title: A two-stage culture system for the induction of antibody-forming cell clones in cultures of normal human blood lymphocytes.
abstract: A two-stage culture method is described for the induction of a specific antibody response to sheep red cells (SRC) in microcultures at limiting dilutions of human peripheral blood lymphocytes (PBL). PBL from normal donors were cultured for 4 days with antigen and EBV using well defined conditions. The cells were then distributed in 10 microliter microcultures at different cell densities in order to estimate the frequency of responding units. The culture wells were tested for the presence of anti-SRC antibody by the spot test. The results show that the expression of antibody-forming cell clones in the second stage microcultures is strictly dependent on the presence of both antigen and EBV during the first stage cultures. The efficiency of the system was improved by the addition of 4% polyethylene glycol (PEG, MW 6000) in the first stage and its removal in the second stage and by the use of human serum (instead of fetal calf) in both stages. This approach permits the separation of different cellular events, occurring when human B cells are stimulated by antigen and represents a useful approach for studying the mechanisms of the specific immune response in man.
authors.affiliation: Department of Immunology, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: A L
authors.initials: AL
authors.lastname: Luzzati
authors.forename: E
authors.initials: E
authors.lastname: Giacomini
authors.forename: P
authors.initials: P
authors.lastname: Frugoni
mesh_terms: D000017:ABO Blood-Group System; D000818:Animals; D000921:Antibody-Producing Cells / Q000378:metabolism*; D000941:Antigens / Q000276:immunology; D002478:Cells, Cultured; D002999:Clone Cells / Q000378:metabolism*; D003470:Culture Media; D005312:Fetal Blood; D006801:Humans; D007158:Immunologic Techniques; D011092:Polyethylene Glycols; D012756:Sheep; D013997:Time Factors
chemical_list: D000017:ABO Blood-Group System; D000941:Antigens; D003470:Culture Media; D011092:Polyethylene Glycols