Bispecific monoclonal anti-MUC-1 X-anti-peroxidase antibody based breast cancer immunoassay

Breast Cancer associated antigen (CA 27.29), a high molecular weight Mucin has been recognized as an important tumor marker and is similar to MUC-1 mu cin described previously. More than 90% of the breast cancers show an incre ased expression of the membrane bound mucin molecule, MUG-I as well as enha nced secretion in serum. Bispecific monoclonal antibodies (BsMAb) are uniqu ely engineered antibodies bearing two different binding site (paratopes) in a single antibody molecule in contrast with the monospecific antibodies th at bear two congruous paratopes. This bifunctional design allowed us to dev elop a BsMAb with one site capable of binding to CA27.29 and the other to a n enzymatic marker (e.g. peroxidase). Two hybrid-hybridoma fusions generated a Trioma and a Quadroma. In order to produce the Trioma, 8-azaguanine resistant (AgR) clone of the desired hybr idoma (B27.29) was selected as a fusion partner for spleen cells immunized with horseradish peroxidase (HRPO). We also developed Second-generation bis pecific monoclonal antibodies produced by fusing the anti-CA27.29 (B27.29) secreting hybridoma to an anti-horseradish peroxidase hybridoma (YP4). The resulting hybrid-hybridoma or quadromas were selected acid BsMAb was purifi ed by affinity chromatography to develop a sandwich assay. The purified fra ction of the BsMAb, presented a higher consistent activity. Even with this semipurified BsMAb, we were able to establish and optimize an assay with a sensitivity of 1Unit/ml of CA27.29 (MUC-1). The final assay format was dete rmined to be 30 min incubation in the first step, 30 min incubation in the second step followed by color reaction for 15 min. The development of bette r methods of purification of BsMAb would further enhance the assay performa nce and shorten the assay time for automation of breast cancer patient mana gement.