Sensitive ELISA for measuring elastin breakdown products in plasma.

SD Kucich et al. (1) described an enzyme-linked immunosorbent assay (EUSA) for soluble degradation products of elastin (elastin-derived peptides, EDP) in plasma. They reported a nonspecific loss of measurable EDP owing to coacervation of the elastin-a physical phenomenon in which certain proteins exhibit reversible aggregation on heating or increasing the ionic strength (2). We modified an ELISA to obviate this phenomenon. EDP used as standards were prepared (2), and the protein concentration was determined (3) (standard: human serum albumin). We used a specific polyclonal antibody to solubilized human lung elastin, raised in rabbits (a gift from Dr. Robert Mecham, Pulmonary Div., Washington Univ. School ofMed., St. Louis, MO). Its specificity was confirmed by double diffusion in agarose and in an ELISA with humanderived elastin (solubilized), fibronectin, neutrophil elastase, collagen, albumin, heparan sulfate, dermatin sulfate, hyaluronic acid, or normal rabbit serum as the antigen. Only solubilized elastin gave a positive reaction. We coated the ELISA plates and generated a standard curve as described (1). In brief, we incubated appropriate dilutions ofthe unknown sample or known concentration of EDP with antibody to EDP (in excess). After 24 h we n mt. unlts/L CV, % transferred the reaction mixtures to U-bottomed ELISA plates coated with EDP, incubated for 1 h at room temperature, then washed with PBS-Tween (per liter, 10 mmol of P04, 150 mmol of NaC1, 1 g of Tween 20). Phosphate ions