A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

Abstract

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.

Fig 1. Characterization of mouse monoclonal antibodies with BIAcore.

Each of four goat anti-mouse IgG Fc ɣ1, ɣ2a, ɣ2b or ɣ2c polyclonal antibodies was coated on the four different channels respectively on a CM5 chip. (A) A representative coating process of goat anti-mouse IgG ɣ1 Fc specific antibodies on the first channel of a CM5 chip. (B) Binding of IgG ɣ1 monoclonal antibody HO14 detected positive by OVA coated ELISA with native OVA protein. The blue curve represents signal of the anti-IgG ɣ2b channel subtracted by the anti-IgG ɣ2c channel. (C) Binding of IgG ɣ1 monoclonal antibody HO14 detected positive by OVA coated ELISA with native OVA protein. The dissociation rate (Kd) and association rate (Ka) between HO1 antibody and native OVA protein were calculated from the corresponding curve.

Fig 2. Two applications of detecting antibody-antigen interaction with BIAcore.

(A) Detecting three native MBP binding antibodies separately using BIAcore. (B) Characterizing the interaction of these three antibodies with MBP using BIAcore. (C) A cartoon for the interaction of these three antibodies with MBP. (D) Pairing of MBP antibodies as coating and detection antibodies for detection of MBP. (E) Detection of interaction between two monoclonal SARS-CoV spike protein specific antibodies with spike and virus receptor ACE2.

Fig 3. Hybridoma cells secreting mouse IgG antibodies also have surface IgG expression.

(A) Staining of surface IgG expression on mouse IgG hybridoma cells with isotype specific antibodies. (B) Detection of secreted and membrane form of IgG heavy chain mRNA expression by RT-PCR.

Fig 4. Hybridoma cell surface IgG expression correlates with antibody secretion.

(A) Cell surface IgG staining of S161.1 hybridoma cell line. (B) Antibody secretion by the sorted surface IgG negative or positive cells from the S161.1 cell line. (C) Cell surface IgG staining of negative secretors or positive secretors derived from single cell colony by limiting dilution method.

Fig 5. Isolation of IgG ɣ2a antibody secreting cells from an IgG ɣ2b hybridoma cell line.

(A) Detection of IgG ɣ2a antibody secreting hybridoma cells in S212, an IgG ɣ2b hybridoma cell line with ELISPOT. (B) Cell surface IgG staining of S212 cells. (C) Enrichment of IgG ɣ2a secreting cells by MACS and single cell sorting of these heavy chain switched cells. (D) Biacore testing of S212 IgG ɣ2b antibody and the switched IgG ɣ2a antibody.

Fig 6. Sorting of native antigen binding IgG hybridomas from a fusion bulk mixture.

(A) Staining of OVA specific hybridoma cell line with fluorescent OVA. (B) Sorting of OVA specific hybridoma clls from a fusion bulk mixture.