An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies.
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.
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Xiaocong
X
Yu
Patricia A
PA
McGraw
Frances S
FS
House
James E
JE
Crowe
D000630:Aminopterin / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000276:immunology; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D002459:Cell Fusion; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral; D018920:Coculture Techniques; D004854:Herpesvirus 4, Human / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D019271:Hypoxanthine / Q000494:pharmacology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D053122:Influenza A Virus, H3N2 Subtype / Q000276:immunology*; D009101:Multiple Myeloma / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology*; D013936:Thymidine / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; C408982:CPG-oligonucleotide; D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D019271:Hypoxanthine; D010042:Ouabain; D000630:Aminopterin; D013936:Thymidine