key: cord-0320913-hpu57396 authors: Li, Yuan; Li, Peipei; Ke, Yuebin; Yu, Xuezhi; Yu, Wenbo; Wen, Kai; Shen, Jianzhong; Wang, Zhanhui title: Rare Monoclonal Antibody Discovery Based on Indirect Competitive Screening of Single Hapten-specific Rabbit Antibody Secreting Cell date: 2022-04-15 journal: bioRxiv DOI: 10.1101/2022.04.14.488422 sha: ef5d7212954495fcb3e7d0bff73d7297be220769 doc_id: 320913 cord_uid: hpu57396 Rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, especially for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody secreting cells (ASCs) selection with 20.6 h and proposed an efficient platform for RmAb production against small molecule with 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent has the great threats for public health. We applied the CSMN to select CAP-specific ASCs and produced one high affinity RmAb, surprisingly showing extremely halophilic properties with an IC50 of 0.08 ng mL-1 in saturated salt solution which has as yet not been shown by other antibodies. Molecular dynamic simulation showed that the negatively charged surface improved the stability of the RmAb structure with additional disulfide bonds compared with mouse antibody. Moreover, the reduced solvent accessible surface area of the binding pocket increased the interactions of RmAb with CAP in a saturated salt solution. Furthermore, the RmAb was used to develop an immunoassay for the detection of CAP in real biological samples with simple pretreatment, shorter assay time, and higher sensitivity. The results demonstrated that the practical and efficient CSMN is suitable for rare RmAb discovery against small molecules. however, developing rabbit hybridomas was rather complicated and lower efficient 48 owing to the unstable rabbit myelomas compared with classic mouse myelomas. 49 Therefore, efforts were originally made to generate rabbit-mouse hybridomas which 50 was also not so successful due to the poor fusion efficiency, genetic instability and 51 decrease in antibody secretion 4 . Immortalization of peripheral B cells with Epstein-Barr 52 virus is another method to induce long-term growth of B cells for antibody production 5 , 53 but the low efficiency of this method has seriously obstructed the efficiency of RmAb 54 preparation. To overcome these issues, several display platforms have been introduced 55 to discovery RmAb combing with the recombinant expression technology, such as yeast 56 surface display, ribosome display, and phage display 6, 7 . However, the variable domain 57 genes of an antibody are randomly combined during the construction of antibody 58 analyzed with fluorescent-based assays in nanowells or microwells, which can be 81 applied for the screening of hybridomas 13 and human ASCs 14 . Various direct staining 82 methods have been developed to identify the ASCs that secrete antibodies of interest. 83 Love et al. applied the ovalbumin-coated slides in microwells to capture the secreted 84 anti-ovalbumin antibody from the corresponding hybridoma and realized an effective 85 screening of hybridomas 15 . Unlike proteins, small molecules that cannot be directly 86 coated in the microwell and always chemically conjugated to proteins as the 87 immunogens or coating antigens due to the small size. With these chemical 88 modifications, not only target molecules alone but also linkers of haptens and amino 89 acid residues on the protein are potential epitopes that can induce the generation of 90 ASCs. Therefore, the strategies that could precise select the ASCs specific to proteins 91 are not yet available to small molecules. 92 In this study, we proposed a competitive screening method, named CSMN, for 93 hapten-specific rabbit ASC selection based on nanowells. Chloramphenicol (CAP) is 94 used as a model analyte, which is widely used for the prevention and treatment of 95 poultry and aquatic diseases. Due to the animal-derived foods with CAP residue posing 96 great health hazards to humans 16, 17 , the use of CAP has been totally banned worldwide. The ASCs loaded in the nanowells with significantly decreased fluorescence intensity 123 at 4-8 h were selected. Other nanowells with a continuous increase in fluorescence 124 intensity due to the secreted antibodies of these loaded ASCs were BSA-specific and 125 these cells were abandoned, which was the key step for the precise identification of 126 ASC against small molecule in the study. The CAP-specific ASCs were selected and lysed at the single cell level to prepare 128 the antibody mRNA. Then, the mRNA was reverse transcribed to cDNA, and the VH 129 and VL were amplified from cDNA with the specific primers. The DNA amplicons of 130 VH and VL were sequenced by the Sanger sequencing method. CAP-specific RmAbs 131 were expressed in vitro by the eukaryotic expression system. 137 The larger nanowell always contributed to higher cellular occupancy 20 ; however, 138 the single-cell occupancy of the larger nanowell with 37% was significantly lower than 139 that of similar nanowell with 52-92% 21-23 . Whereas, the retrieve of single cell is 140 severely difficult due to the very small liquid volume when the size of nanowell is 141 similar to the size of the cells 24 . To ensure both high single-cell occupancy and high 142 retrieve rate, a suitable size of nanowell is critical to for the efficient and precise 143 selection of antigen-specific ASCs. The diameter of rabbit splenocytes has been 144 reported to be approximately 8-13 µm 25 ; thus, nanowells with diameters ranging from 145 20 µm to 100 µm were firstly optimized in this study. To test the single-cell occupancy of the nanowell, the number of loaded cells was 147 adjusted to the half of the well numbers (Fig. S1 ), i.e., 3 × 10 4 cells mL -1 for 100-µm-148 diameter H100, 2.5 × 10 4 cells mL -1 for 40-µm-diameter U40, 1 × 10 4 cells mL -1 for 149 25-µm-diameter U25, and 1.5 × 10 5 cells mL -1 for 20-µm-diameter 370K. The Fig. 2A 150 showed that the cellular occupancies of U40 (90.9%) and H100 (72.8%) were higher 151 than that of 370K (67.6%) and U25 with (77.9%). However, the single-cell occupancy 152 of U25 with 54.3% was highest compared to those of other three nanowells all below 153 46.3% ( Fig. 2A) . Besides, the UFO design with two auricles of U25 with a 25-µm-154 diameter provides a high single-cell retrieve efficiency with a volume of 12 pL. Thus, 155 U25 was chosen for the single rabbit ASC selection. The procedure of splenocytes 156 distribution including cell loading, centrifugation and cell plating was showed in Fig. 157 2B. The number of the loaded rabbit splenocytes in U25 was then optimized with 0.5 × 158 10 4 cells (Fig. 2C ), 1 × 10 4 cells (Fig. 2D) , and 2 × 10 4 cells (Fig. 2E ). As observed in 159 Fig. 2D and 2E, a larger number of splenocytes gave better capture efficiency; however, 160 excessive splenocytes resulted in multiple-cell occupancy in the nanowells (Fig. 2E ). 161 Thus, splenocytes plated on the U25 were at the optimum number of 1 × 10 4 cells, 162 followed by a brief centrifugation to ensure the cells were loaded in the nanowells as 163 shown in Fig. 2D . and produce CAP-specific RmAbs by the CSMN. Before the rabbit splenocyte 208 identification procedure, the rabbit antisera was monitored after the sixth immunization. Seven days after the sixth immunization, the rabbit #6 with the best antisera IC50 value 210 of 0.27 ng mL -1 (Table S1 ) was sacrificed for the splenocytes collection; and then the 211 immunized splenocytes were submitted to the CSMN. The PE-anti-rabbit F(ab')2 antibody was used to stain the secreted CAP-specific 213 antibodies from the ASCs in the U25, which is represented by the five regions shown The result showed that not all the obtained RmAbs could specifically recognize CAP, 258 which may owe to the production of nonspecific antibodies derived from the 259 accumulation of nonspecific products and PCR errors by extensively amplifying. 260 Generally, the production of antibodies for diagnosis mainly focuses on the titer, 261 affinity and specificity. However, some antibodies with high affinity and titer in 262 physiological conditions have poor performance in harsh assay environment originally 263 owing to their unstable structure. From a practical viewpoint, the stability of an 264 antibody is more important to develop a robust immunoassay, and an antibody that is 265 able to tolerate physio-chemical factors, such as organic solvents, acid-base and salt, is 266 preferred and highly demanded. Rabbit antibodies possess an additional disulfide bond 267 that is not found in human or mouse antibodies, which theoretically contributes to their 268 higher stability 32 . Thus, the RmAb3 with higher affinity to CAP, was further purified 269 ( Fig. S6 ) and subsequently assessed for its stability and compared to MmAb for a better 270 application. As shown in Fig. 4A and 4B, the tolerance of RmAb3 to organic solvents in the 272 icELISA were up to 20% for methanol and 40% for acetonitrile, which were obviously 273 higher than those of MmAb with 10% methanol and only 2.5% acetonitrile (Fig. S7A 274 and S8B). The optimum pH values for RmAb3 and MmAb were comparable, both 275 between 6.5-7.4 ( Fig. 4C and S7C) . Surprisingly, the RmAb3 showed the best 276 performance in high sodium strength saturated salt solution (Fig. 4D) , which was 277 approximately 30-fold higher than that of MmAb with the optimum sodium strength of used as a coating antigen at the concentration of 37.55 ng mL -1 (Fig. 4H ), all better than 290 those of the MmAb with 174 ng mL -1 , 0.28 ng mL -1 and 500.0 ng mL -1 (Fig. S7H ). Furthermore, the stability test proved the RmAb3 was an extremely halophilic protein, 292 which has been rarely reported for antibody. We consider that the outstanding stability S10B, S11C, S11B and S12C). Structural flexibility of proteins, as enzymes, has been 392 proven to be essential for their catalytic stability [43, 44] . Therefore, we further (Fig. 6D-6I) . This (Fig. 6S-6U ). In contrast, the nitro of CAP stably inserted into the 419 binding pocket in control and physiological salt solutions (Fig. 6M-6R) , which 420 significantly decreased of the interactions between MmAb and CAP and noticeably 421 impaired their affinity to CAP in saturated salt solution (Fig. 6V-6X) . 422 We next compared the interactions of mAbs-CAP at optimum salt solutions for a 423 better understanding of the RmAb halophilic mechanism, using the RmAb3-CAP in 424 saturated salt solution and the MmAb-CAP in physiological salt solution, respectively. The binding surface area of the RmAb3 is 333.529 Å 2 , which is larger than that of the Fig. 6L and 6W) , which leads the nitro of 431 CAP towards the "arch" built by the loop of CDRL3 (Fig. 6I) , while, another hydrogen 432 bond formed by Tyr104 with CAP drives the CAP into the "arch". Besides, the 433 hydrophobic force between CAP and the RmAb3 also make a significant contribution 434 to the interactions (Fig. 6L ). In the case of the MmAb, three hydrogen bonds are formed 435 between the Trp47/Thr106/Arg59 and CAP, leading the nitro of CAP to vertically insert 436 into the binding pocket (Fig. 6W) . The hydrophobic force between the CAP and the 437 MmAb is also important, although it is much weaker than that between the CAP and 438 the RmAb3. Moreover, the interactions between the CAP and the RmAb3 are mainly 439 from the VL, but from the VH in the case of the MmAb-CAP. The observation of the 440 RmAb3 recognition mechanism was corresponding to the previous X-ray 441 crystallography finding that the rabbit light chain and particular CDRL3 was the main 442 contributors to the antibody and antigen interaction 41 . In a word, the binding free energy Table 1 , the recoveries of the icELISA with the RmAb3 477 ranging from 87.9% to 121.2% with intra-assay CVs below 11.7% and inter-assay CVs showing a good agreement of two methods (Table S5) . Additionally, investigation into 481 the specificity of one immunoassay is also crucial to ensure the accuracy of the 482 subsequent application. As shown in Table S2 , the developed icELISA showed ignored 483 cross-reactivities to three analogs of TAP, FF, and FFA below 0.01%. These results titer and affinity of the prepared the RmAb3 is 3-fold higher than that of the best MmAb 495 produced so far. More importantly, the RmAb3 was found to be extremely halophilic, 496 which has not been reported previously. We found that the unique structure, negatively The optimal nanowell with the highest single-cell occupancy was pretreated with 556 1 mL of anhydrous ethanol and centrifuged at 1000 rpm for 10 min to drain the bubbles. Assessment of specific binding proteins suitable for the detection of paralytic shellfish 663 poisons using optical biosensor technology Simultaneous raising of rabbit 665 monoclonal antibodies to fluoroquinolones with diverse recognition functionalities via 666 single mixture immunization Rabbit monoclonal antibodies: 668 generating a fusion partner to produce rabbit-rabbit hybridomas Rabbit-mouse hybridomas secreting 671 intact rabbit immunoglobulin Mutant EL-673 4 thymoma cells polyclonally activate murine and human B cells via direct cell 674 interaction Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the 677 impact of kappa allotype-correlated variation in cysteine content on antibody libraries 678 selected by phage display The rabbit 680 antibody repertoire as a novel source for the generation of therapeutic human antibodies Phage display and other peptide display technologies Potent RBD-specific 686 neutralizing rabbit monoclonal antibodies recognize emerging SARS-CoV-2 variants 687 elicited by DNA prime-protein boost vaccination Generation and reactivity analysis of human recombinant monoclonal 691 antibodies directed against epitopes on HLA-DR Heterofunctional 694 Particles as Single Cell Sensors to Capture Secreted Immunoglobulins and Isolate 695 Single-cell droplet microfluidic 698 screening for antibodies specifically binding to target cells Multifunctional microwell arrays for single cell level 701 functional analysis of lymphocytes Miniaturized 703 single-cell technologies for monoclonal antibody discovery A microengraving 706 method for rapid selection of single cells producing antigen-specific antibodies Screening of chloramphenicol 709 residues in broiler chickens slaughtered in an industrial poultry abattoir in Mashhad Based point-of-care immunoassays: 713 Recent advances and emerging trends Development of a monoclonal 715 antibody based-ELISA for the detection of chloramphenicol in shrimp, feed and milk 716 samples and validation by LC-MS/MS coupled with immunoaffinity clean-up Preparation and characterization of size-controlled 719 glioma spheroids using agarose hydrogel microwells Microwell arrays reveal cellular heterogeneity during the clonal expansion of 722 transformed human cells Large-scale single-cell trapping and imaging using microwell 724 arrays Concurrent detection of secreted products from human lymphocytes by microengraving: 727 cytokines and antigen-reactive antibodies Tuning the 729 Surface Interactions between Single Cells and an OSTE+ Microwell Array for 730 Enhanced Single Cell Manipulation Epstein-Barr virus reprograms human B lymphocytes immediately in the prelatent 734 phase of infection Factors affecting early antibody secreting 737 cell maturation into long-lived plasma cells Receptor dissociation and B-cell activation. B Cell Receptor 739 Signaling Improving single-cell encapsulation 741 efficiency and reliability through neutral buoyancy of suspension High-throughput and automation 744 advances for accelerating single-cell cloning, monoclonality and early phase clone 745 screening steps in mammalian cell line development for biologics production A study based on four 748 immunoassays: Hepatitis C virus antibody against different antigens may have unequal 749 contributions to detection A 751 microengraving method for rapid selection of single cells producing antigen-specific 752 antibodies Crystal structure of a conformation-dependent rabbit 754 IgG Fab specific for amyloid prefibrillar oligomers Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo 758 From rabbit antibody repertoires to rabbit monoclonal 760 antibodies Molecular bases of protein halotolerance Production of a specific 764 monoclonal antibody and a sensitive immunoassay for the detection of diphacinone in 765 biological samples Hapten design and 767 monoclonal antibody to fluoroacetamide, a small and highly toxic chemical Enhancing protein stability with 770 genetically encoded noncanonical amino acids Halophilic enzymes: proteins with a grain of 773 salt Affinity improvement of a cancer-targeted antibody through alanine-induced 776 adjustment of antigen-antibody interface Generation 778 and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev Magnetic assisted fluorescence 781 immunoassay for sensitive chloramphenicol detection using carbon dots@ CaCO3 782 nanocomposites GROMACS: fast, flexible, and free A Kirkwood-Buff derived force field for sodium 787 chloride in water Comparison 789 of simple potential functions for simulating liquid water LINCS: a linear constraint solver for 792 molecular simulations Particle mesh Ewald: An N⋅ log (N) method for 794 Ewald sums in large systems g_mmpbsa A GROMACS 796 tool for high-throughput MM-PBSA calculations 598 The CAP-specific RmAb3 from the ASCs were characterized and compared to the 599 CAP-specific MmAbs by antibody titer, affinity, specificity, stability and tolerance of 600 sodium strength, methanol, acetonitrile, and pH. All measurements were conducted for 601 three replicates. 602 The antibody titer is represented by antibody dilution, and the antibody affinity is The authors declare no competing financial interests.