A competitive chemiluminescent enzyme-linked immunosorbent assay for the determination of RMP-7 in human blood

• Published in: Journal of pharmaceutical and biomedical analysis 1996-09, Vol.14 (12), p.1653-1662
• Main Authors: Jordan, Tracy, Walus, Lee, Velickovic, Alex, Last, Thomas, Doctrow, Susan, Liu, Hanjiu
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase; Amino Acid Sequence; Antibodies; Avidin; Biological and medical sciences; Biotin; Bradykinin - agonists; Bradykinin - analogs & derivatives; Bradykinin - blood; Bradykinin agonist; Competitive chemiluminescent ELISA; Enzyme-Linked Immunosorbent Assay; Hemocyanins; Humans; Luminescent Measurements; Medical sciences; Miscellaneous; Pharmacology. Drug treatments; Reference Standards; Sensitivity and Specificity
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/0731-7085(96)01805-5

RMP-7, a bradykinin agonist, is a synthetic nonapeptide designed to enhance the delivery of therapeutics to the central nervous system. A sensitive, competitive chemiluminescent enzyme-linked immunosorbent assay (ELISA) for quantifying RMP-7 in human blood samples has been developed. Rabbit antibodies against RMP-7 were produced using the conjugate of RMP-7 to keyhole limpet hemocyanin through glutaraldehyde. Biotinylated RMP-7, conjugated via N-hydroxysuccinimide ester, was used as the tracer. A premixed solution of biotinylated alkaline phosphatase and avidin was used to quantify the tracer, with a dioxetane-based compound as the chemiluminescent substrate. The method involves treating blood samples with organic solvents to precipitate proteins, evaporating the supernatants to dryness, reconstituting residues in PBS and assaying the buffer solutions with the ELISA. The assay, using 1.0 ml of whole blood, has precision and accuracy within ± 20% over the concentration range 25–800 pg ml −1. There are no significant endogenous interferences. The assay has been successfully used to support clinical trials of RMP-7.