Single B cell technologies for monoclonal antibody discovery
Authors: Alessandro Pedrioli, Annette Oxenius
Journal: Trends in Immunology (2021)

Monoclonal antibodies (mAbs) are among the most important type of biologic drugs on the pharmaceutical market, as well as for diagnostic purposes. Presently, more than 100 mAbs have been approved by the US FDA against a variety of diseases such as cancer, infectious diseases, autoimmune diseases, and neurological disorders.Primary antigen-specific B cells are the main source for obtaining antigen-specific mAb sequences, particularly using human specimens such as peripheral blood mononuclear cells. Also, the humanization of mAbs derived from other species (e.g. mice, rats, and rabbits) has become easier and more efficient.Currently, single B cell screening systems bear multiple advantages over other systems, such as display technologies. Particularly, the in vivo development of mAbs favors the safety profile and the overall developability. It also has reduced off-target binding to the human proteome.Single B cell technologies have significantly evolved, becoming faster and higher throughput than before. Nonetheless, hybridoma technology, the first technique in this field, still represents an important methodology and is well known within the scientific community.At present no gold standard exists in the field, relying on a broad variety of different single B cell systems for mAb discovery – each with its advantages and disadvantages. Monoclonal antibodies (mAbs) are often selected from antigen-specific single B cells derived from different hosts, which are notably short-lived in ex vivo culture conditions and hence, arduous to interrogate. The development of several new techniques and protocols has facilitated the isolation and retrieval of antibody-coding sequences of antigen-specific B cells by also leveraging miniaturization of reaction volumes. Alternatively, mAbs can be generated independently of antigen-specific B cells, comprising display technologies and, more recently, artificial intelligence-driven algorithms. Consequently, a considerable variety of techniques are used, raising the demand for better consolidation. In this review, we present and discuss the major techniques available to interrogate antigen-specific single B cells to isolate antigen-specific mAbs, including their main advantages and disadvantages. Monoclonal antibodies (mAbs) are often selected from antigen-specific single B cells derived from different hosts, which are notably short-lived in ex vivo culture conditions and hence, arduous to interrogate. The development of several new techniques and protocols has facilitated the isolation and retrieval of antibody-coding sequences of antigen-specific B cells by also leveraging miniaturization of reaction volumes. Alternatively, mAbs can be generated independently of antigen-specific B cells, comprising display technologies and, more recently, artificial intelligence-driven algorithms. Consequently, a considerable variety of techniques are used, raising the demand for better consolidation. In this review, we present and discuss the major techniques available to interrogate antigen-specific single B cells to isolate antigen-specific mAbs, including their main advantages and disadvantages. Highly specific mAbs functionally surpass polyclonal antibodies (pAbs) (see Glossary), which are a heterogeneous and undefined mixture of mAbs [1.Kohler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.Nature. 1975; 256: 495-4927Crossref PubMed Google Scholar,2.Lipman N.S. et al.Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources.ILAR J. 2005; 46: 258-268Crossref PubMed Google Scholar]. mAbs are broadly used as highly specific diagnostic tools, and as therapeutic modalities in the context of viral infections, tumors, autoimmune diseases, metabolic diseases, neurological diseases, and transplantation [3.Wilson P.C. Andrews S.F. Tools to therapeutically harness the human antibody response.Nat. Rev. Immunol. 2012; 12: 709-719Crossref PubMed Scopus (92) Google Scholar,4.Weiner G.J. Building better monoclonal antibody-based therapeutics.Nat. Rev. Cancer. 2015; 15: 361-370Crossref PubMed Scopus (358) Google Scholar]. Particularly in the field of immuno-oncology, mAbs, and variants thereof, namely single chain variable fragments (scFvs), antibody–drug conjugates (ADCs), bispecific antibodies, etc. are gaining importance as leading therapeutic biologic drugs [4.Weiner G.J. Building better monoclonal antibody-based therapeutics.Nat. Rev. Cancer. 2015; 15: 361-370Crossref PubMed Scopus (358) Google Scholar,5.Walsh G. Biopharmaceutical benchmarks 2018.Nat. Biotechnol. 2018; 36: 1136-1145Crossref PubMed Scopus (337) Google Scholar]. Since the USA FDA approval of Orthoclone OKT3 (anti-CD3 mAb) in 1986 for kidney transplantation rejection (the first therapeutic mAb), more than 100 mAbs have been approved by the FDA [6.Mullard A. FDA approves 100th monoclonal antibody product.Nat. Rev. Drug Discov. 2021; 20: 491-495Crossref PubMed Scopus (17) Google Scholar]. Furthermore, the current COVID-19 pandemic highlights how this class of biologics can be crucial in the context of a therapeutic setting and how important the rapid and efficient development of biologics can be [7.Taylor P.C. et al.Neutralizing monoclonal antibodies for treatment of COVID-19.Nat. Rev. Immunol. 2021; 21: 382-393Crossref PubMed Scopus (53) Google Scholar]. Moreover, mAbs can potentially play key roles in prophylactic settings as well (passive immunization), especially when considering pathogens such as respiratory syncytial virus, for which effective vaccines are still unavailable [8.Slifka M.K. Amanna I.J. Passive immunization.Plotkin's Vaccines. 2018; : 84-95.e10Crossref Google Scholar]. Following the classical hybridoma technology (the first technology developed to isolate mAbs), many other methodologies have been developed and refined to improve or even radically change (e.g., phage display technology) the discovery of mAbs (Box 1) [3.Wilson P.C. Andrews S.F. Tools to therapeutically harness the human antibody response.Nat. Rev. Immunol. 2012; 12: 709-719Crossref PubMed Scopus (92) Google Scholar,9.Mondon P. et al.Human antibody libraries: a race to engineer and explore a larger diversity.Front. Biosci. 2008; 13: 1117-1129Crossref PubMed Scopus (45) Google Scholar,10.Walker L.M. Burton D.R. Passive immunotherapy of viral infections: 'super-antibodies' enter the fray.Nat. Rev. Immunol. 2018; 18: 297-308Crossref PubMed Scopus (139) Google Scholar]. Table 1 (Key table) lists some of the major published protocols that use primary B cells for the identification of antigen-specific antibodies, including their specific features, such as time expenditure, costs, ability to target difficult antigens, affinity of retrieved mAbs, ability to isolate rare binders, throughput, and ease of use.Box 1The advantage of B cell mAb selectionFrom a clinical point of view, there are several advantages to using human B cell-derived mAbs over other systems, such as phage display. Because of their in vivo development and affinity maturation, they show a rather high specificity towards their targets, usually proteins, allowing a tailored response with low off-target binding to other host proteins [15.Carter P.J. Potent antibody therapeutics by design.Nat. Rev. Immunol. 2006; 6: 343-357Crossref PubMed Scopus (855) Google Scholar]. Related to this first characteristic, they also usually bear low immunogenicity upon in vivo administration [115.Le Basle Y. et al.Physicochemical stability of monoclonal antibodies: a review.J. Pharm. Sci. 2020; 109: 169-190Abstract Full Text Full Text PDF PubMed Google Scholar]. Thus, hybridoma-derived mAbs have had, on average, a better general developability profile (a crucial aspect, considered the 'Achilles heel' of most biologics) compared to mAbs derived from, for example, phage display libraries [116.Spencer S. et al.Solubility evaluation of murine hybridoma antibodies.MAbs. 2012; 4: 319-325Crossref PubMed Scopus (36) Google Scholar, 117.Nixon A.E. et al.Drugs derived from phage display: from candidate identification to clinical practice.MAbs. 2014; 6: 73-85Crossref PubMed Scopus (0) Google Scholar, 118.Kaleli N.E. et al.Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues.Proteins. 2019; 87: 607-618Crossref PubMed Scopus (10) Google Scholar, 119.Sun A. Benet L.Z. Late-stage failures of monoclonal antibody drugs: a retrospective case study analysis.Pharmacology. 2020; 105: 145-163Crossref PubMed Scopus (9) Google Scholar, 120.Garripelli V.K. et al.Developability assessment for monoclonal antibody drug candidates: a case study.Pharm. Dev. Technol. 2021; 26: 11-20Crossref PubMed Scopus (1) Google Scholar]. This is also due to their native post-translational modification profile, which decreases the possibility of aggregation in vitro and importantly, in vivo [121.Jefferis R. Posttranslational modifications and the immunogenicity of biotherapeutics.J Immunol Res. 2016; 20165358272Crossref PubMed Scopus (63) Google Scholar,122.Jenkins N. et al.Post-translational modifications of recombinant proteins: significance for biopharmaceuticals.Mol. Biotechnol. 2008; 39: 113-118Crossref PubMed Scopus (139) Google Scholar].The theoretical size of the human B cell repertoire is between 1012 and 1018 different B cell clones (defined as the mathematical possibility of different V(D)J recombinations) [123.Elhanati Y. et al.Inferring processes underlying B-cell repertoire diversity.Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2015; 37020140243Crossref PubMed Scopus (84) Google Scholar]. However, in reality the number of different B cell clones within an individual is lower (around 1×107–2×107 different circulating B cell clonotypes) [124.Soto C. et al.High frequency of shared clonotypes in human B cell receptor repertoires.Nature. 2019; 566: 398-402Crossref PubMed Scopus (90) Google Scholar]. This might be due to several factors, such as the sample size of each donor used to estimate the clonal repertoire, out-of-frame mutations inserted during V(D)J recombination, and the formation of self-reactive BCRs which eventually undergo clonal deletion [123.Elhanati Y. et al.Inferring processes underlying B-cell repertoire diversity.Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2015; 37020140243Crossref PubMed Scopus (84) Google Scholar, 124.Soto C. et al.High frequency of shared clonotypes in human B cell receptor repertoires.Nature. 2019; 566: 398-402Crossref PubMed Scopus (90) Google Scholar, 125.Rees A.R. Understanding the human antibody repertoire.MAbs. 2020; 121729683Crossref PubMed Scopus (11) Google Scholar]. Yet, one must acknowledge that in display technologies, such as phage display, the repertoire size of naïve B cell libraries is usually above 109 possible combinations, outpacing de facto the natural B cell repertoire size in a given sample [126.Lim C.C. et al.Development of a phage display panning strategy utilizing crude antigens: isolation of MERS-CoV nucleoprotein human antibodies.Sci. Rep. 2019; 9: 6088Crossref PubMed Scopus (9) Google Scholar,127.Sheets M.D. et al.Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6157-6162Crossref PubMed Scopus (0) Google Scholar]. This aspect can be partially compensated by the possibility of obtaining PBMC-derived B cells from many different healthy donors or patients.An important aspect of retrieving antibody-coding sequences from (single) B cells is the reliable PCR-based amplification of VH and VL genes, their pairing, and sequencing. A variety of different degenerate primers/primer sets (usually for nested PCR) have been designed and are available for the most commonly analyzed species, especially humans and mice. In our experience, the primer sets published by von Boehmer et al. allow to consistently amplify murine Ig genes [51.von Boehmer L. et al.Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat. Protoc. 2016; 11: 1908-1923Crossref PubMed Scopus (60) Google Scholar]. For human Ig genes, the primer sets published by Friedensohn et al. have also demonstrated good results [128.Friedensohn S. et al.Synthetic standards combined with error and bias correction improve the accuracy and quantitative resolution of antibody repertoire sequencing in human naive and memory B cells.Front. Immunol. 2018; 9: 1401Crossref PubMed Scopus (0) Google Scholar].Table 1Key table. Platforms used to discover antigen-specific mAbs from single B cellsbThe different techniques discussed in this review were compared by considering several relevant parameters such as time requirement, cost, possibility of screening difficult antigens (Box 3), chances of identifying rare binders, throughput, and ease of screening., aColor legend: green: good performance, yellow: medium performance, red: low performancea Color legend: green: good performance, yellow: medium performance, red: low performanceb The different techniques discussed in this review were compared by considering several relevant parameters such as time requirement, cost, possibility of screening difficult antigens (Box 3), chances of identifying rare binders, throughput, and ease of screening. From a clinical point of view, there are several advantages to using human B cell-derived mAbs over other systems, such as phage display. Because of their in vivo development and affinity maturation, they show a rather high specificity towards their targets, usually proteins, allowing a tailored response with low off-target binding to other host proteins [15.Carter P.J. Potent antibody therapeutics by design.Nat. Rev. Immunol. 2006; 6: 343-357Crossref PubMed Scopus (855) Google Scholar]. Related to this first characteristic, they also usually bear low immunogenicity upon in vivo administration [115.Le Basle Y. et al.Physicochemical stability of monoclonal antibodies: a review.J. Pharm. Sci. 2020; 109: 169-190Abstract Full Text Full Text PDF PubMed Google Scholar]. Thus, hybridoma-derived mAbs have had, on average, a better general developability profile (a crucial aspect, considered the 'Achilles heel' of most biologics) compared to mAbs derived from, for example, phage display libraries [116.Spencer S. et al.Solubility evaluation of murine hybridoma antibodies.MAbs. 2012; 4: 319-325Crossref PubMed Scopus (36) Google Scholar, 117.Nixon A.E. et al.Drugs derived from phage display: from candidate identification to clinical practice.MAbs. 2014; 6: 73-85Crossref PubMed Scopus (0) Google Scholar, 118.Kaleli N.E. et al.Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues.Proteins. 2019; 87: 607-618Crossref PubMed Scopus (10) Google Scholar, 119.Sun A. Benet L.Z. Late-stage failures of monoclonal antibody drugs: a retrospective case study analysis.Pharmacology. 2020; 105: 145-163Crossref PubMed Scopus (9) Google Scholar, 120.Garripelli V.K. et al.Developability assessment for monoclonal antibody drug candidates: a case study.Pharm. Dev. Technol. 2021; 26: 11-20Crossref PubMed Scopus (1) Google Scholar]. This is also due to their native post-translational modification profile, which decreases the possibility of aggregation in vitro and importantly, in vivo [121.Jefferis R. Posttranslational modifications and the immunogenicity of biotherapeutics.J Immunol Res. 2016; 20165358272Crossref PubMed Scopus (63) Google Scholar,122.Jenkins N. et al.Post-translational modifications of recombinant proteins: significance for biopharmaceuticals.Mol. Biotechnol. 2008; 39: 113-118Crossref PubMed Scopus (139) Google Scholar]. The theoretical size of the human B cell repertoire is between 1012 and 1018 different B cell clones (defined as the mathematical possibility of different V(D)J recombinations) [123.Elhanati Y. et al.Inferring processes underlying B-cell repertoire diversity.Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2015; 37020140243Crossref PubMed Scopus (84) Google Scholar]. However, in reality the number of different B cell clones within an individual is lower (around 1×107–2×107 different circulating B cell clonotypes) [124.Soto C. et al.High frequency of shared clonotypes in human B cell receptor repertoires.Nature. 2019; 566: 398-402Crossref PubMed Scopus (90) Google Scholar]. This might be due to several factors, such as the sample size of each donor used to estimate the clonal repertoire, out-of-frame mutations inserted during V(D)J recombination, and the formation of self-reactive BCRs which eventually undergo clonal deletion [123.Elhanati Y. et al.Inferring processes underlying B-cell repertoire diversity.Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2015; 37020140243Crossref PubMed Scopus (84) Google Scholar, 124.Soto C. et al.High frequency of shared clonotypes in human B cell receptor repertoires.Nature. 2019; 566: 398-402Crossref PubMed Scopus (90) Google Scholar, 125.Rees A.R. Understanding the human antibody repertoire.MAbs. 2020; 121729683Crossref PubMed Scopus (11) Google Scholar]. Yet, one must acknowledge that in display technologies, such as phage display, the repertoire size of naïve B cell libraries is usually above 109 possible combinations, outpacing de facto the natural B cell repertoire size in a given sample [126.Lim C.C. et al.Development of a phage display panning strategy utilizing crude antigens: isolation of MERS-CoV nucleoprotein human antibodies.Sci. Rep. 2019; 9: 6088Crossref PubMed Scopus (9) Google Scholar,127.Sheets M.D. et al.Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6157-6162Crossref PubMed Scopus (0) Google Scholar]. This aspect can be partially compensated by the possibility of obtaining PBMC-derived B cells from many different healthy donors or patients. An important aspect of retrieving antibody-coding sequences from (single) B cells is the reliable PCR-based amplification of VH and VL genes, their pairing, and sequencing. A variety of different degenerate primers/primer sets (usually for nested PCR) have been designed and are available for the most commonly analyzed species, especially humans and mice. In our experience, the primer sets published by von Boehmer et al. allow to consistently amplify murine Ig genes [51.von Boehmer L. et al.Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat. Protoc. 2016; 11: 1908-1923Crossref PubMed Scopus (60) Google Scholar]. For human Ig genes, the primer sets published by Friedensohn et al. have also demonstrated good results [128.Friedensohn S. et al.Synthetic standards combined with error and bias correction improve the accuracy and quantitative resolution of antibody repertoire sequencing in human naive and memory B cells.Front. Immunol. 2018; 9: 1401Crossref PubMed Scopus (0) Google Scholar]. In this Review, we focus on these most frequently used technologies based on single B cell retrieval to identify antigen-specific mAbs. This includes the hybridoma technology, single B cell cultures using conventional laboratory equipment, surface staining techniques, single B cell cultures in miniaturized equipment, as well as single B cell replica methodologies and protocols based on high throughput genetic analysis of single B cells (Figure 1, Figure 2). With the aim of providing a sound overview that supports the selection of a suitable technology when embarking on mAb discovery and isolation, these technologies are explained at a technical level, followed by a discussion of their key advantages and disadvantages (Table 1). In a first section, the technologies are presented and discussed in the context of conventional cell cultures, while the second section comprises microfluidics-based technologies.Figure 2Single B cell screening technologies using miniaturized equipment.Show full caption(A) Single B cell screening methodologies. Usually, ASCs are used with these technologies, as all rely on secreted antibodies. Alternatively, activated memory B cells can also be used. We divided methodologies into open and closed assays. Among the open assays is the Beacon (Berkeley Lights) technology, the microfluidic chamber, the microengraved systems, and the microcapillary system. Closed assays comprise all the methodologies relying on encapsulation of single cells in water-in-oil droplets. Both open and closed technologies mainly rely on the detection of antigen-specific antibodies via either colocalization of secreted antibodies and beads displaying antigen on their surface, or soluble antigen that is bound by anti-IgG-captured secreted antibodies either via colocalization of different fluorophores or via generation of a Förster resonance energy transfer (FRET) signal. (B) B cell replica. Primary B cells are encapsulated in a water-in-oil droplet and paired VH–VL sequences are amplified in one sequence using oligos containing complementary antibody constant region sequences. In order to do so, cells are lysed and both the VH and VL single B cell-mRNAs anneal to the encapsulated oligos. Upon emulsion breaking, the library is then prepared through RT-PCR and cloned into suitable expression vectors. This library is then transformed into expression cells such as yeast, phage, or mammalian cells. The obtained cell population is then further used in conventional single cell screenings such as surface staining with fluorescently labeled antigens or ELISA of antibody secreted from these single engineered host cells. (C) Single B cell analysis (VH–VL paired). Primary B cells are encapsulated together with beads bearing barcoded-oligos and subsequently lysed, allowing the oligos to anneal with single-cell total mRNAs. Upon emulsion breaking, these oligos are then amplified and prepared for next-generation sequencing. After data analysis the antibody clones are selected mainly based on clonotype expansion (particular VH–VL pairing), but also potentially considering other characteristics (see main text). Antibody sequences of the most expanded clones (or taking into account additional features) are then synthesized, cloned into suitable plasmids, and recombinant monoclonal antibodies are generated; these are then tested with conventional techniques, for example, ELISA. This figure was created using BioRender (https://biorender.com). Abbreviation: mAb, monoclonal antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Single B cell screening methodologies. Usually, ASCs are used with these technologies, as all rely on secreted antibodies. Alternatively, activated memory B cells can also be used. We divided methodologies into open and closed assays. Among the open assays is the Beacon (Berkeley Lights) technology, the microfluidic chamber, the microengraved systems, and the microcapillary system. Closed assays comprise all the methodologies relying on encapsulation of single cells in water-in-oil droplets. Both open and closed technologies mainly rely on the detection of antigen-specific antibodies via either colocalization of secreted antibodies and beads displaying antigen on their surface, or soluble antigen that is bound by anti-IgG-captured secreted antibodies either via colocalization of different fluorophores or via generation of a Förster resonance energy transfer (FRET) signal. (B) B cell replica. Primary B cells are encapsulated in a water-in-oil droplet and paired VH–VL sequences are amplified in one sequence using oligos containing complementary antibody constant region sequences. In order to do so, cells are lysed and both the VH and VL single B cell-mRNAs anneal to the encapsulated oligos. Upon emulsion breaking, the library is then prepared through RT-PCR and cloned into suitable expression vectors. This library is then transformed into expression cells such as yeast, phage, or mammalian cells. The obtained cell population is then further used in conventional single cell screenings such as surface staining with fluorescently labeled antigens or ELISA of antibody secreted from these single engineered host cells. (C) Single B cell analysis (VH–VL paired). Primary B cells are encapsulated together with beads bearing barcoded-oligos and subsequently lysed, allowing the oligos to anneal with single-cell total mRNAs. Upon emulsion breaking, these oligos are then amplified and prepared for next-generation sequencing. After data analysis the antibody clones are selected mainly based on clonotype expansion (particular VH–VL pairing), but also potentially considering other characteristics (see main text). Antibody sequences of the most expanded clones (or taking into account additional features) are then synthesized, cloned into suitable plasmids, and recombinant monoclonal antibodies are generated; these are then tested with conventional techniques, for example, ELISA. This figure was created using BioRender (https://biorender.com). Abbreviation: mAb, monoclonal antibody. The ex vivo survival of B cells is generally difficult to sustain, which poses a challenge for the identification of B cells expressing an antigen-specific antibody (immunoglobulin). To increase their lifespan, immortalization procedures have been developed, such as the fusion of primary B cells with immortalized myeloma cells (hybridoma technology), or immortalization of human B cells by Epstein–Barr virus (EBV) infection. Alternatively, B cell culture conditions have been refined to: (i) either maintain antibody-secreting B cells long enough in culture to screen their secreted antibodies for specificity or function; or (ii) culture times have been minimized by enrichment of antigen-specific B cells. In this section, we describe and discuss these technologies and provide a graphical illustration in Figure 1. The hybridoma technology is probably the best-known immortalization technique of mouse B cells and since its development, has revolutionized mAb discovery. The key principle of this technology is to immortalize short-lived antibody-secreting cells (ASCs) by fusion with myeloma cells, generating immortal clones whose supernatant can be screened for the presence of antigen-specific antibodies [11.Parray H.A. et al.Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives.Int. Immunopharmacol. 2020; 85106639Crossref PubMed Scopus (21) Google Scholar]. Most commonly, immunized mice are rechallenged with the antigen of interest and a few days later, spleen cells, containing ASCs, are used for the immortalization procedure (Box 2). A variety of additional protocols have been developed that use primary ASCs derived from other organs and compartments, such as bone marrow, lymph nodes, and peripheral blood mononuclear cells (PBMCs) [12.Basalp A. Yucel F. Development of mouse hybridomas by fusion of myeloma cells with lymphocytes derived from spleen, lymph node, and bone marrow.Hybrid Hybridomics. 2003; 22: 329-331Crossref PubMed Scopus (0) Google Scholar]. The original protocol relied on PEG-mediated fusion of splenic short-lived murine ASCs, naturally expressing the enzyme hypoxanthine–guanine phosphoribosyltransferase (HGPRT+), with immortalized murine myeloma cells (HGPRT-) [1.Kohler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.Nature. 1975; 256: 495-4927Crossref PubMed Google Scholar,13.de Almeida R. et al.Enhanced immunization techniques to obtain highly specific monoclonal antibodies.MAbs. 2018; 10: 46-54Crossref PubMed Scopus (6) Google Scholar]. This protocol has been further developed to accommodate the use of ASCs derived from different species [11.Parray H.A. et al.Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives.Int. Immunopharmacol. 2020; 85106639Crossref PubMed Scopus (21) Google Scholar].Box 2Immunization protocolThis first step of immunizing host species with a specific antigen of interest is still common to many B cell screening systems, relying heavily on in vivo generated antigen-specific B cells from different species. Alternatively, B cells can be selected from healthy volunteers, individuals, or animals known to be seropositive for a specific antigen of interest. Different improvements of the immunization strategy, such as adjuvants used along with the antigen of interest and optimized immunization schedules, have been developed to elicit strong humoral responses, including those against difficult antigens such as membrane proteins, GPCRs, ion channels, and carbohydrates [131.Dodd R.B. et al.Therapeutic monoclonal antibodies to complex membrane protein targets: antigen generation and antibody discovery strategies.BioDrugs. 2018; 32: 339-355Crossref PubMed Scopus (8) Google Scholar, 132.Chiarella P. Fazio V.M. Mouse monoclonal antibodies in biological research: strategies for high-throughput production.Biotechnol. Lett. 2008; 30: 1303-1310Crossref PubMed Scopus (0) Google Scholar, 133.Aucouturier J. et al.Adjuvants designed for veterinary and human vaccines.Vaccine. 2001; 19: 2666-2672Crossref PubMed Scopus (0) Google Scholar]. Usually, researchers still rely on the classical prime–boost strategy, using specific boosting schedules with respect to the number of boosts and time intervals between them. The antigen of interest used for immunization has to be carefully selected because the antigen must remain in the original conformation