A sensitive assay for the quantitation of soluble elastin (original) (raw)
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.
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.
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.
Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures
Biochemistry, 1988
Insoluble elastin accumulation, elastin mRNA translational efficiencies, and elastin mRNA levels were evaluated in cultures of neonatal rat aortic smooth muscle cells grown for several days in consecutive passages. When the products of in vitro translation were immunoprecipitated with an antio-elastin antibody, a single 79 000-Da protein was obtained. Northern blot analysis also indicated an elastin mRNA species corresponding to approximately 4.2 kilobases. Insoluble elastin accumulation increased in cells cultured for 7-21 days in first through fourth passages, while with one exception, relative levels and translational activity of elastin mRNA decreased with time in culture. The data indicated that a simple relationship between elastin accumulation and elastin mRNA levels was not evident.
Properties of chick tropoelastin
Biochimica et biophysica acta, 1973
I. Tropoelastin was purified from the aortas of copper-deficient chicks. The amino acid composition of this protein is very similiar to that previously described for porcine tropoelastin. The protein contains more than 4 ° residues of lysine per thousand total residues and no desmosine or isodesmosine. 2. Tropoelastin is easily solubilized by elastase. The rate of elastolysis was found to increase if tropoelastin was first preincubated in solutions containing sodium dodecyl sulfate, but not dipalmityllecithin or cholesterol. 3-Sodium dodecyl sulfate elicits a marked conformational change in tropoelastin. The circular dichroism spectrum of tropoelastin in the presence of sodium dodecyl sulfate was typical of proteins containing significant amounts of a-helix. 4. Most of the lysyl residues in tropoelastin appear to equilibrate with aldehyde functions in the formation of Schiff base products. The potential for such reactions is a prerequisite in the formations of elastin cross-links. Approx. 8o% of the lysyl groups in the protein appeared capable of forming Schiff base products.
Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells
The Journal of biological chemistry, 1989
Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-...
2009
Elastic fibres play an important role in the pulmonary development process. The overall goal of this work was to quantify elastin in chicken lungs, using domestic fowl (Gallus gallus) as an experimental model, from the 14 th day of hatching until the 42 nd after hatchling. As analytical methodologies we used high-performance liquid chromatography (HPLC) and a colorimetric method based on the use of a stain that has been only applied in histological techniques. The HPLC analysis was carried out in a reversed phase system with a binary gradient and detection by UV at 254 nm to quan- tify desmosine and isodesmosine, as the elastin cross links, in the studied samples. The colorimetric method was used by applying a commercial kit ("Fastin™ Elastin Assay") which quantifies soluble tropoelastin and insoluble elastin made soluble after treatment with hot water solution of oxalic acid. The results obtained by HPLC were statistically different if comparing the incubation period with...
Circulation Research, 1998
The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to g/mL range. Similar concentrations of an elastin peptide preparation (-elastin) were previously demonstrated to induce, among other biological actions, a dose-and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10 Ϫ13 to 10 Ϫ7 mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca 2ϩ ] i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium-and dose-dependent vasorelaxation and to increase endothelial [Ca 2ϩ ] i , whereas PVGV and VGVA produced these effects only at low concentration (10 Ϫ11 mol/L). A likely candidate for mediating the elastin peptide-related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.
Biochimica et biophysica acta, 1975
ClostrIdium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.19) was purified by batchwise separation with DEAE-cellulose followed by affinity chromatography on a column of alkali-treated elastin. The N-terminal amino acid profile of elastin isolated from bovine ligamentum nuchae using this enzyme preparation was compared with that of a duplicate sample purified with a mixture of collagenase I and II (Yoshida, E, and Noda, H. (1965) Biochim. Biopsys. Acta 105, 562-574). An approx. three-fold decrease in the molar concentration of N-terminal residues and a considerable reduction in their number was obtained by using the former enzyme preparation.