A high-efficiency cloning system for single hapten-specific B lymphocytes that is suitable for assay of putative growth and differentiation factors.
Fluorescein (FLU)-specific murine splenic B lymphocytes from nonimmunized adult mice were prepared by the hapten-gelatin fractionation technique and cultured singly or in very small numbers in 10-microliters culture wells. Growth and differentiation to antibody-secreting status were promoted by polymeric FLU-conjugated antigens with or without added T-lymphocyte-derived conditioned media or purified cytokines. In some cultures, 3T3 fibroblasts or CBA/N thymocytes provided a source of filler cells. Anti-FLU antibody formation was detected by a sensitive enzyme-linked immunosorbent assay (ELISA). With an optimal number (around 300) of 3T3 cells per well, up to 77% of the B cells could be induced to produce detectable antibody. The ELISA permitted detection of antibody formation in essentially all wells where B-cell proliferation occurred, and it was more efficient in detecting antibody-forming clones than the hemolytic plaque assay, whether filler cells were present or not. When 10 B cells rather than 1 were included per well, the ELISA, detecting absorbance in standard fashion, provided a useful method for assessment of B-cell growth- and differentiation-promoting factors (BGDF). It was found that 3T3 cells gave less background stimulation than thymus cells, permitting the detection of as little as 1/100th as much BGDF as with thymocytes, thus offering a dynamic range of up to 30 between control absorbance in the absence of factors and the optimal factor level. Use of 3T3 cells also avoids a potential lymphokine cascade. The system has confirmed that interleukin-2 acts as a BGDF, but it has failed to establish an effect of interferon-gamma on B cells. It has also shown the inactivity of a variety of hemopoietic growth factors on B lymphocytes. This system thus promises to be a useful tool in the further analysis of B-lymphocyte activation.
D000818:Animals; D000917:Antibody Formation; D000921:Antibody-Producing Cells / Q000648:ultrastructure; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001681:Biological Assay / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts; D006133:Growth Substances / Q000032:analysis* / Q000494:pharmacology; D006241:Haptens; D006462:Hemolytic Plaque Technique; D015847:Interleukin-4; D008222:Lymphokines / Q000032:analysis* / Q000494:pharmacology; D051379:Mice
D006133:Growth Substances; D006241:Haptens; D008222:Lymphokines; D015847:Interleukin-4