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

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 appro...

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Bibliographic Details
Published in:PloS one 2015-08, Vol.10 (8), p.e0136613-e0136613
Main Authors: Liu, Haolin, White, Janice, Crawford, Frances, Jin, Niyun, Ju, Xiangwu, Liu, Kangtai, Jiang, Chengyu, Marrack, Philippa, Zhang, Gongyi, Kappler, John W
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal, Murine-Derived - chemistry
Antibodies, Monoclonal, Murine-Derived - immunology
Antigens
Antigens - chemistry
Antigens - immunology
B-Lymphocytes - chemistry
B-Lymphocytes - immunology
Binding
Biomedical research
Care and treatment
Cell culture
Cloning
Crystallography
Development and progression
Dilution
Enzyme-linked immunosorbent assay
Flow cytometry
Fluorescence
Histology
Hybridomas
Hybridomas - chemistry
Hybridomas - immunology
Identification methods
Immunoglobulin G
Immunoglobulin G - chemistry
Immunoglobulin G - immunology
Immunoglobulins
Immunology
In vivo methods and tests
Isotypes
Laboratory animals
Lymphocytes B
Methods
Mice
Molecular biology
Monoclonal antibodies
Multiple myeloma
Patient outcomes
Proteins
Risk factors
Surface plasmon resonance
X-ray crystallography
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0136613