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A rapid method for antigen-specific hybridoma clones isolation Xiuqing Li, Hongfen Bian, Siming Yu, Wei Xiao, Jianying Shen, Caifeng Lan, Kenan Zhou, Caihong Huang, Lei Wang, Dan Du, Yuehe Lin, and Yong Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04595 • Publication Date (Web): 30 Dec 2017 Downloaded from http://pubs.acs.org on December 31, 2017

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Analytical Chemistry

A rapid method for antigen-specific hybridoma clones isolation Xiuqing Li†,§, Hongfen Bian †,§, Siming Yu‡, Wei Xiao†, Jianying Shen⊥, Caifeng Lan†, Kenan Zhou†, Caihong Huang†, Lei Wang†, Dan Du# , Yuehe Lin# and Yong Tang†, ∇,* †

Department of Bioengineering, Guangdong Province Engineering Research Center for antibody drug and immunoassay, Jinan University, Guangzhou 510632, PR China. ‡ Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China ⊥ Guangzhou Highway Engineering Company, Guangzhou 510075, PR China. # School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164, United States ∇Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China ABSTRACT: Using enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigenspecific hybridoma cells are extremely time-consuming and labor-intensive. The work presented here features a simple and rapid cell surface-fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular OleylPEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently-labeled anti-mouse IgG-Fc antibody. After adding a methylcellulose semi-solid medium, positive clones are easily picked using a pipette. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against of both malachite green (MG) and porcine epidemic diarrhea virus (PEDV) were selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating over two-fold more hybridoma clones in less than 25% corresponding processing time. In brief, the CSFIA-based method is high efficient and low cost with simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in monoclonal antibody preparation. al cells including microfluidic devices17-22, cell-based microarrays23-25, enzyme-linked immunospot (ELISPOT)26 and hemolytic plaque assays27,28. However, they do not allow for both the high-throughput analysis of positive product and/or the recovery of living cells for clone expansion. The GenetixClonePix FL is a powerful way for selecting antigen-specific clones from hybridoma fusions29. This method uses fluorescent antigens to stain positive clones in semisolid medium and is based on the net-like complex of the anti-

Due to their high specificity and affinity toward target antigens, monoclonal antibodies (mAbs) are indispensable tools15 as well as therapeutic agents6,7 for biomedical basic research and clinical applications. MAbs are secreted from hybridoma cells8, which are created by fusing normal antibody producing splenic B-cells with either immortal myeloma cells or another immortalized cell line9. At present, hybridoma technology is the most commonly used method for generating monoclonal antibodies. In conventional assays, positive hybridoma cells are selected by using ELISA to detect the liquid supernatant of microwells that contain fusion cells. If there are two or more cell clones in a microwell, the positive cells in this microwell must be subcloned to further identify the real positive one. In the subcloning process, a given number candidate cells are delivered into certain number separate microwells to form single cell clones using a limiting dilution method. After the adequate confluence grown from a single cell clone is obtained, the clone’s culture supernatant is retested using ELISA. At least three subcloning cycles are needed to demonstrate and establish a positive monoclonal cell strain. This procedure typically takes more than four weeks for any given hybridoma. In the whole process, strong positive clones are likely missed because of cell clone competition and limiting dilution. Due to its time-consuming and low-efficient nature, there is an urgent need for quicker methods for isolating a desired hybridoma cell. Two alternatives for sorting cells into microtiter plate are available using semi-solid medium10-12and fluorescence-activated cell sorting (FACS)13-16. Both methods have improved the efficiency of screening by serial dilution, but the resulting cultures usually require additional testing by ELISA or equivalent methods to verify secretion and specificity. Other methods have also been studied for the analysis of individu-

Figure 1. Schematic diagram of cell surface-fluorescence immunosorbent assay (CSFIA) based on the specific binding of antigens anchored to the cell surface and antibodies secreted from the cells.

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Figure 2. Schematic diagram of our rapid CSFIA-based method for positive hybridoma clones.

ciently applied to the isolation of specific positive hybridoma cell clones.

gen-antibody. As this method only allows the fluorescent dye to attach around the cell clones and not directly on the cell surface, the resulting clone fluorescence often has low quality. Moreover, it is often hard to distinguish the positive and negative cell clones when they connect together. With these limitations in mind, an intelligent and innovative cell surface-fluorescence immunosorbent assay (CSFIA)30,31has recently been reported for the rapid stain of hybridoma cells as long as they secret antibodies. However, it cannot differentially detect antigen-specific monoclonal antibodies. Given this, a second generation CSFIA was developed in this work, featuring rapid selection of antigen-specific hybridoma cells. In this approach, antigens are first linked with Oleyl-PEG4000-NHS32-34and added to candidate cell clones deriving from the fusion of myeloma cells and splenic cells. After incubation, the antigens linked with Oleyl-PEG4000NHS are subsequently anchored onto the hybridoma cell surface. Specific antibodies secreted from the hybridoma cells are then captured by the antigens on the cell surface with high efficiently. The antigen-specific positive hybridoma cells could be fluorescently stained using fluorescently-labeled antimouse IgG Fc (Figure 1). After adding methylcellulose semisolid medium, the positive clones could be picked easily using a pipettor. These positive cell clones could then be used to produce monoclonal antibodies after expansion (Figure 2). Positive hybridoma cell clones secreting specific antibodies against malachite green (MG) and porcine epidemic diarrhea virus (PEDV) were both selected with high efficiency using this method. Compared with the ELISA-based method, our CSFIA-based method obtained more than twice of positive hybridoma clones in less than 25% of the corresponding processing time. This work demonstrates that the antigen-specific cell surface-fluorescence immunosorbent assay can be effi-

METHODS AND MATERIALS Materials. GibicoRPMI-1640, Tween-20,NHydroxysuccinimide, N-(3-Dlmethylaminopropyl)-N’ethylcarbodiimide hydrochloride, bovine serum albumin (BSA), and ovalbumin (OVA) were purchased from Asegene (Guangzhou, China). Geiner bio-one cell culture plates were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. (Beijing, China). Sodium chloride (NaCl), potassium chloride (KCl), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), and potassium carbonate (K2CO3) were purchased from GZ Chemical Reagent (Guangzhou, China). 96-well polystyrene plates were purchased from JET BIOFIL (Guangzhou, China). Methylcellulose and fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO, USA).Oleyl alcohol (99+%) was purchased from Alfa Aesar (Ward Hill, USA). Hydroxyl-poly(ethylene glycol)-succinic acid was bought from Jenkem Technology (Beijing, China）. 1-(3-(Dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) from Aladdin chemistry company (Shanghai, China). Malachite green and antigen synthesis were provided by Shenzhen Three Square Biotechnology Co., Ltd (Shenzhen, China). Goat antimouse IgG H＆L (HRP) was purchased from Abcam (Cambridge, MA, USA). Fluorescein (FITC) AffiniPure Sheep AntiMouse IgG Fc Fragment Specific was purchased from Jackson (West Grove, PA, USA). Deionized water with a resistivity of 18.2 MΩ•cm was ob-tained from Millipore (Milli-Q grade, Billerica, MA, USA) and used throughout this study.


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Analytical Chemistry Myeloma cells (SP2/0) were purchased from Shanghai Cell Biology (Shanghai, China). Anti-3-amino-2oxazolidinone (AOZ) derivative (Anti-CPAOZ) cells (2H11) secreting monoclonal antibodies against CPAOZ were prepared by our laboratory. Anti-creatine kinase MB (AntiCKMB) cells (7C1) secreting monoclonal antibodies against CKMB were also prepared by our laboratory. Apparatus. The absorbance of the HRP-based ELISA was measured using a Synergy H1 Hybrid MultiModemicroplate reader (Bio-Tek Instruments, Inc.Winooski, VT, USA). Antigens and modified antigens absorption peaks were detected using a UV-VIS spectrophotometer (Thermo, Waltham, MA, USA.).Fluorescent cell images were acquired on a ZEISS AXIOVERT A1 phase-contras fluorescence microscope (ZEISS, Germany.) with an objective lens (10×objective). Synthesis of Oleyl-PEG4000-NHS. In this work, we synthesized Oleyl-O-poly(ethylene glycol)-succinyl-Nhydroxy-succinimidyl esters(Oleyl-PEG4000-NHS) in our lab. The synthetic route was schematically presented in scheme 1. Briefly, In the synthesis process, Oley alcohol was first transferred to Oley bromine by nucleophilic substitution reaction, and then reacted with hydroxyl-poly(ethylene glycol)-succinic acid to obtain Oleyl-O-poly(ethylene glycol)-succinic acid. Finally, N-Hydroxyl succinimide was covalently bound to the above structure by reacting with the terminal carboxyl group of the PEG chain, yielding Oleyl-PEG4000-NHS. Detailed synthetic methods were described in Supporting Information (ST1). Characterization results of Oleyl-PEG4000-NHS and its corresponding intermediates are presented in Supporting Information (Figures. S1 and S2).

clones. After incubation, cells were gently washed twice with serum-free medium. Cell clones were subsequently incubated with 0.1 mg/ml Oleyl-PEG4000-NHS-Ag and 0.015 mg/ml FITC-labeled anti-mouse IgG Fc at 37 ℃ in a 5% CO2 incubator for 1 h. After gently washing twice with serum-free medium, cell clones were observed under a ZEISS AXIOVERT A1 phase-contrast fluorescence microscope and photographed. Screening method optimization. Using this new method we would eventually see fluorescent signals on the positive cells, the main factors affecting fluorescence intensity are the incubation time and concentrations of Oleyl-PEG4000-NHSAntigen and FITC-labeled anti-mouse IgG Fc. Optimization of these conditions was performed with CPAOZ cells. Details are shown in the Supporting Information (ST3). Comparison of CSFIA method and traditional ELISA method. Balb/c mice were immunized with immune antigen BSA-malachite green (BSA-MG), and then the spleen lymphocytes were harvested and fused with SP2/0 mouse myeloma cells35. The fusion cells were then screened with antigen OVA-MG based on both the CSFIA and traditional ELISA methods. After detected using CSFIA, cell clones were added to the methylcellulose semi-solid medium. The positive clones were then rapidly picked using a pipette. The selected positive cell clones can be used to produce monoclonal antibodies after direct expansion. When using the ELISA approach, positive hybridoma cell clones were generated using three subcloning cycles based on the limiting dilution method. All animal experiments were performed using protocols approved by Laboratory Animal Ethics Committee Jinan University. The procedure for preparing anti-PEDV hybridoma cell clones was the same as reported above.

RESULTS AND DISCUSSION Identification of Oleyl-PEG4000-NHS-Antigen using UV-VIS spectrophotometry. Biocompatible anchor for membrane (BAM) contains two main components: Oleyl groups to act as hydrophobic cell membrane anchors and polyethylene glycol (PEG) to impart hydrophilicity. The chemical structure of the anchor is designed to have various reactive groups at the PEG terminals, allowing them to attach to either physiologically active substance or material surfaces. OleylPEG4000-NHS is a type of amphiphilic molecule that can couple with proteins and anchor itself to the cell membrane. In order to verify the coupling of Oleyl-PEG4000-NHS to an antigen, all relative chemical compounds were inspected using UV-VIS scanning spectroscopy. Figure S3a presents the UV-VIS spectroscopy analysis results. The absorption peaks were observed at 261 nm, 280 nm, 270 nm and 265 nm for Oleyl-PEG4000-NHS, BSA, Oleyl-PEG4000-NHS-BSA and Oleyl-PEG4000-NHS-BSACPAOZ, respectively. As shown in Figure S3b-d, the corresponding UV absorption peaks of Oleyl-PEG4000-NHS-OVA-MG, OleylPEG4000-NHS-CKMB and Oleyl-PEG4000-NHS-PEDV were at 271 nm, 271 nm and 265 nm, respectively. Performance of the CSFIA for the detection of antigen specific positive hybridoma clones. In order to verify the feasibility of the CSFIA method, hybridomacells secreting antibody against CPAOZ and CKMB were detected with Oleyl-PEG4000-NHS-BSA-CPAOZ. As shown in Figure 3a and b, bright green anti-CPAOZ cells can be clearly observed in the fluorescent images, while the image for anti-CKMB cells does not show fluorescent signals (Figure 3c). When they

Scheme 1. Synthetic route of Oleyl-O-poly(ethylene glycol)succinyl-N-hydroxy-succinimidyl ester (Oleyl-PEG4000NHS). Preparation of Oleyl-PEG4000-NHS-Antigen (Ag1,Ag2,Ag3,and Ag4)(Detailed Protocol in Supporting Information, ST2) Development of rapid CSFIA method for the detection of antigen-specific, positive hybridoma clones. AntiCPAOZ cells and anti-CKMB cells were mixed and then diluted to an appropriate concentration. The mixture was cultured for 4~6 days at 37 ℃ in a 5% CO2 incubator to form cell



were detected with Oleyl-PEG4000-NHS-CKMB, anti-CKMB cells exhibited bright green fluorescence (Figure S4 a and b). Almost no fluorescence can be observed in anti-CPAOZ cells (Fig. S4c). Therefore, these results confirmed good specificity of the antigen-specific CSFIA method.

Performance of positive hybridoma cell clone selection from fusion cell using CSFIA-based method. The achievement of using this CSFIA method to screen fusion cells was verified with the selection of hybridoma cells against MG and PEDV. As shown in Figures 4 and 5, positive cell clones marked by red cycles exhibited bright green fluorescence. In

Figure 3. Representative fluorescent image of hybridoma cell clones after incubation with Oleyl-PEG4000-NHS-BSACPAOZ. (a) Anti-CPAOZ cells. (b) Anti-CPAOZ cells and anti-CKMB cells. (c) Anti-CKMB cells. Top rows are bright field images and bottom rows are dark field fluorescent images.

Figure 4. Fluorescence imaging of fusion cells fused with myeloma cells and splenic cells immunized with malachite green (MG), which were then detected by cell surfacefluorescence immunosorbent assay. (a) Anti-MG positive clone. (b) Anti-MG positive and negative clones. (c) Anti-MG negative clones.

CSFIA method optimization. The incubation time of Oleyl-PEG4000-NHS-Antigen and FITC-labeled anti-mouse IgG Fc was optimized using 0.1 mg/ml Oleyl-PEG4000-NHSBSA-CPAOZ and 0.015 mg/ml FITC-labeled anti-mouse IgG Fc. When the incubation time was increased to 30 min, green fluorescence was clearly observed (Figure S5b). This indicated a near-complete immunereaction due to extended incubation time. Incubated with longer periods (40, 50, 60 and 70 min here), cells were still alive and showed gradual green fluorescence enhancement. The cells incubatedfor 60mins exhibited almost same fluorescent intensity level as the cells incubate for 70 min (Figure S5e). These results indicate that an incubation time of approximately 1 h is enough for maximum antigen anchoring and immunereactivity. Then the concentration of the Oleyl-PEG4000-NHSAntigen was optimized. As shown in Figure S6, increasing the Oleyl-PEG4000-NHS-BSA-CPAOZ concentration resulted in an enhanced green fluorescence. Cells incubated with a concentration of 0.05 mg/ml (Figure S6d) appeared to have the maximum fluorescence. On the other hand, incubation with concentrations higher than 0.05 mg/ml resulted in nearly unchanged fluorescence intensities. These results indicate that a concentration of approximately 0.05 mg/ml is the optimal Oleyl-PEG4000-NHS-BSA-CPAOZ concentration. We also optimized the concentration of FITC-labeled anti-mouse IgG Fc. As shown in Figure S7, increasing the FITClabeled anti-mouse IgG Fc concentration resulted in the enhanced green fluorescence. Cells incubated with a concentration of 0.015 mg/ml (Figure S7e) appeared to have the maximum fluorescence. These results indicate that a concentration of approximately 0.015 mg/ml is the optimal FITC-labeled anti-mouse IgG Fc concentration.

Figure 5. Fluorescent images of fusion cells fused with myeloma cells and splenic cells immunized with porcine epidemic diarrhea virus (PEDV), which were then detected by cell surface-fluorescence immunosorbent assay. (a) Anti-PEDV positive clone. (b) Anti-PEDV positive and negative clones. (c) Anti-PEDV negative clones.


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Analytical Chemistry Table 1 Comparison of MG and PEDV fusion cell screening results obtained using surface-fluorescence assay and traditional methods Spleen cells Selection periAntigen Methods Positive clones Working days for fusion od(days) 7 CSFIA 2. 1 66 30 9 MG ELISA . 1 7 30 90 38 CSFIA 1 7 67 30 10 PEDV ELISA 1 7 12 80 48

antigen-specific CSFIA is a rapid and highly efficient screening method for positive hybridoma cell clones. This method has great potential in the applications of monoclonal antibody preparation of both complete and incomplete antigens.

comparison, negative cell clones marked by white cycles showed no fluorescence. The results indicate that this method can distinguish between positive and negative cell clones, even though they appear together (Figure 4a and b). In parallel evaluation of CSFIA and traditional ELISA methods. Fusion cells were screened by either the CSFIA or traditional ELISA methods. Results from these two methods are shown in Table 1. There were more than twice amount of positive cell clones selected using CSFIA when compared to traditional ELISA. Moreover, the CSFIA method required much shorter processing time. In a word, this new CSFIA selection method is simple, intuitive, and efficient, and provides a powerful approach for antigen-specific positive hybridoma clones isolation in monoclonal antibodies preparation. Although the monoclonal antibody preparation technology has been established for several decades, the most widely used preparation technology of monoclonal antibody is still based on ELISA detection and limited dilution method. The indirect ELISA was used to detect whether the cells in the wells secreted specific antibodies or not and the cells needed to subcloned at least three times in order to obtain positive single cell clones. The process is cumbersome and inefficient. Based on the above factors, we developed a simple and rapid antigen-specific hybridoma cell clone selection method based on the Cell Surface Fluorescence Assay (CSFIA). This method allows to stain positive cells directly. We utilized this method to detect hybridoma cell clones that were formed by fusing myeloma and splenic cells immunized with either malachite green (MG) or Porcine epidemic diarrhea virus (PEDV). Strong fluorescent signals were observed on the positive cell membrane, but no autofluorescence from negative cells was found. There was no visible nonspecific binding between the fluorescently-labeled anti-mouse IgG Fc and negative cells. The positive clone number selected by this antigen-specific CSFIA method was twice as that of the traditional ELISA method. Meanwhile, the new method required a much shorter working time.The experimental results show that the novel method is rapid, high efficient and labor-saving, which is suitable for a variety of antigens. For isolating positive clones in this method, the cell clones are picked up with pipettes after adding methylcellulose semi-solid medium, which is not very convenient. Cell selection equipment with automation picking system could be developed in the future. CONCLUSIONS In summary, we developed a simple and rapid antigenspecific positive hybridoma cell clone selection method based on a cell surface-fluorescence assay (CSFIA). Using a combination of lipid-labeled antigens and fluorescently-labeled antimouse IgG Fc, we can quickly stain and easily pick up positive cell clones from uncertain fusion clones. The developed

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Experimental details, additional images and tables as presented in the text (PDF)

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] ORCID: 0000-0002-9954-4563 Author Contributions †These authors contributed equally. Yong Tang, Yuehe, Dan Du, LinXiuqing Li and Hongfen Bian designed research; LinXiuqing Li, Hongfen Bian, Siming Yu, Wei Xiao, Jianying Shen, Caifeng Lan, Kenan Zhou, Caihong Huang and ShitingYu performed research; LinXiuqing Li, Hongfen Bian, and Jianying Shen analyzed data; Yong Tang, Yuehe, Dan Du, LinXiuqing Li and Hongfen Bian wrote the paper. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (2016YFD0500600), the Technology Research Program of Guangzhou City (201508020100) and Chinese Fundamental Research Founds for the Central Universities (21616306).

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