Generation of a murine monoclonal antibody to normal mammary epithelium using mice rendered immune-tolerant to malignant mammary epithelium.

A monoclonal antibody (MAb) that distinguishes normal from malignant mammary epithelia in tissue or cell lines was generated using a procedure that involved immune-tolerization before immunization. Immune-tolerance to two transformed mammary epithelial cell lines (MCF.7 and MDA.MB.231 cell lines combined) was induced in neonatal mice within 24 hr of birth. Successful induction of immune-tolerance was determined by an indirect immunohistological method, testing sera from mice against the tolerogen (i.e., the MCF.7 and MDA.MB.231 cell lines). Mice lacking antibodies in their sera against the immune-tolerogen were subsequently immunized with an extract of normal breast epithelium. One mouse was selected for hybridoma production based on evidence of serum antibody that showed reactivity with normal mammary epithelial cells (MEC) but not with invasive breast carcinoma cells, as determined by an indirect immunohistological method. Spleen cells from the selected mouse were fused with a mouse myeloma cell line to generate MAb. After extensive screening, one MAb was further studied on the basis of reactivity with normal MEC in tissue and absence of staining of malignant MEC in tissue or tumorigenic MEC lines. This test of specificity of reactivity revealed that the antigen detected by the specific antibody was expressed on the apical plasma membrane of normal glandular epithelia that included breast, cervix, colon, lung, pancreas, and stomach, but not on their malignant counterparts in tissue sections. The antigen recognized by the MAb was termed luminal epithelial antigen with an apparent MW of 92 KD (LEA.92). This study illustrates the practical usefulness of the immune-tolerization/immunization approach in the generation of antibodies with particular specificity requirements, as in the identification of an antigen that is differentially expressed in two tissues (e.g., normal and malignant) which otherwise have a multiplicity of antigens in common.