Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the pro duction of polyclonal and monoclonal antibodies (Mabs). We also produced Ma bs against intracellular secretory glycoproteins of the bovine subcommissur al organ (SCO). These Mabs were used for immunodetection of secretory prote ins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were d esigned to detect and quantify soluble secretory materials in different sam ples, to study coexistence of epitopes, and to elucidate whether epitopes f or Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs reco gnizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well a s in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types o f ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moi ety of the secretion, (2) they coexist in the same molecular forms and, wit h few exceptions, they did not overlap, (3) they were not repeated in the s ecretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against no n-repeated sequences of RF-glycoproteins that have not been conserved in ve rtebrate phylogeny. Microsc. Res. Tech. 52:510-519, 2001. (C) 2001 Wiley-Li ss, Inc.