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Monoclonal antibodies are an important tool in the study of botryllid ascidians' immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.

We describe here the development of a new hybridoma cell line, CF12F6, that produces a specific antibody to Botryllus schlosseri (a colonial tunicate). The monoclonal antibody was isotyped as IgG1 (by enzyme-linked immunosorbent assay), and the cellular localization of the antigenic epitope that reacts specifically with CF12F6 was confined to the cells of the pyloric gland of the zooid (by immunohistochemistry). The pyloric gland participates in osmoregulation, digestion, glycogen storage, and various other secretion functions that will be studied further by the use of monoclonal antibody CF12F6, the first in botryllid ascidians that recognizes cells of a whole, single organ.