An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

• Published in: Journal of immunological methods 2008-07, Vol.336 (2), p.142-151
• Main Authors: Yu, Xiaocong, McGraw, Patricia A., House, Frances S., Crowe, James E.
• Format: Article
• Language: English
• Subjects: Aminopterin - pharmacology; Antibodies; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Antibodies, Viral - biosynthesis; Antibodies, Viral - immunology; B-Lymphocytes - drug effects; B-Lymphocytes - immunology; Biological and medical sciences; Cell Fusion; Cell Line, Tumor; Cell Transformation, Viral; Coculture Techniques; Epstein-Barr virus; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Herpesvirus 4, Human - drug effects; Herpesvirus 4, Human - physiology; Humans; Humoral; Hybridomas; Hybridomas - immunology; Hypoxanthine - pharmacology; Immunity; Immunoglobulin G - biosynthesis; Immunoglobulin G - immunology; Influenza A Virus, H3N2 Subtype - immunology; Molecular immunology; Monoclonal; Multiple Myeloma - immunology; Oligodeoxyribonucleotides - pharmacology; Ouabain - pharmacology; Respiratory syncytial virus; Respiratory Syncytial Virus, Human - immunology; Techniques; Thymidine - pharmacology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2008.04.008

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.