Monoclonal antibodies (MAbs) are being used with explosive frequency and in ever-increasing amounts in bio-medical research and may play an important role in the future diagnosis and treatment of human diseases. This project proposes to design a system for the formation of hybridomas secreting human MAbs using in vitro immunization and to design a method to grow these hybridomas in vitro in large numbers. To reduce the problems of contamination of MAb preparations with microbes, endotoxins and foreign proteins and to enhance the efficiency of hybrid formation, all phases hybridoma growth and formations will be done in a completely serum-free defined medium which contains less than 15 ug/ml of protein. In vitro immunization will be carried out using alcohol-fixed human B lymphoma cells as antigen using a technique that has been used for the successful formation of murine hybrids in vitro. Large-scale growth in serum-free medium of antigen-specific hybrids will be done using large spinner flasks. Purification of MAbs from these cultures will be done by ultrafiltration, ammonium sulfate precipitation and dialysis, resulting in preparations of over 90% purity at minimal cost.
