Quantitation of elastin in human urine and rat pleural mesothelial cell matrix by a sensitive avidin-biotin ELISA for desmosine.
A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of desmosine, a cross-linked amino acid specific to fibrous elastin. Competition between solid phase-bound desmosine-protein conjugate and free desmosine for binding to monospecific anti-desmosine antiserum constituted the underlying principle of the assay. The conjugation of desmosine to different protein carriers was carried out with the 1-ethyl-3-(dimethylamino-propyl)carbodiimide (ECDI); rabbits were immunized with desmosine-bovine serum albumin and micro-titer plates were coated with desmosine-egg albumin. An avidin-biotin peroxidase system was used to reveal anti-desmosine antibodies bound to the desmosine-protein conjugate. As both conjugates revealed new non-specific common epitopes on the carrier proteins, prior absorption of the anti-desmosine antiserum on rabbit albumin polymerized with ECDI was required to remove the antibodies directed against these neo-antigens. The absorption procedure resulted in an increased specificity and sensitivity. Values ranging from 0.07 to 4 ng of desmosine/well could be detected and this sensitivity was greater than that obtained in previous immunoassays for desmosine. In order to assess the specificity of the test, samples containing aminoacids and urine hydrolysates were included in an assay. Some cross-reactivity was observed with the desmosine precursor lysinonorleucine and the desmosine isomer isodesmosine but, in contrast the very low cross-reactivity observed with collagen hydrolysate was similar to that exhibited by albumin hydrolysate. Analysis of urine samples from 118 normal male volunteers showed, firstly, that urinary creatinine measurement was a good indicator of the amount of urine which could be safely introduced in the assay without risk of non-specific interference by other organic compounds and, secondly, that the desmosine/creatinine ratio was a reliable index for an in vivo assessment of degraded elastin excretion. The assay also allowed quantitation of elastin fiber biosynthesis in the connective tissue matrix of cultured rat pleural mesothelial cells. This ELISA for demosine is a simple technique which should be useful for further in vivo or in vitro investigations of fibrous elastin tissue metabolism.
Laurent
P
P
Unité de Recherche sur la Biopathologie, INSERM U 139, CHU Henri Mondor, Créteil, France.
Magne
L
L
De Palmas
J
J
Bignon
J
J
Jaurand
M C
MC
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001360:Avidin; D001667:Binding, Competitive; D002478:Cells, Cultured; D003094:Collagen / Q000276:immunology; D003238:Connective Tissue / Q000032:analysis*; D003895:Desmosine / Q000032:analysis*; D004549:Elastin / Q000032:analysis* / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000032:analysis; D006801:Humans; D010994:Pleura / Q000166:cytology; D051381:Rats; D013696:Temperature
D000596:Amino Acids; D003895:Desmosine; D001360:Avidin; D003094:Collagen; D004549:Elastin