A rapid and scalable method for selecting recombinant mouse monoclonal antibodies

Abstract

Background: Monoclonal 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.

Results: Increased 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.

Conclusions: This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

Figure 1

Overview of a scalable procedure to select recombinant mouse monoclonal antibodies. Purified ectodomain fragments of zebrafish cell surface and secreted proteins expressed in mammalian cells were normalized, pooled and used to immunize mice. Hybridomas were generated by cell fusion, and supernatants screened for positives using ELISA. The rearranged light and heavy antibody chains were amplified from RNA extracted from small numbers of hybridoma cells by reverse transcription-polymerase chain reaction and cloned into a single expression plasmid. The recombinant monoclonal antibodies were produced by transfecting mammalian cells before testing for fixation sensitivity and wholemount staining.

Figure 2

Cloning of the 5F11 monoclonal antibody. (a) Reverse transcriptase-polymerase chain reaction (PCR) amplification of the variable light (V_Lκ) and heavy (V_H) antibody chains from the 5F11 hybridoma. (b) Assembly of the 5F11 V_Lκ and V_H regions by fusion PCR through a joining fragment resulting in a 2.4 kbp product. (c) Schematic of the plasmid encoding the recombinant antibody with the fusion PCR product cloned between NotI and AscI. (d) Western blot run under reducing (left panel) or non-reducing (right panel) conditions: tissue culture medium (Med), supernatant of cells producing the recombinant 5F11 antibody (Rec) or tissue culture medium spiked with the 5F11 monoclonal antibody purified from the parent hybridoma (Hyb). Staining of the recombinant 5F11 [Rec 5F11, (e)] is indistinguishable from that of the parent hybridoma [Hyb 5F11, (f)], red. Nuclei are counterstained in blue (DAPI). Abbreviations: cns = central nervous system; e = eye; CMV = cytomegalovirus promoter; L = leader sequence; C = constant region; V = variable region.

Figure 3

Sensitivity to formalin treatment and affinity measurement of recombinant antibodies. (a) Recombinant antibodies SI1-Unc5b and SI2-Unc5b were tested for formalin fixation sensitivity by ELISA following antigen treatment with a 4% formalin solution. (b-e) The dissociation rates of the recombinant (red line) antibodies were compared to the original hybridoma antibodies (blue line) using surface plasmon resonance. Antibodies are: (b) SI3-Flrt3, (c) SI4-Jamc.2, (d) SI2-Unc5b and (e) SI5-Lrrn1. In order to facilitate comparison, binding at saturation was normalized to 100% and rebinding was minimized by immobilizing low levels of target protein and washing at 37°C using high flow rates (100 μL/min).

Figure 4

Recombinant monoclonal antibodies recapitulate their gene expression patterns on wholemount fixed zebrafish embryos. (a-c) Dorsal views of a 24 hpf zebrafish embryo showing flrt3 gene expression by in situ hybridization on the midbrain side of the mid-hindbrain boundary (a) and antibody staining with SI3-Flrt3 [green in (b), white in (c)]. (d-f) Lateral view of a dissected 24 hpf eye showing expression of the unc5b gene in the dorsal retina (d) and antibody staining with SI2-Unc5b [green in (e), white in (f)]. (b, e) red = DAPI nuclear stain. Scale bars = 50 μm.