Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood.
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.


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.
Tamm
Natalia N
NN
HyTest Ltd., Turku, Finland. natalia.tamm@hytest.fi
Seferian
Karina R
KR
Semenov
Alexander G
AG
Mukharyamova
Kadriya S
KS
Koshkina
Ekaterina V
EV
Krasnoselsky
Mihail I
MI
Postnikov
Alexander B
AB
Serebryanaya
Daria V
DV
Apple
Fred S
FS
Murakami
MaryAnn M
MM
Katrukha
Alexey G
AG
D000918:Antibody Specificity / Q000276:immunology; D002850:Chromatography, Gel; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D020097:Natriuretic Peptide, Brain / Q000097:blood* / Q000276:immunology*; D011498:Protein Precursors / Q000097:blood* / Q000276:immunology*
D000939:Epitopes; D011498:Protein Precursors; D020097:Natriuretic Peptide, Brain