Hybrid hybridomas producing bispecific antibodies to CEA and peroxidase isolated by a combination of HAT medium selection and fluorescence activated cell sorting.
A combination of fluorescence-activated cell sorting and HAT medium selection has been used to establish bispecific antibody (biAbs)-producing hybrid hybridomas. For this purpose hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient mutants were isolated from a hybridoma line (D11-DG2) producing anti-CEA antibodies by 8-azaguanine treatment. The resulting HAT-sensitive hybrid cells were stained with the fluorescence marker tetramethyl rhodamine isothiocyanate (TRITC) and fused by polyethylene glycol (PEG) with HAT-non-sensitive unstained hybrid cells producing antibodies to horseradish peroxidase (POD). Fluorescent fused hybrid hybridomas as well as non-fused stained anti-CEA cells were separated from the unstained anti-POD cells using a fluorescent activated cell sorter (FACS). Finally, non-fused enzyme-deficient anti-CEA cells were eliminated by cultivation in HAT selection medium which permits only an outgrowth of HAT-resistant hybrid hybridoma cells containing the genes for producing both antibodies.
Jantscheff
P
P
Institute of Immunobiology, University of Freiburg, Germany.
Winkler
L
L
Karawajew
L
L
Kaiser
G
G
Böttger
V
V
Micheel
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001377:Azaserine; D002272:Carcinoembryonic Antigen / Q000276:immunology* / Q000302:isolation & purification; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase / Q000276:immunology* / Q000302:isolation & purification; D006825:Hybridomas / Q000276:immunology*; D019271:Hypoxanthine; D007042:Hypoxanthines; D051379:Mice; D012235:Rhodamines; D013936:Thymidine
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D003470:Culture Media; D005456:Fluorescent Dyes; D007042:Hypoxanthines; D012235:Rhodamines; D019271:Hypoxanthine; C009434:tetramethylrhodamine isothiocyanate; D001377:Azaserine; D006735:Horseradish Peroxidase; D013936:Thymidine