A sensitive assay for the quantitation of soluble elastin (original) (raw)
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Connective Tissue Research, 1991
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.
Detection of elastin by immunoelectronmicroscopy
Histochemistry, 1987
Elastin components have been identified in chick aorta by different immunoelectronmicroscopic procedures (peroxidase-antiperoxidase, immunoferritin and immunogold) using affinity purified antibodies to chick tropoelastin. The PAP method used in a preembedding procedure stained the outer portion of amorphous elastin and the microfibrils very intensively. The surface of the cells was also slightly stained. On the contrary immunogold labelling on Epon or Lowicryl embedded sections produced a strong decoration only of amorphous elastin, while microfibrils remained almost completely unlabelled. The result is not due to loss of antigenicity of microfibrils during embedding, since similar data were obtained with immunoferritin in a preembedding procedure. Experiments performed under different stringency conditions showed that the products of the peroxidase reaction diffuse and redistribute in the tissue, indicating that the positive staining of microfibrils and cell surface is an artifact. The value of different immunological reagents and procedures in studying the fine mapping of elastin components is discussed.
Connective Tissue Research, 1999
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 e-amino groups to aldehydes. lmmunofluorescence 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.
A high molecular weight species of soluble elastin
Biochemical and Biophysical Research Communications, 1976
The isolation of a high molecular weight species (130–140,000 daltons) of soluble elastin from the aortas of lathyritic chicks is described. In comparison to chick tropoelastin, the higher molecular weight material possesses an increased amount of acidic and hydroxyl amino acids and in contrast to tropoelastin, contains histidine, methionine and cystine residues. This molecular weight species of soluble elastin is susceptible to proteolytic degradation and can be shown to readily breakdown to lower molecular weight components including tropoelastin.
Generation of a Monoclonal Antibody to Detect Elastin-like Polypeptides
Biomacromolecules, 2019
The identification and use of antibodies dominates the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. One emerging class of peptide biologics is known as the Elastin-like polypeptide (ELP), which are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific towards ELPs and ELP fusion proteins. Purified antibody was evaluated in ELISA, Western Blotting, and immunofluorescence assay. The optimal anti-ELP mAb proved highly reactive and specific towards ELPs. Moreover, they were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase separate in response to heating; furthermore, when incubated at great excess to ELP the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that novel murine mAbs can be raised against purified ELPs. This new reagent will enable purification and experimental detection, and characterization of these biopolymers.
The Anatomical Record, 1990
Light microscope histochemistry and immunohistochemistry, and routine electron microscopy techniques were performed to analyse elastin distribution and structure in the human liver compared with that in baboon and mouse. In man and baboon, elastic fibers stained by iron hematoxylin or orcinolnew fuchsin seemed to be solitary and were few in number; in the mouse they were thinner but abundant, both in the portal tract and in hepatic veins. Orcein or resorcin-fuchsin stains, employed after oxidation of tissue sections, revealed a network comprising elastic, elaunin, and oxytalan fibers, which was also demonstrated by immunofluorescence with anti-elastin antibody in man and baboon. At the ultrastructural level, the elastic fibers of the human portal tract corresponded to discontinuous patches of amorphous material intermingled with few microfibrils. These contrasted with the thinner elastic fibers of baboon and mouse liver which had a core of amorphous material. In man and baboon, these fibers meshed into slender bundles of microfibrils often exhibiting small spots of amorphous material (elaunin fibers) and terminated as isolated microfibrils (oxytalan fibers). Immunoelectron microscopy of elastin carried out on baboon liver tissue labelled the amorphous material and also its microfibrillar component. Immunoperoxidase deposits were also associated with isolated bundles of microfibrils in the baboon portal stroma. Immunolabelling and elastic stains disclosed an important elastin portal network located around vascular, biliary structures and interspaced with collagen bundles. The structural polymorphism of elastin, assembling different relative amounts of amorphous material and microfibrils, might have a relationship with the required elasticity in a given species.
The use of tritiated elastin for the determination of subnanogram amounts of elastase
Analytical Biochemistry, 1977
A procedure to quantitate trace amounts of elastase in tissue or cell homogenate preparations is described. The procedure is a modification of a method employing NaB3H4-reduced elastin and it does not restrict assay volume. The assay is specific and can distinguish between pancreatic elastase and trypsin or chymotrypsin, both of which solubilize small amounts of the substrate. Pancreatic elastase remains active in this assay system for at least 4 weeks.