Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.
Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.
Kipriyanov
S M
SM
Diagnostics and Experimental Therapy Programme, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Kupriyanova
O A
OA
Little
M
M
Moldenhauer
G
G
D000595:Amino Acid Sequence; D000818:Animals; D018941:Antigens, CD19 / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000032:analysis* / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis* / Q000276:immunology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D018941:Antigens, CD19; D000954:Antigens, Surface; D007141:Immunoglobulin Fc Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins