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Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the epsilon-amino groups to aldehydes. Immunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin.

Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.

Immunization of rabbits with either porcine tropoelastin, lung alpha-elastin, or aortic alpha-elastin resulted in the production of antibodies against the respective antigens. The assay of antibody activity with a radioimmunoassay indicated a high degree of cross reactivity between these three soluble elastin preparations. Through the use of competitive protein binding experiments it was possible to detect antigen-specific differences between tropoelastin and the two alpha-elastins. No antigenic differences were observed between lung or aortic alpha-elastin by any of the assay procedures uded in this investigation. Absorption of the various antisera with either insolubilized tropoelastin, lung elastin or aortic elastin allowed the preparation of non-crossreactive antibodies and provided further evidence for the antigenic differences between tropoelastin and the soluble alpha-elastins. The implications of the findings, as they relate to our current understanding of the molecular and structural arrangement of elastin and the elastin precursor, are discussed.

Highly purified elastin was prepared from bovine ligamentum nuchae by hot alkali extraction. Antisera to the protein, developed against suspensions or acid solubilized preparations, were obtained from rabbits and guinea pigs. Of a large number of proteins, only insoluble elastin (from the aortae of several animal species) and tropoelastin, the putative soluble precursor, reacted with the antisera as determined by double antibody radioimmunoassay. Nanogram quantities of insoluble elastin can be detected following solubilization of the protein by brief acid treatment. Tropoelastin can be determined in the microgram range. The radioimmunoassay should permit determination in tissues, in fluids, and in in vitro cultures of soluble, newly synthesized elastin, mature insoluble elastin, and intermediate forms of the fibrous protein which may have heretofore escaped detection. A sensitive assay for elastin should also permit the acquisition of information concerned with connective tissue turnover during development, aging, and disease.

Antibodies to human fetal aortic elastin were isolated from sheep immunized with alpha-elastin peptides. In preliminary tests of specificity using passive hemagglutination, partial cross-reactivity was demonstrated with alpha-elastin from other species. However, in a double antibody radio-immunoassay alpha-elastin peptides from other mammalian species failed to compete with 125I-labelled human alpha elastin. These results suggest the existence of at least two different antigenic sites on the elastin molecule. One, a high affinity site, demonstrates species specificity at low antigen/antibody concentrations. The other, a low affinity site, is common to mammalian elastins and is demonstrated at high antibody/antigen concentrations. In the radioimmunoassay the antibodies showed considerably less avidity for adult human alpha-elastin than for the fetal antigen. This implies that the species specific site is age-dependent and probably involves the cross-linking region of the elastin molecule. Using the sheep antiserum immunohistochemical staining of elastic tissue has been developed. This should prove to be a useful technique for studying polymeric elastin in intact tissue by light microscopy and at the ultrastructural level.

The connective tissue protein elastin is largely responsible for maintaining the elasticity of major blood vessels and lung tissue. Comparatively few studies have been made of the immunologic properties of elastin because of its high degree of insolubility and because of its apparent limited capacity to elicit precipitating antibodies. Recent studies have focused on the possible role of elastolytic enzymes, particularly leukocyte elastase, in the pathogenesis of chronic obstructive lung disease, but there are no reports in the literature concerning the antigenic properties of peptides released by digestion of insoluble elastin with leukocyte elastase. In the present study, we have obtained antibodies in rabbits to peptides prepared by digestion of dog and human lung and aortic elastin with either oxalic acid or leukocyte elastase. By hemagglutination assays, we have shown generally that: (1) within a given species the peptides from aorta and lung cross-react strongly with one another, (2) there was rather poor cross-reactivity between peptides from different species (3) the peptides obtained by oxalic acid digestion cross-react poorly with those obtained by elastase digestion, (4) the antibodies to the peptides cross-reacted with the insoluble elastin from which the peptides were derived. These elastin-specific antibodies may be useful for ultrastructural localization of elastin and identification of fragments derived from elastin in the sera of humans and experimental animals.

By radioimmunologic techniques, it has been possible to detect two serologically distinct elastin antibody fractions in antiserum prepared against bovine ligamentum nuchae elastin; a species specific fraction which bound only bovine elastin, and a population of antibodies with affinity for elastin from porcine aorta, bovine ligamentum nuchae and aorta, rabbit and hamster lung, and human aorta. Bovine specific antibodies were detected when bovine alpha-elastin was used as specific ligand whereas interspecies reactivity was detected when radiolabeled nonbovine solubilized elastins were used in the assay. Two distinct antibody populations were confirmed using immunoabsorption techniques. Thus, the elastin radioimmunoassay can be modified to detect bovine or other elastins depending upon the choice of radiolabeled antigen. The assay permits determination and quantification of elastin synthesis and degradation in a wide range of species without having to raise specific antibodies to each.