A FACS-microfluidics pipeline for rapid discovery of high-affinity rabbit monoclonal antibodies against hapten molecules
Abstract
Single B-cell technology has become increasingly important for discovering of rabbit monoclonal antibody (RabmAb) with naturally paired heavy- and light-chain variable-region genes (VH/VL), which offers many advantages for detecting hapten contaminants in food safety testing, clinical diagnostics, and environmental monitoring. However, the efficient recovery of naturally paired VH/VL genes from single B cells remains challenging. To address this challenge, we developed an integrated pipeline that couples fluorescence-activated cell sorting (FACS) with a closed-nanoliter microfluidic system for automated single-cell cDNA preparation, thereby bridging hapten-specific B-cell enrichment and antibody gene recovery. Using chloramphenicol (CAP) as a model hapten, FACS identified antigen-specific memory B cells (MBCs) representing 1.10% of the IgG+ population, and the pipeline recovered 22 paired VH/VL sequences, of which 10 were confirmed as antigen-specific, representing a high positive hit rate of 45.5%. The entire process from cell sorting to RabmAb validation was completed within one week, eliminating conventional manual steps of cell lysis, reverse transcription, and sample transfer that are prone to RNA loss and contamination. The lead candidate, RabmAb-4E, demonstrated exceptional analytical performance in an indirect competitive ELISA, with an IC50 of 0.01 ng/mL, negligible cross-reactivity (<0.001%) against structurally related analogues, and robust tolerance to high ionic strength (0-6.0 M NaCl), broad pH range (4.0-8.5), and organic solvent (0-60% methanol). Recoveries from spiked skim milk and chicken samples ranged from 90.28% to 117.53%, confirming its practical utility in food matrix analysis. Molecular docking further suggested that a shape and chemical complementary within the paratope pocket in which multiple noncovalent interactions including hydrogen bonds, π-π stacking, and hydrophobic contacts, cooperatively stabilize the antibody-CAP complex. This work establishes a FACS-microfluidics pipeline that not only overcomes the technical barriers to hapten-specific RabmAb discovery but also provides a rapid, efficient, and scalable platform for generating high-performance antibodies against hapten molecules.
References
(1) Chappey, O.; Sandouk, P.; Scherrmann, J. Monoclonal-Antibodies in Hapten Immunoassays. Pharm. Res. 1992, 9 (11), 1375–1379.
(2) Starr, C. G.; Tessier, P. M. Selecting and engineering monoclonal antibodies with drug-like specificity. Curr. Opin. Biotechnol. 2019, 60, 119–127.
(3) Kuai, M.; Shi, Z.; Li, B.; Pan, Y.; Tang, M.; Gao, X.; Chen, D.; Lü, P. Rabbit monoclonal antibodies: Synergistic innovation and breakthrough based on B-cell development mechanism and single B-cell technology. Colloids and Surfaces B: Biointerfaces 2026, 257, 115179.
(4) Li, P.; Pan, Y.; Li, Y.; Zhang, Y.; Lv, B.; Wen, K.; Yu, X.; Shen, J.; Wang, Z. Single-Cell-Derived Rabbit mAb-Based Immunoassay for Simultaneous Detection of Florfenicol and Florfenicol Amine. Anal. Chem. 2026, 98 (29), 21280–21291.
(5) Li, Y.; Kong, Y.; Yu, X.; Yu, W.; Wen, K.; Shen, J.; Wang, Z. Characteristics of rabbit hapten-specific and germline-based BCR repertoires following repeated immunization. One Health Adv. 2023, 1 (1), 17.
(6) Weber, J.; Peng, H.; Rader, C. From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp Mol Med 2017, 49 (3), e305–e305.
(7) Zhang, Z.; Liu, H.; Guan, Q.; Wang, L.; Yuan, H. Advances in the Isolation of Specific Monoclonal Rabbit Antibodies. Front. Immunol. 2017, 8, 494.
(8) Tiller, T. Single B cell antibody technologies. N Biotechnol 2011, 28 (5), 453–457.
(9) Kretzschmar, T.; von Rüden, T. Antibody discovery: phage display. Curr. Opin. Biotechnol. 2002, 13 (6), 598–602.
(10) Slavny, P.; Hegde, M.; Doerner, A.; Parthiban, K.; McCafferty, J.; Zielonka, S.; Hoet, R. Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects. Front. Immunol. 2024, 15, 1469329.
(11) Li, B.; Fouts, A. E.; Stengel, K.; Luan, P.; Dillon, M.; Liang, W.; Feierbach, B.; Kelley, R. F.; Hoetzel, I. In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation. mAbs 2014, 6 (2), 437–445.
(12) Ponsel, D.; Neugebauer, J.; Ladetzki-Baehs, K.; Tissot, K. High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation. Molecules 2011, 16 (5), 3675–3700.
(13) Gao, C.; Guo, L.; Yang, H.; Zhang, Y.; Yu, X.; Wen, K.; Yu, W.; Liang, X.; Shen, J.; Pan, Y.; Wang, Z. Discovery of Nanobodies against Tetramethylenedisulfotetramine by Integrating Next-Generation Sequencing and Moderate Random Mutagenesis. Anal. Chem. 2026, 98 (17), 13012–13027.
(14) Pedrioli, A.; Oxenius, A. Single B cell technologies for monoclonal antibody discovery. Trends in Immunology 2021, 42 (12), 1143–1158.
(15) Starkie, Dale. O.; Compson, J. E.; Rapecki, S.; Lightwood, D. J. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells. PLoS One 2016, 11 (3), e0152282.
(16) Duncombe, T. A.; Tentori, A. M.; Herr, A. E. Microfluidics: reframing biological enquiry. Nat. Rev. Mol. Cell Biol. 2015, 16 (9), 554–567.
(17) Prakadan, S. M.; Shalek, A. K.; Weitz, D. A. Scaling by shrinking: empowering single-cell “omics” with microfluidic devices. Nat. Rev. Genet. 2017, 18 (6). https://doi.org/10.1038/nrg.2017.15
(18) Han, L.; Zi, X.; Garmire, L. X.; Wu, Y.; Weissman, S. M.; Pan, X.; Fan, R. Co-detection and sequencing of genes and transcripts from the same single cells facilitated by a microfluidics platform. Sci Rep 2014, 4, 6485.
(19) White, A. K.; VanInsberghe, M.; Petriv, O. I.; Hamidi, M.; Sikorski, D.; Marra, M. A.; Piret, J.; Aparicio, S.; Hansen, C. L. High-throughput microfluidic single-cell RT-qPCR. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (34), 13999–14004.
(20) Irimia, D.; Tompkins, R. G.; Toner, M. Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device. Anal. Chem. 2004, 76 (20), 6137–6143.
(21) Anjos-Afonso, F.; Buettner, F.; Mian, S. A.; Rhys, H.; Perez-Lloret, J.; Garcia-Albornoz, M.; Rastogi, N.; Ariza-McNaughton, L.; Bonnet, D. Single cell analyses identify a highly regenerative and homogenous human CD34+hematopoietic stem cell population. Nat. Commun. 2022, 13 (1), 2048.
(22) Xu, Y.; Mizuno, T.; Sridharan, A.; Du, Y.; Guo, M.; Tang, J.; Wikenheiser-Brokamp, K. A.; Perl, A.-K. T.; Funari, V. A.; Gokey, J. J.; Stripp, B. R.; Whitsett, J. A. Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis. JCI Insight 2016, 1 (20), e90558.
(23) Titcombe, P. J.; Wigerblad, G.; Sippl, N.; Zhang, N.; Shmagel, A. K.; Sahlstrom, P.; Zhang, Y.; Barsness, L. O.; Ghodke-Puranik, Y.; Baharpoor, A.; Hansson, M.; Israelsson, L.; Skriner, K.; Niewold, T. B.; Klareskog, L.; Svensson, C. I.; Amara, K.; Malmstrom, V.; Mueller, D. L. Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis. Arthritis Rheumatol. 2018, 70 (12), 1933–1945.
(24) Zhang, L.; Wang, S.; Xiong, J.; Xv, Y.; Xie, J.; Wang, Z.; Wu, C.; Jiang, H.; Shen, J. A free elution immunoassay for chloramphenicol in chicken and egg after magnetic ZIF-8 adsorption separation. One Health Adv. 2023, 1 (1), 15.
(25) Wang, L.; Zhang, Y.; Gao, X.; Duan, Z.; Wang, S. Determination of Chloramphenicol Residues in Milk by Enzyme-Linked Immunosorbent Assay: Improvement by Biotin-Streptavidin-Amplified System. J. Agric. Food Chem. 2010, 58 (6), 3265–3270.
(26) Li, Y.; Ji, B.; Chen, W.; Liu, L.; Xu, C.; Peng, C.; Wang, L. Production of new class-specific polyclonal antibody for determination of fluoroquinolones antibiotics by indirect competitive ELISA. Food Agric. Immunol. 2008, 19 (4), 251–264.
(27) Li, Y.; Li, P.; Ke, Y.; Yu, X.; Yu, W.; Wen, K.; Shen, J.; Wang, Z. A rare monoclonal antibody discovery based on indirect competitive screening of a single hapten-specific rabbit antibody secreting cell. Analyst 2022, 147 (13), 2942–2952.
(28) Liang, L.-L.; Long, J.-Y.; Zhang, X.-T.; Gong, M.-D.; Xu, W.-B.; Wang, X.-Q.; Liu, Z.-Y. Preparation of rabbit monoclonal antibody against T-2 toxin and development of enzyme-linked immunosorbent assay in milk, feed and pork samples. Microchemical Journal 2024, 207, 111977.
(29) Engels, N.; Koenig, L. M.; Heemann, C.; Lutz, J.; Tsubata, T.; Griep, S.; Schrader, V.; Wienands, J. Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells. Nat. Immunol. 2009, 10 (9), 1018–1764.
(30) Gross, A.; Schoendube, J.; Zimmermann, S.; Steeb, M.; Zengerle, R.; Koltay, P. Technologies for Single-Cell Isolation. Int. J. Mol. Sci. 2015, 16 (8), 16897–16919.
(31) Satish, H. P. S.; Zeglinski, K.; Uren, R. T.; Nutt, S. L.; Ritchie, M. E.; Gouil, Q.; Kluck, R. M. NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells. mAbs 2022, 14 (1), 2106621.
(32) Briones, J.; Espulgar, W.; Koyama, S.; Takamatsu, H.; Tamiya, E.; Saito, M. A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis. Sci Rep 2021, 11 (1), 12995.
(33) Liu, Y.; Chen, X.; Zhang, Y.; Liu, J. Advancing single-cell proteomics and metabolomics with microfluidic technologies. Analyst 2019, 144 (3), 846–858.
(34) Luo, X.; Chen, J.; Ataei, M.; Lee, A. Microfluidic Compartmentalization Platforms for Single Cell Analysis. Biosensors-Basel 2022, 12 (2), 58.
(35) Mahendra, A.; Haque, A.; Prabakaran, P.; Mackness, B. C.; Fuller, T. P.; Liu, X.; Kathuria, S.; Wang, Y.-H.; Amatya, N.; Yu, X.; Hopke, J.; Hoffmann, D.; Bric-Furlong, E.; Zhang, N.; Cho, H.-S.; Zhang, R.; Sancho, J.; Saleh, J.; Rao, S. P.; Wendt, M.; Chowdhury, P. S. Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire. Commun. Biol. 2022, 5 (1), 1157.
(36) Huang, J.; Qing, Y.; Ma, X.; Lin, A.; Li, J. Rapid detection of monensin via time-resolved fluorescence immunoassay based on rabbit monoclonal antibodies from single-cell BCR sequencing. Microchem J. 2026, 225, 118187.
(37) Ozawa, T.; Piao, X.; Kobayashi, E.; Zhou, Y.; Sakurai, H.; Andoh, T.; Jin, A.; Kishi, H.; Muraguchi, A. A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity. PLoS One 2012, 7 (12), e52383.
(38) Sompunga, P.; Pruksametanan, N.; Rangnoi, K.; Choowongkomon, K.; Yamabhai, M. Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology. Talanta 2019, 201, 397–405.
(39) Condado-Morales, I.; Dingfelder, F.; Waibel, I.; Turnbull, O. M.; Patel, B.; Cao, Z.; Rose Bjelke, J.; Nedergaard Grell, S.; Bennet, A.; Hummer, A. M.; Raybould, M. I. J.; Deane, C. M.; Egebjerg, T.; Lorenzen, N.; Arosio, P. A comparative study of the developability of full-length antibodies, fragments, and bispecific formats reveals higher stability risks for engineered constructs. mAbs 2024, 16 (1), 2403156.
(40) Petrus, M. L. C.; Kiefer, L. A.; Puri, P.; Heemskerk, E.; Seaman, M. S.; Barouch, D. H.; Arias, S.; van Wezel, G. P.; Havenga, M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol 2019, 103 (21), 8875–8888.
(41) Rangnoi, K.; Rüker, F.; Wozniak-Knopp, G.; Cvak, B.; O’Kennedy, R.; Yamabhai, M. Binding Characteristic of Various Antibody Formats Against Aflatoxins. ACS Omega 2021, 6 (39), 25258–25268.
(42) Unverdorben, F.; Richter, F.; Hutt, M.; Seifert, O.; Malinge, P.; Fischer, N.; Kontermann, R. E. Pharmacokinetic properties of IgG and various Fc fusion proteins in mice. mAbs 2016, 8 (1), 120–128.
(43) Xiao, J.; Qin, L.; Zhao, D.; Huang, N.; Xu, W.; Zhang, L.; Pan, X.; Han, S.; Ding, M.; Li, L.; Le, T.; Peng, D. Monospecific and ultrasensitive detection of ofloxacin: A computational chemistry-assisted hapten screening strategy and analysis of molecular recognition mechanism. J. Hazard. Mater. 2024, 465, 133221.
(44) Lu, F.; Wu, X.; Zhang, F.; Wu, J.; Yuan, Z.; Wang, B.; Tan, G.; Guo, S. Comparison of single-chain variable fragments and monoclonal antibody against dihydroartemisinin. J. Immunol. Methods 2024, 532, 113728.
(45) Kim, Y. J.; Cho, Y. A.; Lee, H. S.; Lee, Y. T. Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal. Chim. Acta 2003, 494 (1–2), 29–40.
(46) Zhang, Q.; Wang, L.; Ahn, K. C.; Sun, Q.; Hu, B.; Wang, J.; Liu, F. Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion. Anal. Chim. Acta 2007, 596 (2), 303–311.
(47) Wang, Z.; Zhang, H.; Ni, H.; Zhang, S.; Shen, J. Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens. Anal. Chim. Acta 2014, 820, 152–158.
(48) Wang, Z.; Sheng, Y.; Duan, H.; Yu, Q.; Shi, W.; Zhang, S. New haptens synthesis, antibody production and comparative molecular field analysis for tetracyclines. RSC Adv. 2014, 4 (96), 53788–53794.
(49) Kamata, N.; Enomoto, A.; Ishida, S.; Nakamura, K.; Kurisaki, J. I.; Kaminogawa, S. Comparison of pH and ionic strength dependence of interactions between monoclonal antibodies and bovine beta-Lactoglobulin. Biosci. Biotechnol. Biochem. 1996, 60 (1), 25–29.
(50) Pisetsky, D. S.; Shaffer, R.; Armstrong, D. D.; Spencer, D. M. The interaction of anti-DNA antibodies with DNA antigen: Evidence for hysteresis for high avidity binding. Clinical Immunology 2021, 231, 108848.
(51) Livesay, D.; Linthicum, S.; Subramaniam, S. pH dependence of antibody: hapten association. Molecular Immunology 1999, 36 (6), 397–410.
(52) Pan, R.; Sampson, J. M.; Chen, Y.; Vaine, M.; Wang, S.; Lu, S.; Kong, X.-P. Rabbit Anti-HIV-1 Monoclonal Antibodies Raised by Immunization Can Mimic the Antigen-Binding Modes of Antibodies Derived from HIV-1-Infected Humans. Journal of Virology 2013, 87 (18), 10221–10231.
(53) Kasahara, K.; Kawade, R.; Nakakido, M.; Matsunaga, R.; Akiba, H.; Entzminger, K. C.; Maruyama, T.; Okumura, S. C. J.; Caaveiro, J. M. M.; Kuroda, D.; Tsumoto, K. Unveiling the structural mechanisms behind high affinity and selectivity in phosphorylated epitope-specific rabbit antibodies. J. Biol. Chem. 2024, 300 (12), 107989.
(54) Liu, N.; Song, S.; Lu, L.; Nie, D.; Han, Z.; Yang, X.; Zhao, Z.; Wu, A.; Zheng, X. A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue. Food Agric. Immunol. 2014, 25 (4), 523–534.
(55) Xu, N.; Xu, L.; Ma, W.; Kuang, H.; Xu, C. Development and characterisation of an ultrasensitive monoclonal antibody for chloramphenicol. Food Agric. Immunol. 2015, 26 (3), 440–450.
(56) Liu, C.; Deng, D.; Xu, D.; Wu, K.; Yang, H.; Zhao, K.; Li, J.; Deng, A. Development of a monoclonal antibody based-ELISA for the detection of chloramphenicol in shrimp, feed and milk samples and validation by LC-MS/MS coupled with immunoaffinity clean-up. Anal. Methods 2019, 11 (4), 507–516.
(57) Wang, Z.; Wang, M.; Fu, X.; Qian, J.; Wang, M.; Tan, G. Novel hapten design, highly sensitive monoclonal antibody production, and immunoassay development for rapid screening of illegally added chloramphenicol in cosmetics. J. Immunol. Methods 2024, 525, 113604.
(58) Swofford, C. A.; Nordeen, S. A.; Chen, L.; Desai, M. M.; Chen, J.; Springs, S. L.; Schwartz, T. U.; Sinskey, A. J. Structure and specificity of an anti‐chloramphenicol single domain antibody for detection of amphenicol residues. Protein Sci 2022, 31 (11), e4457.
Metrics
DOI:
Submission ID:
Downloads
Posted
How to Cite
Download Citation
Declaration of Competing Interests
The authors declare no competing interests to disclose.
Copyright
The copyright holder for this preprint is the author/funder.
All rights reserved. This work is protected by copyright. No part of this work may be reproduced, distributed, or transmitted in any form or by any means without the prior written permission of the copyright holder.