[Production of human monoclonal autoantibodies that react with islet-cell-surface antigens by EBV-transformed lymphocytes: new method of preselection of ICSA producing lymphocyte by protein A magnetic microspheres].
Because of the low titer of ICSA, as well as the polyclonality of serum, it has been difficult to determine whether there is a variety of autoantibodies, each reacting with a different organ, or whether the autoimmune response is more restricted, with only a limited number of autoantibodies recognizing common antigenic determinants in the different organs. To study this and other questions, we sought to prepare human monoclonal autoimmune ICSA. Peripheral-blood lymphocytes from patients with freshly onset IDDM or other autoimmune abnormalities were transformed by EB virus. And rapid screening on In 111 cells by an ELISA were used to identify monoclonal ICSA. But, ICSA titer was very low even in freshly onset IDDM patients, and reduced very quickly. ICSA positive wells at cloning were extremely rare. So, the methods that enhance the efficiency of cloning were required. Firstly, ultrasensitive Enzyme Immunoassay for ICSA were developed. Secondly, new preselection method of ICSA producing lymphocyte by Protein A Magnetic Microspheres were employed. Thirdly, in vitro immunization was partly employed to enhance the frequency of ICSA producing lymphocytes. Fourthly, EBV-hybridoma technique was employed to stabilize the ICSA producing clone. The efficiency of cloning was increased from 0.07% to 24% by the new preselection method. The sensitivity of the fluorometric enzyme immunoassay for ICSA was 140pg/ml. 21 times sensitivity to usual ELISA was obtained. We describe sure methods for production of Human Monoclonal Autoantibodies that react with Islet-Cell-Surface Antigens.
Tsushima
S
S
Second Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D000954:Antigens, Surface / Q000276:immunology*; D001323:Autoantibodies / Q000032:analysis* / Q000096:biosynthesis; D002469:Cell Separation / Q000379:methods; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D007515:Islets of Langerhans / Q000276:immunology*; D008213:Lymphocyte Activation; D008280:Magnetics; D008863:Microspheres; D051381:Rats; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001323:Autoantibodies; D013205:Staphylococcal Protein A