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.