A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning

Abstract

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40 years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12 days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4 days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

“…B cells may also be a source of antigen-specific antibodies for clinical diagnostics or for therapeutic applications. Existing methods to identify antigen-specific B cells and generate monoclonal antibodies include ex vivo B cell culture approaches, which could promote the survival and expansion of certain B cell subsets, screening of the culture supernatants to identify B cell reactivity and fluorescent-activated cell sorting (15)(16)(17)(18)(19)(20). An essential element in the process of selecting antigen-specific B cells is detection of antibodies with a certain degree of specificity.…”

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

“…Constructs encoding antibody heavy and light chains were built from gBlock DNA fragments (Integrated DNA Technologies, Redwood City, CA, USA) and assembled using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) [36]. The κ-acceptor vector was built using a murine κ chain leader sequence (Figure 1), which was separated by a NotI restriction site from the human κ-chain constant region (IGKC*01) in the pcDNA3.4 backbone plasmid (Invitrogen, Carlsbad, CA, USA).…”

Platelet Derived Growth Factor Receptor β (PDGFRβ) is a Host Receptor for the human malaria parasite adhesin TRAP

“…Production of recombinant proteins in suspension HEK293F cells cultures was performed, as previously described (28,36,37) . Briefly, each expression construct contained codon-optimized sequences encoding a tissue plasminogen activator signal peptide (38) , the full extracellular domain or subdomains from P. yoelii TRAP (PyTRAP) (44,45) and is absent from TRAP in vivo (46) .…”

“…For PfTRAP blocking experiments monoclonal antibodies recognizing PfTRAP were produced as previously described (37) . Briefly, BALB/cJ mice were immunized intramuscularly three times, three weeks apart, with 20 µg of PfTRAP full ectodomain formulated in Adjuplex (Sigma Aldrich).…”

“…Proteins were purified by Ni 2+ -affinity chromatography followed by size-exclusion chromatography. Subsequently, a BirA biotin-protein ligase kit (Avidity, LLC; Aurora ,CO, USA) was used to biotinylate protein samples, as previously described (37) .…”