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 se lection has been used to establish bispecific antibody (biAbs)-produci ng hybrid hybridomas. For this purpose hypoxanthine-guanine phosphorib osyl transferase (HGPRT)-deficient mutants were isolated from a hybrid oma line (D11-DG2) producing anti-CEA antibodies by 8-azaguanine treat ment. The resulting HAT-sensitive hybrid cells were stained with the f luorescence marker tetramethyl rhodamine isothiocyanate (TRITC) and fu sed by polyethylene glycol (PEG) with HAT-non-sensitive unstained hybr id cells producing antibodies to horseradish peroxidase (POD). Fluores cent fused hybrid hybridomas as well as non-fused stained anti-CEA cel ls were separated from the unstained anti-POD cells using a fluorescen t activated cell sorter (FACS). Finally, non-fused enzyme-deficient an ti-CEA cells were eliminated by cultivation in HAT selection medium wh ich permits only an outgrowth of HAT-resistant hybrid hybridoma cells containing the genes for producing both antibodies.