A rapid and scalable method for selecting recombinant mouse monoclonal antibodies.
BACKGROUND
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.


RESULTS
Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.


CONCLUSIONS
This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK.
Cécile
C
Crosnier
Nicole
N
Staudt
Gavin J
GJ
Wright
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015027:Zebrafish / Q000196:embryology / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins