43] Antibodies to desmosine

Abstract

Publisher Summary With the exception of eggshell membrane, elastin is the only known protein to contain desmosine. Because desmosine levels in elastin are constant within species, this cross-linking amino acid can be used to quantitate mature, insoluble elastin. This chapter presents the procedure of radioimmunoassay specific for desmosine. For the radioimmunoassay, 100/μl of diluted antiserum is added to a solution containing desmosines. This is mixed and allowed to incubate at room temperature for 1 hr. Fifty microliters of 125 I-labeled Bolton-Hunter desmosine probe are added, mixed, and incubated at 4 ° for 18 hr. All assays are done in duplicate. Immunoglobulin-bound probe can be determined by either of two procedures with identical results. (1)The mixture is filtered with vacuum through prewashed (phosphate buffer) cellulose nitrate filters and rinsed twice with 1 ml of cold phosphate buffer; the radioactivity of the filters is measured. (2)To each mixture is added 50 μl of a 1% suspension of S. aureus. After vortexing, the suspension is diluted with 2 ml of phosphate buffer and centrifuged at 2000 rpm for 10 min. The supernatant is removed by aspiration, and the radioactivity is measured in the pellet.