Efficient selection of high-affinity B cell hybridomas using antigen-coated magnetic beads.
A method for the selection of antigen-specific B cell hybridomas using antigen-coated magnetic beads is described. Stable B cell hybridoma cell lines directed against human thyroglobulin were incubated with thyroglobulin-coated beads. 2 h of incubation at 4 degrees C using bead-to-cell ratios of at least 3:1 were found to be the optimal conditions for rosette formation. Rosettes were efficiently isolated with a strong magnet. Rosette formation was antigen-specific since irrelevant hybridoma cell lines could not form rosettes, nor could BSA-coated or uncoated beads form rosettes. Free antibodies produced by the hybridoma cells were able to block rosette formation. Blocking of rosette formation permitted the identification of different and overlapping epitopes recognized by four different hybridomas. Using six stable hybridoma cell lines with different affinities for thyroglobulin, rosette formation appeared to be dependent on the affinity of the immunoglobulin membrane receptor for antigen. A correlation was observed between the affinity of the secreted antibodies and the capacity of the hybridomas to form rosettes, suggesting that this method is suitable for the selection of hybridomas producing antibodies with a high affinity for the antigen. Antigen-coated magnetic beads were found to be suitable for the efficient selection of thyroglobulin-specific hybridoma cells from bulk cultures shortly after fusion. A 300-fold enrichment of thyroglobulin-specific cells was obtained using this method.
Ossendorp
F A
FA
Division of Tumor Biology, Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Amsterdam.
Bruning
P F
PF
Van den Brink
J A
JA
De Boer
M
M
D000818:Animals; D000915:Antibody Affinity; D001402:B-Lymphocytes / Q000166:cytology; D001667:Binding, Competitive; D002469:Cell Separation / Q000379:methods*; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D006825:Hybridomas; D007163:Immunosorbent Techniques; D008280:Magnetics; D051379:Mice; D008807:Mice, Inbred BALB C; D012397:Rosette Formation; D013696:Temperature; D013954:Thyroglobulin / Q000276:immunology
D000939:Epitopes; D013954:Thyroglobulin