Novel and Sensitive Noncompetitive Enzyme Immunoassay to Measure Peptides by Biotinylation

Abstract

A novel and sensitive noncompetitive enzyme immunoassay for angiotensin I as a peptide model is described. Angiotensin I in buffer containing bovine serum albumin or in plasma was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. the biotinylated angiotensin I was trapped onto anti-angiotensin I IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. the biotinylated angiotensin I eluted was reacted with anti-angiotensin I Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. the detection limit of angiotensin I was 13 fg (10 amol)/tube and 6.5 ng/1 of plasma, which was 10 to 480-fold lower than that previously reported by competitive radioimmunoassay and competitive enzyme immunoassay. and other peptides could also be measured more sensitively by the present method than by competitive radioimmunoassay.

Key Words:

• Enzyme immunoassay
• Peptide
• Angiotensin I
• Biotinylation