Detection of cellular retinoic acid-binding protein in chick embryonic tissues by monoclonal antibodies

Abstract

We describe the production of monoclonal antibodies (MAbs) directed against cellular retinoic acid-binding protein (CRABP) and their application for the quantitation and localization of CRABP during the development and growth of chick embryo. Three MAbs, classified as D-10 and H-6 (IgG₁-isotype), and G-4 (IgM-isotype), exhibited the highest degree of immunoreactivity for chick embryo CRABP. The antibodies showed partial reactivity to CRABP from rat testis. None of the MAbs showed cross-reactivity with cellular retinol-binding protein or fatty acid-binding protein which are structurally similar to CRABP. The antigen-specificity was confirmed by immunoblot analysis as well as by fast protein liquid chromatographic analysis. The radioimmunoassay developed for MAb (D-10) provided a detectability range of 0.5-5.0 ng of CRABP in the standard displacement curves. An abundance of CRABP was found in embryonic skin, brain, testis and eye. Several other tissues (heart, lung, liver), previously reported to have undetectable levels of CRABP, showed significant amounts of the binding protein. The levels of CRABP peaked in early (4–6-day-old) and late (11–14-day-old) stages of embryo development. Immunolocalization of CRABP in chick embryo skin demonstrates a specific intense staining for the antigen in the dense areas of mesenchyme cells (mesodermal layers); little or no staining was apparent in the differentiated cells of epidermis and peridermis as well as in the loose connective tissues. The MAbs are useful not only in the purification of CRABP as an affinity absorbent, but also in the elucidation of the possible role of CRABP in the transfer of the ligand to its nuclear receptors and in the morphogenetic gradient formation of RA in chick embryo tissues.