Received on November 16, 2007
Accepted on June 10, 2008

Proteomics and Protein Markers

Novel Immunoassay for Quantification of Brain Natriuretic Peptide and the Propeptide in Human Blood

¹ HyTest Ltd., Turku, Finland
² Department of Biochemistry, Moscow State University, Moscow, Russia
³ Moscow Research Institute of Medical Ecology, Moscow, Russia
⁴ 67 City Hospital, Moscow, Russia
⁵ Moscow State Medico-Stomatological University, Moscow, Russia
⁶ Hennepin County Medical Center, University of Minnesota School of Medicine, Minneapolis, MN

^* To whom correspondence should be addressed. E-mail: natalia.tamm{at}hytest.fi.

BACKGROUND: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis artifacts.

METHODS: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11–22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5.

RESULTS: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the "single-epitope sandwich assay"), which requires only a short BNP fragment (fragment 11–22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays.

CONCLUSIONS: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.