12 hours at 37 °C. There are two groups of each type of cell, and each group has two wells.
  • Another group is set up: add FITC-free Ag (Free-Ag, 1mM) for 1h before incubating HAW-1 cells with FAg, wash with 1 ⁇ PBS buffer and incubate with FAg at 37°C for 2 hours in the dark.
  • FITC-free Ag Free-Ag, 1mM
  • the inventor also verified the above conclusions through flow cytometry experiments. Set up four groups: the first group is a negative control group; the second group is adding Free-FAg and FAg; the third group is only adding FAg as a positive control group; the fourth group is adding FAg and NGO, each group has two wells.
  • the four SelK monoclonal antibody hybridoma (HAW-1) cells at 106 cells / well were incubated for 12 hours at 37 °C, then the second, third and fourth groups of cells and FAg (28 ⁇ M) in the dark at 37 [deg.] C After incubating for 2 hours, the fourth group of cells were treated with NGO (80 ⁇ g/mL) at room temperature for 5 minutes; the second group of cells was treated with FITC-free Ag (Free-Ag, 1mM) in advance for 1h, 1 ⁇ PBS buffer ( 0.01M, pH 7.4) Wash twice.
  • NGO 80 ⁇ g/mL
  • FITC-free Ag Free-Ag, 1mM
  • the ELISA experiment process is as follows:
  • Coating liquid pH 9.6: Na 2 CO 3 (1.59 g), NaHCO 3 (2.93 g), add double distilled water to 1L.
  • a solution sodium acetate 13.6g, citric acid 1.6g, 30% (v/v) H 2 O 2 0.3mL, add double distilled water to 500mL.
  • Solution B EDTA-Na 2 0.2g, citric acid 0.95g, glycerol 50mL, TMB (tetramethylbenzidine) (dissolved in DMSO was 10mg / mL before adding) 0.2g, double distilled water was added to 500mL.
  • Color development add 100 ⁇ L of TMB color development solution (first add 50 ⁇ L of A solution, then 50 ⁇ L of B solution), protect from light at room temperature for 10 minutes.
  • Termination Add 50 ⁇ L of termination solution to each well to terminate the color reaction.
  • Glue preparation Prepare 12% separation gel and 5% concentrated gel in advance, soak in electrophoresis solution and store in a refrigerator at 4°C for later use.
  • Electrophoresis After loading the sample, connect to the power supply correctly, and perform electrophoresis: Concentrate gel 65V 40min, Separate gel 120V 1.5h.
  • Transfer membrane Prepare the transfer membrane solution in advance and put it in a refrigerator at 4°C for later use, and activate the PVDF membrane with methanol. According to a certain order, correctly stack the sponge, filter paper, film, and PVDF membrane together to avoid air bubbles between them. Put the installed transfer splint in the transfer tank in the correct device, add 1L of transfer fluid, connect the power supply, and transfer the film (constant voltage 120V 1.5h or constant current 100mA 12h). Due to the heat generated during the film transfer process, the film transfer tank needs to be placed in an ice bath environment.
  • Sealing pre-prepared with a sealing solution (5% skimmed milk powder), soak the converted PVDF membrane with the sealing solution, and put it on a horizontal shaker (85-100 rpm) and seal at room temperature for 1 hour.
  • a sealing solution 5% skimmed milk powder

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Abstract

一种基于荧光传感器的单克隆抗体分泌细胞筛选方法，属于生物技术、免疫分析和纳米材料应用领域。该方法构建了荧光标记多肽与纳米氧化石墨烯基于FRET的荧光传感器，将其加入杂交瘤细胞中，实现了高亲和力单克隆抗体分泌细胞的筛选。此方法只需要一步就可以筛选出高亲和力单克隆抗体分泌细胞，简化了传统筛选方法的复杂过程，具有方便快速、节约人力、物力、财力的优点。

Description

基于荧光传感器的单克隆抗体分泌细胞筛选方法 技术领域
本发明属于生物技术和免疫分析领域，特别涉及一种基于荧光传感器的单克隆抗体分泌细胞筛选方法。
背景技术
2004年，纳米石墨烯片层被首次分离出来，并由于其独特的结构和优异的性能成为国内外学者的研究热点。纳米氧化石墨烯(Nanographene oxide，NGO)是纳米石墨烯材料经过强氧化后再分解而得，其表面含有含氧基团如羟基、烷氧基、羧基等，这些活性基团的存在使NGO在水溶液中的分散稳定性显著提高，并使其具有亲水性和生物相容性，可与多种生物分子相互作用。除此之外，最让人关注的是纳米氧化石墨烯高效的能量吸收和贮藏功能。
荧光共振能量转移(fluorescence resonance energy transfer,FRET)是一项成熟灵敏的分子相互作用监测技术，是检测活体中生物大分子纳米级距离和纳米级距离变化的有力工具。FRET是相互接近的两个荧光分子间产生能量转移的一种现象，当一个荧光基团(供体Donor)的发射光谱与另一个基团(受体Acceptor)的吸收光谱有一定重叠，在两者合适的距离下就可以观察到荧光能量由供体向受体转移的现象。纳米氧化石墨烯对能量具有高效的转移和吸收作用，当NGO与荧光分子相互靠近到纳米级间距时，将劫取荧光能量，发生快速荧光萃灭效应，而当NGO与荧光分子解离，则不发生荧光能量转移效应，荧光即可重现。因此，NGO可作为一种灵敏的荧光传感器材料，用于设计和构建基于FRET的检测技术体系。
目前，单克隆抗体(monoclonal antibody，mAb)在生物制药、免疫分析和生物制备中已得到广泛应用。杂交瘤技术仍然是生产制备mAb的主流方法，其基本步骤包括：1)用特定的抗原免疫动物；2)动物B淋巴细胞与骨髓瘤细胞融合；3)分泌单克隆抗体杂交瘤细胞筛选；4)抗体分泌细胞克隆化；5)mAb特性鉴定。其中，细胞筛选与克隆化是制备理想mAb的关键环节。即传统的单克隆抗体细胞筛选方法过程比较繁琐，有两次筛选过程，第一次是使用选择培养基筛选出杂交瘤细胞；第二次就是进一步筛选出能产生我们需要的抗原特异 性抗体的杂交瘤细胞。但是大量杂交瘤细胞样品需要在短时间内确认是否为抗体分泌细胞，并且要鉴定高亲和力抗体分泌细胞，以决定杂交瘤细胞的取舍。因此，必须建立方便快速和廉价的高亲和力单克隆抗体分泌细胞的筛选方法。当前，酶联免疫分析(ELISA)、单细胞显微分析和荧光细胞分选(FACS)等方法相继建立，但仍未能尽如人意，高亲和力单克隆抗体分泌细胞筛选方法一直是获得高亲和力mAb的技术瓶颈。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种基于荧光传感器的单克隆抗体分泌细胞筛选方法。
本发明的目的通过下述技术方案实现：一种基于荧光传感器的单克隆抗体分泌细胞筛选方法，包括如下步骤：
(1)将纳米氧化石墨烯(NGO)与荧光标记抗原(FAg)混合，超滤，得到NGO-FAg荧光传感器；
(2)将单克隆抗体杂交瘤细胞与步骤(2)的NGO-FAg荧光传感器混合，分选强荧光细胞，即筛选出高亲和力单克隆抗体分泌细胞。
步骤(1)中所述的荧光标记抗原是通过将异硫氰酸荧光素(FITC)标记在免疫抗原(Ag)的多肽N端得到。
所述的免疫抗原优选为硒蛋白K特定的抗原表位肽，其氨基酸序列为：MVYISNGQVLDSRNQSPWRV，分子量为2852Da。
所述的异硫氰酸荧光素的激发光波长为488nm，发射光波长为520nm。
步骤(1)中所述的NGO的片径大小为50～200nm，比表面积为50～100m 2/g；优选片径大小为86nm，比表面积为90m 2/g。
步骤(1)中所述的NGO在体系中的浓度为1.0～1.5μg/mL；优选为1.25μg/mL。
步骤(1)中所述的FAg在体系中的浓度为80～120nM；优选为100nM。
步骤(1)中所述的FAg的丰度与NGO的表面积之比为1×10 7～8×10 7/μm 2；优选为2.3×10 7/μm 2。
步骤(1)中所述的混合是采用浓度为0.01M、pH＝7.4的1×PBS缓冲液混合1～5min。
步骤(1)中所述的超滤采用的超滤离心管为4mL、截留分子5～10KD的millipore超滤离心管。
步骤(1)中所述的超滤的步骤为：采用1×PBS缓冲液(0.01M、pH＝7.4)离心3次，每次3mL，收集滤液并调整终体积至1mL。
所述的离心为4℃条件下4000rpm离心10min。
步骤(2)中所述的单克隆抗体杂交瘤细胞优选为以硒蛋白K特定的抗原表位肽进行动物免疫得到的SelK单克隆抗体杂交瘤细胞。
步骤(2)中所述的单克隆抗体杂交瘤细胞与NGO-FAg荧光传感器混合的时间为5～20min；优选为10min。
步骤(2)中所述的单克隆抗体杂交瘤细胞先经过复苏培养，扩增达到10 6个后，再与NGO-FAg荧光传感器混合。
步骤(2)中所述的分选强荧光细胞为采用分选型流式细胞仪进行细胞分选。
本发明相对于现有技术，具有如下的优点及效果：
1.本发明利用纳米氧化石墨烯与荧光标记抗原多肽制备基于FRET的荧光传感器，将其加入杂交瘤细胞中，通过细胞分泌的高亲和力mAb抢夺Ag，导致FAg与NGO产生空间位阻或解离，从而荧光重现(图1)。因此，根据细胞荧光信号强弱可区分和筛选高亲和力抗体分泌细胞。本发明的方法只需要一步就可以筛选出高亲和力单克隆抗体分泌细胞，可以简化传统筛选方法的复杂过程，具有方便快速、节约人力、物力、财力的优点。
附图说明
图1是本发明的原理示意图。
图2是实施例1中，不同浓度NGO对FAg荧光猝灭效应检测结果图；其中，A为荧光光谱图；B为荧光强度曲线图；C为加入SelK mAb后荧光恢复结果图；D为NGO对FAg荧光猝灭动力学曲线图。
图3是实施例1中，pH和温度对NGO-FAg的影响结果图；其中，A为pH对NGO-FAg的影响；B为温度对NGO-FAg的影响结果图。
图4是实施例2中，NGO-FAg在不同效价SelK mAb条件下的荧光恢复强度检测结果图；其中，A为荧光光谱图；B为相对荧光强度对SelK mAb效价的线性拟合图；C为检测SelK mAb标准曲线图。
图5是实施例2中，加入不同效价SelK mAb与NGO-FAg共聚物的荧光强度变化检测结果图。
图6是实施例2的SelK mAb特异选择性分析结果图。
图7是实施例3的共聚焦激光荧光图；其中，A为HAW-1细胞+FAg；B为RPMI-8226细胞+FAg；C为HAW-1细胞+FAg+NGO；D为RPMI-8226细胞+FAg+NGO；E为HAW-1细胞+Free-Ag+Fag。
图8是实施例3的流式细胞图。
图9是实施例4的流式细胞分选及抗体效价亲和力检测结果图；其中，A为流式细胞分选图，a和b为强荧光细胞，c和d为弱荧光细胞，e和f为无荧光细胞；B为间接ELISA方法检测不同时间的抗体效价结果图；C为Western Blot检测结果图。
具体实施方式
下面将结合实施方式和附图对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本发明，而不应视为限制本发明的范围。未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1构建荧光共振能量转移传感器
(1)定制荧光标记抗原
采用化学合成方法定制(上海昕浩生物科技有限公司)硒蛋白K特定的抗原表位肽(Ag，氨基酸序列为：MVYISNGQVLDSRNQSPWRV，分子量：2852Da)。固相多肽合成过程中，将异硫氰酸荧光素(FITC，激发光波长：488nm；发射光波长：520nm)标记在多肽N端，定制获得荧光标记抗原(FAg)5mg。
(2)构建NGO-FAg体系
在步骤(1)得到的FAg中分别加入一系列用量的NGO(购买于南京先丰纳米材料科技有限公司，片径大小在50～200nm范围内，平均片径为86nm，厚度为0.8～1.6nm，单层比为96％，纯度为98％，比表面积(BET)：90平方米/克。在纯水中浓度为0.5mg/mL时呈棕黄色悬液)，NGO在体系中的浓度分别为0μg/mL、0.25μg/mL、0.5μg/mL、0.75μg/mL、1.0μg/mL、1.25μg/mL、1.5μg/mL、2.0μg/mL，FAg在体系中的浓度为100nM，混合1分钟，超滤除去游离FAg(采用4mL、截留分子5～10KD的millipore超滤离心管，反复用1×PBS缓冲液离心(4℃条件下4000rpm离心10min)3次，每次3mL，收集并调整终体积到1mL)。
此外，发明人通过在向上述不同浓度NGO下构建的NGO-FAg体系中加入效价为160U/mL的硒蛋白K单克隆抗体(SelK mAb)来观察NGO的最适浓度。 用荧光分光光度计检测荧光强度以及扫描荧光光谱(图2)，结果显示，荧光强度随着NGO浓度的增大而降低，当NGO浓度达到1.25μg/mL以后再增加NGO浓度，荧光强度基本不再有很大的降低；在NGO浓度为1.25μg/mL时，加入待检测的SelK mAb后荧光恢复的最好。因此，1.25μg/mL为NGO最适浓度，混合1分钟对100nM的FAg荧光猝灭效率达到90％以上。
SelK抗体制备过程：
1)将杂交瘤细胞(硒蛋白K特定的抗原表位肽进行动物免疫得到，购自德国分子诊断公司Epigenomics，我们将其编号为HAW-1)37℃培养一周，收集合并杂交瘤细胞培养上清液(分泌SelK抗体)，4℃冷藏(不超过2周)。
2)取50mL上清液于离心管中，逐滴加等量的饱和硫酸铵水溶液(SAS)，滴加过程中采用磁力搅拌器(200～300rmp)回旋轻轻混合，避免产生气泡。
3)将离心管静置1小时或在4℃下过夜，偶尔旋转轻轻混合，让抗体充分沉淀。
4)将上述混悬液在4℃、离心力为4000×g条件下离心1小时，小心除去上清液，以免扰动沉淀物。
5)用半饱和硫酸铵水溶液(SSAS)反复洗涤沉淀物5次，每次向含有沉淀物的离心管中沿管壁滴加SSAS 20mL，轻轻旋转1分钟使沉淀物重悬，然后在4℃、离心力为4000×g条件下离心5分钟。(注：在洁净玻璃容器中，将20mL饱和硫酸铵与等量超纯水混合即得半饱和硫酸铵水溶液，简称SSAS)。
6)将试管倒置于吸水纸上充分沥干，然后加入1～3mL 1×BBS或1×PBS(0.01M、pH 7.4)溶液，用滴管轻轻上下吹吸使沉淀物完全溶解，避免产生气泡。
7)将分子截留量为8kD的透析袋预先浸泡在1×BBS或1×PBS溶液中，一端用透析袋夹封闭，检查无渗漏，将上述溶解的抗体溶液转移到透析袋后，将另一端封闭，然后置于装有50倍体积容量1×PBS溶液的容器中，搅拌透析过夜，每隔2～4小时更换1×PBS溶液1次，共5次，直至透析袋内的溶液变为澄清液体。
8)将彻底透析后的抗体溶液收集，分装、冷冻干燥，-20℃保存。
(3)为了探究温度和pH对NGO-FAg体系的影响，利用荧光分光光度计分别检测FAg、NGO-FAg在不同pH(3.0～11.1)和不同温度(4～37℃)的荧光强度(图3)。结果显示，pH和温度对FAg的荧光强度具有很大的影响，而NGO-FAg荧光强度较稳定，在温度为4～37℃和PH为5.5～8.0条件下基本不受影响。
实施例2 NGO-FAg对SelK mAb反应性及特异性分析
(一)以实施例1制备的NGO-FAg为体系，首先在1×PBS缓冲液(0.01M、pH 7.4)中将FAg和NGO提前预混合，体系中FAg和NGO的浓度分别为100nM和1.25μg/mL，然后向NGO-FAg混合体系分别加入不同效价(0.25U/mL、0.5U/mL、0.75U/mL、1.0U/mL、1.25U/mL、2.5U/mL、5.0U/mL、10U/mL、20U/mL、40U/mL、80U/mL、160U/mL)的高亲和力SelK抗体(实施例1中的制备方法制备得到)，室温孵育10min，最后用荧光分光光度计检测荧光强度(图4)。此外，为了探究体系中加入SelK mAb后荧光恢复强度与时间的关系，还利用荧光分光光度计分别记录了不同时间点(0min、2min、4min、6min、8min、10min)的荧光强度(图5)。
结果显示，荧光强度随着SelK抗体效价的增大和时间的延长逐渐增强，在10min内基本达到稳定状态。并且，在0～20U/mL效价的范围内具有良好的线性关系(R 2＝0.9937)。
(二)与此同时，发明人还做了干扰实验和特异性分析，分别将KCl(1mM)、CaCl 2(0.5mM)、甘氨酸(0.2mM)、谷氨酰胺(1mM)、牛血清白蛋白BSA(2.5μg/mL)、Glucose葡萄糖(10mM)、胎牛血清FBS(2.5μg/mL)、β-actin mAb(2.5μg/mL)等作为干扰物质和效价为20U/mL的SelK mAb加入实施例1制备的NGO-FAg。结果发现(图6)，加入干扰物质荧光强度基本没有什么变化，而加入效价为20U/mL的SelK mAb后荧光有明显的增强，这表明构建的NGO-FAg对SelK mAb的反应具有很好的特异性和抗干扰特性。
实施例3 NGO-FAg对SelK单克隆抗体分泌细胞的选择性及细胞成像
(一)SelK单克隆抗体杂交瘤细胞(HAW-1)分泌SelK mAb，而人多发性骨髓瘤细胞(RPMI-8226细胞)不分泌SelK mAb，作为阴性对照(SelK单克隆抗体杂交瘤细胞从德国分子诊断公司Epigenomics购买；RPMI-8226细胞购置于ATCC，cat#：ml-cs-0071)。将SelK单克隆抗体杂交瘤细胞和RPMI-8226分别在DMEM培养基和1640培养基中培养，37℃下以10 5个细胞/孔温育12小时。每种细胞有两组，每组有两个孔。用1×PBS缓冲液(0.01M，pH 7.4)洗涤两次后，将它们与FAg(28μM)于37℃在暗处温育2小时，之后用NGO(80μg/mL)将两组细胞中的一组处理5分钟，然后用1×PBS缓冲液(0.01M、pH 7.4)洗涤所有细胞5次，每次3min，再用4％(m/v)多聚甲醛固定20分钟。用1×PBS缓冲液(0.01M、pH 7.4)洗涤两次后用30％(v/v)甘油封片，并用共聚焦激 光荧光显微镜成像(图7)。同时，设置另外一组：将HAW-1细胞与FAg温育前加入不含FITC的Ag(Free-Ag，1mM)处理1h，1×PBS缓冲液洗涤后与FAg于暗处37℃温育2小时。
结果显示，与FAg温育2小时后的SelK单克隆抗体杂交瘤细胞表面有明显的荧光，加NGO处理之后仍有明显的荧光，且随时间延长荧光增强；而与FAg温育2小时后的RPMI-8226细胞表面有较弱的荧光，加入NGO后基本没有荧光。结果证明，FAg能与SelK mAb特异性结合，使得NGO无法与SelK mAb争夺FAg，通过细胞表面荧光强度能简单快速灵敏分辨出单克隆抗体分泌细胞，而FAg与RPMI-8226细胞表面的其他物质非特异性结合，NGO的加入与其争夺FAg，从而引起荧光一定程度的猝灭。
(二)除此之外，发明人还通过流式细胞实验同样验证了上述的结论。设置四组：第一组是阴性对照组；第二组是加入Free-FAg和FAg；第三组是只加入FAg作为阳性对照组；第四组是加入FAg和NGO，每组两个孔。将四组SelK单克隆抗体杂交瘤(HAW-1)细胞以10 6个细胞/孔在37℃下孵育12小时，然后将第二、三、四组细胞与FAg(28μM)于暗处37℃温育2小时，之后于室温下用NGO(80μg/mL)处理第四组细胞5min；第二组细胞提前加入不含FITC的Ag(Free-Ag，1mM)处理1h，1×PBS缓冲液(0.01M、pH 7.4)洗涤两次。用1×PBS缓冲液(0.01M、pH 7.4)洗涤四组细胞5次，每次3min，用1×PBS缓冲液(0.01M、pH 7.4)制成细胞悬液，最后用流式细胞仪检测细胞的平均荧光强度(图8)。
结果显示，第二组用不含FITC的Free-Ag提前处理的SelK单克隆抗体杂交瘤细胞荧光强度明显比第三组只加入FAg的阳性对照组细胞弱，可能的原因是不含FITC的Free-Ag提前占据细胞表面的SelK mAb位点，从而导致FAg无法与SelK mAb特异性结合。而第四组加入NGO处理后的细胞与阳性对照组细胞的荧光强度基本一样，这与前面的结论一致。
实施例4利用NGO-FAg筛选高亲和力SelK单克隆抗体分泌细胞
将SelK单克隆抗体杂交瘤(HAW-1)细胞和人多发性骨髓瘤细胞(RPMI-8226)混合培养(混合细胞在37℃下孵育12小时，1×PBS缓冲液(0.01M、pH 7.4)洗涤两次后，将混合培养后的细胞加入NGO-FAg，于37℃在暗处温育10min，然后，用1×PBS缓冲液(0.01M、pH 7.4)洗涤5次，每次3min，用1×PBS制成细胞悬液。用分选型流式细胞仪分选出强荧光、弱荧光、阴性三 种细胞群，之后将三种细胞群在37℃、CO 2浓度为5％(v/v)的细胞培养箱中培养一周，然后通过间接ELISA和Western Blot检测三种细胞群上清液中SelK mAb的分泌水平和亲和力(图9)。
ELISA实验过程如下：
一)试剂配方：
(1)包被液(pH为9.6)：Na 2CO 3(1.59g)、NaHCO 3(2.93g)，加双蒸水至1L。
(2)PBST：1×PBS(2L)+0.05％(v/v)吐温20(1mL)。
(3)终止液(10％(v/v)H 2SO 4)：取54.25mL 98％(v/v)的浓硫酸逐滴加入445.75mL双蒸水中，边加边搅拌，混匀冷却后备用。
(4)TMB显色液(避光保存)
A液：醋酸钠13.6g、柠檬酸1.6g、30％(v/v)的H 2O 2 0.3mL，加双蒸水至500mL。
B液：EDTA-Na 2 0.2g、柠檬酸0.95g、甘油50mL、TMB(四甲基联苯胺)(用DMSO溶解为10mg/mL后再加入)0.2g，加双蒸水至500mL。
二)实验步骤
(1)包被：用包被液把实施例1制备的抗原表位肽(Free-Ag)稀释至2μg/mL。将稀释后的抗原多肽加到96孔酶联板，每孔100μL，4℃过夜。取出后用PBST洗涤三次，每次3min。
(2)封闭：将5％脱脂奶粉加到96孔酶联板中，每孔加入200μL，然后将酶联板放入37℃恒温培养箱封闭90min。取出后用PBST洗涤三次，每次3min。
(3)孵育一抗：将上述三种不同细胞上清液分别加入96孔酶联板中，每孔加100μL，然后将酶联板放入37℃恒温培养箱孵育60min。取出后用PBST洗涤三次，每次3min。
(4)孵育二抗：参照抗体说明书用PBST稀释HRP标记的兔二抗(CST，cat#7074S)(1:1000)，然后将稀释后的二抗加入96孔酶联反应板中，每孔加100μL。37℃恒温培养箱孵育40-45min。取出后用PBST洗涤五次，每次3min。
(5)显色：加入100μL TMB显色液(先加A液50μL，后加50μL B液)，室温避光10min。
(6)终止：每孔分别加入50μL终止液终止显色反应。
(7)利用酶标仪读取450nm吸光度值(OD 450)，通过绘制的标准曲线计算分析数据。
Western Blot实验过程：
(1)制胶：预先配置好12％的分离胶和5％的浓缩胶，用电泳液浸泡放4℃冰箱保存备用。
(2)上样：将备用的预制胶正确的装置在电泳槽内，加入电泳液。从冰箱中取出蛋白样品解冻及混匀，用移液枪小心的吸取适量的蛋白样品按一定的顺序依次加到泳道中。
(3)电泳：上样后，正确接上电源，进行电泳：浓缩胶65V 40min，分离胶120V 1.5h。
(4)转膜：预先配置好转膜液放4℃冰箱备用，用甲醇活化PVDF膜。按一定的顺序，将海绵、滤纸、胶片、PVDF膜正确的叠合在一起，避免之间存在气泡。将安装好的转膜夹板在正确的装置在转膜槽内，加入1L的转膜液，接上电源，进行转膜(恒压120V 1.5h或恒流100mA 12h)。因转膜过程产热，转膜槽需放入冰浴环境中。
(5)封闭：预先配置好封闭液(5％脱脂奶粉)，将转好的PVDF膜用封闭液浸泡，放到水平摇床上(85～100rmp)室温封闭1h。
(6)孵育一抗：分别加入上述三种细胞上清液(一抗)置于37℃摇床过夜孵育。
(7)孵育二抗：孵育一抗后用TBST洗涤三次，每次5min。用5％(v/v)BSA参照抗体说明书按比例稀释HRP标记的二抗(CST，cat#7074S)，室温孵育1h。
(8)化学发光：将配置好的ECL发光液滴加到PVDF膜上，再使用数码凝胶图像处理系统曝光。
结果显示，通过分选型流式细胞仪分选出三群细胞：强荧光细胞(图9A的a-b)；弱荧光细胞(图9A的c-d)和无荧光细胞(图9A的e-f)。进一步检测结果证明(图9的B和C)，强荧光细胞上清液中SelK mAb的分泌水平最高，亲和力最强，弱荧光细胞上清中SelK mAb分泌水平和亲和力则相对较弱，无荧光细胞则检测不到SelKmAb的分泌。因此，可以通过上述方法筛选出高亲和力SelK单克隆抗体分泌杂交瘤细胞。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020082962-appb-000001

Claims (10)

  1. 一种基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于包括如下步骤：
    (1)将纳米氧化石墨烯与荧光标记抗原混合，超滤，得到NGO-FAg荧光传感器；
    (2)将单克隆抗体杂交瘤细胞与步骤(2)的NGO-FAg荧光传感器混合，分选强荧光细胞，即筛选出高亲和力单克隆抗体分泌细胞。
  2. 根据权利要求1所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    步骤(1)中所述的荧光标记抗原是通过将异硫氰酸荧光素标记在免疫抗原的多肽N端得到；
    步骤(1)中所述的纳米氧化石墨烯的片径大小为50～200nm，比表面积为50～100m 2/g。
  3. 根据权利要求2所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    所述的免疫抗原为硒蛋白K特定的抗原表位肽，其氨基酸序列为：MVYISNGQVLDSRNQSPWRV，分子量为2852Da；
    所述的异硫氰酸荧光素的激发光波长为488nm，发射光波长为520nm；
    步骤(1)中所述的纳米氧化石墨烯的片径大小为86nm，比表面积为90m 2/g；
    步骤(2)中所述的单克隆抗体杂交瘤细胞为以硒蛋白K特定的抗原表位肽进行动物免疫得到的SelK单克隆抗体杂交瘤细胞。
  4. 根据权利要求1所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    步骤(1)中所述的纳米氧化石墨烯在体系中的浓度为1.0～1.5μg/mL；
    步骤(1)中所述的荧光标记抗原在体系中的浓度为80～120nM；
    步骤(1)中所述的荧光标记抗原的丰度与荧光标记抗原的表面积之比为1×10 7～8×10 7/μm 2。
  5. 根据权利要求4所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    步骤(1)中所述的荧光标记抗原在体系中的浓度为1.25μg/mL；
    步骤(1)中所述的荧光标记抗原在体系中的浓度为90nM；
    步骤(1)中所述的荧光标记抗原的丰度与荧光标记抗原的表面积之比为2.3×10 7/μm 2。
  6. 根据权利要求1所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    步骤(1)中所述的超滤采用的超滤离心管为4mL、截留分子5～10KD的millipore超滤离心管；
    步骤(1)中所述的混合是采用浓度为0.01M、pH＝7.4的1×PBS缓冲液混合1～5min。
  7. 根据权利要求6所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：步骤(1)中所述的超滤的步骤为：采用1×PBS缓冲液(0.01M、pH＝7.4)离心3次，每次3mL，收集滤液并调整终体积至1mL。
  8. 根据权利要求7所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：所述的离心为4℃条件下4000rpm离心10min。
  9. 根据权利要求1所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：步骤(2)中所述的单克隆抗体杂交瘤细胞与NGO-FAg荧光传感器混合的时间为5～20min。
  10. 根据权利要求1所述的基于荧光传感器的单克隆抗体分泌细胞筛选方法，其特征在于：
    步骤(2)中所述的单克隆抗体杂交瘤细胞先经过复苏培养，扩增达到10 6个后，再向其中加入NGO-FAg荧光传感器；
    步骤(2)中所述的分选强荧光细胞为采用分选型流式细胞仪进行细胞分选。
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