A rapid and scalable method for selecting recombinant mouse monoclonal antibodies

Abstract

BackgroundMonoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.ResultsIncreased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.ConclusionsThis method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

Developing high-quality mouse monoclonal antibodies for neuroscience research – approaches, perspectives and opportunities

“…However, we are now generating recombinant NeuroMabs by post facto cloning from hybridomas, using a protocol developed by Wright and colleagues [46; 47]. This archiving of NeuroMabs is independent of cryopreserved hybridomas and is a route to enhancing their unambiguous identification by sequence-based methods such as MS-based sequencing of the native or recombinant mAb preparation [12].…”

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

“…The in vivo use of the mammalian immune system combined with hybridoma generation to produce monoclonal antibodies was first established in 1975 [3]. Because the maturation of the antibody producing B-lymphocytes isolated to produce the immortal hybridoma cell lines occurs in vivo and includes somatic hypermutation, in vivo approaches tend to produce high affinity antibodies at the expense of production time and cost [3,4]. Alternatively, the in vitro use of phage display monoclonal antibody libraries can dramatically decrease production time; however, due to the lack of the hypermutation maturation process, these antibodies tend to have lower binding affinity and often fail when used by cell and developmental biologists in immunofluorescence and immunohistochemistry protocols requiring multiple sample washings [5].…”

“…Further, the use of chemically synthesized peptides for immunogens, although cost effective and rapidly produced, often do not adequately replicate the natively folded protein resulting in the production of antibodies that recognize only the denatured form of the protein (i.e. western blots) and are of limited utility in cell based assays [4]. …”

“…However, due to the presence of highly immunogenic glycans present in the lysates, these studies generally inefficient at producing high quality antibodies to specific targets using standard ELISA based screening methods [6]. Recent efforts have refined the approach using pools of purified protein fragments for in vivo immunization combined with ELISA based screening and recombinant DNA methods to rapidly clone the heavy and light chains into mammalian expression vectors to produce multiple high quality antibodies from a single mouse [4]. Similarly, in work to define targetable antibodies to membrane proteins associated with triple negative breast cancer, researchers immunized mice with intact breast cancer cells and were able to generate over 4,000 hybridoma colonies that were screened by FACS to identify antibodies that were able to bind MDA-MB-231 triple negative breast cancer cells [7].…”

Advances in Monoclonal Antibodies Production and Cancer Therapy

“…Currently, for the isolation of high affinity mAbs by somatic hypermutation and affinity maturation technique, the direct molecular cloning of identical pairs of antibody light chain lambda variable (VLλ), heavy chain (IgH) variable (VH) and light chain kappa variable (VLκ) genes from single antigen-specific plasma/plasmablast cells (ASPCs) with the help of polymerase chain reaction (PCR) is an alternative technique being used for the development of mAb from immunized animals [37][38][39][40][41][42] [43][44][45][46][47][48][49][50][51][52][53].…”