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

Abstract

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.

Broadly neutralizing antibodies with few somatic mutations and hepatitis C virus clearance

“…Screening was performed against E1E2 variants 1a53 (autologous) and 1a154 (H77) (heterologous) for subject 117 and 1a53 (heterologous) and 1a38 (autologous) variants for subject 110. Cells from wells with supernatants reacting with HCV antigens were fused with HMMA2.5 myeloma cells using an electrofusion technique (40). After fusion, hybridoma cell lines were cloned by limited dilutions and single-cell fluorescence-activated cell sorting and expanded in post-fusion medium as previously described (39).…”

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

“…After 1 wk, culture supernatants were screened by ELISA for binding to recombinant, postfusion RSV A2 F protein and FFL_001. Cells from positive wells were fused with HMMA2.5 myeloma cells by electrofusion (26). Fused cells were plated in 384-well plates in growth medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT Media Supplement, Sigma), and 7 μg/mL ouabain (Sigma).…”

Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization

“…4,22 Even though electrofusion of biological cells is potentially a useful method, achieving the sufficient efficiency still requires extensive trial-and-error studies. [23][24][25][26] One of the earliest approaches to improve electrofusion efficiency was the use of hypotonic electrofusion buffer that resulted in the considerable fusion efficiency increase. [27][28][29][30][31][32][33][34] To ensure the improvement of fusion efficiency in hypotonic buffer the duration and the osmolarity of the hypotonic treatment has to molarity of solutions was determined with Knauer vapor pressure osmometer K-7000 (Knauer, Wissenschaftliche Gerätebau, Germany).…”