An ELISA assay efficiently detects clonal antibody formation by single, hapten-specific B lymphocytes.
Spleen cells from non-immunized adult mice were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B cells. These B cells were cultured either singly or in very small numbers in 10 microliter microcultures with 0.1 microgram/ml of the T cell-independent (TI) antigen FLU-E. coli lipopolysaccharide (FLU-LPS). Cultures were either filler cell-free, or supported by the addition of 10(5) CBA/N thymus cells per well. At 4-6 days, culture supernatants were assayed for the presence of anti-FLU antibody either by an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). With the filler cell-free cultures, B cell proliferation was scored microscopically before removal of culture supernatant. The cultured cells from each well were assayed for their capacity to form directly hemolytic FLU-specific plaques. In the filler cell-free system, the ELISA was much more sensitive than the plaque assay in identifying antibody-forming cell (AFC) clones, with over 10% of fractionated B cells yielding clones secreting detectable antibody, though with a low mean optical density (OD). This value represented over 80% of the proliferating clones. In the more efficient, filler cell-supported system, the difference between the 2 read-out methods was smaller. Here, one half of the hapten-specific B cells formed AFC clones, the highest cloning efficiency yet reported for an antigen-driven system. Comparative studies showed the RIA to be only marginally more sensitive than the ELISA, and not nearly as convenient for routine use.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D000915:Antibody Affinity; D000921:Antibody-Producing Cells / Q000032:analysis; D001402:B-Lymphocytes / Q000276:immunology*; D002458:Cell Fractionation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D019793:Fluorescein; D005452:Fluoresceins; D005780:Gelatin; D006241:Haptens / Q000276:immunology; D006462:Hemolytic Plaque Technique; D008297:Male; D051379:Mice; D011863:Radioimmunoassay; D013154:Spleen / Q000166:cytology; D013950:Thymus Gland / Q000276:immunology
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D006241:Haptens; D005780:Gelatin; D019793:Fluorescein