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

Abstract

Bovine ReissnerЈs fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins 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 designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF-and SCO-glycoproteins in solutions (ELISA) as well as 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 of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory 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 non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.

Endocannabinoid system in the adult rat circumventricular areas: An immunohistochemical study

“…All these biologically active substances produced in the SCO may be released into the CSF to participate in its homeostasis. Although we did not perform double immunohistochemistry, it appears quite evident that virtually all secretory cells of the rat SCO that were immunostained by a specific monoclonal antibody like 4H6 used in this study (Fernández‐Llebrez et al, 2001; present results) also stained with antisera against both DAGLs. Thus, it is likely that the rat SCO displays the machinery to produce 2‐AG.…”

Continuous delivery of a monoclonal antibody against Reissner’s fiber into CSF reveals CSF-soluble material immunorelated to the subcommissural organ in early chick embryos

“…Twenty-three monoclonal antibodies (MAbs) against bovine RF (2A5, 2A8, 3B1, 3E6, 3F1, 1F4, 1F12, 5G4, 1H3) and SCO secretory material (4A6, 5B9, 4B11, 2B12, 2C2, 1C12, 5D8, 1D9, 5E2, 4F7, 3F8, 5G7, 3G12, 4H6; Perez et al 1995Perez et al , 1996Fernandez-Llebrez et al 2001b) were screened to find those that immunoreacted with chick SCO secretory material. SCO explants at E13 were dissected out under a dissecting microscope (MZ6, Leica Microsystems, Wetzlar, Germany) under sterile conditions, maintained in culture (DMEM-F12 supplemented with 10% fetal calf serum; FCS) for 2 h (see Hoyo-Becerra et al 2005 for details) and then incubated with various MAbs (hybridoma culture supernatant, concentration 50 μg/ml) for 1 h followed by Alexa-568-labelled anti-mouse IgG for 30 min.…”

Bovine floor plate explants secrete SCO-spondin

“…A monoclonal antibody against the SCO glycoproteins (Mab-4H6; Fernández-Llebrez et al 2001) and Mab-(4H6)-sepharose columns were generously provided by Dr. E. Miranda, Faculty of Sciences, Málaga University, Spain. Chromatography conditions were as described by Estivill-Torrús et al (1998).…”