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Proteomics and Protein Markers |
1 Bio-Rad Laboratories, Marnes-La-Coquette, France.
2 CNRS UMR 5160, Montpellier, France.
3 I2T, Montpellier, France.
4 Hôpital Arnaud de Villeneuve, Montpellier, France.
5 Hôpital Lapeyronie, Département de Biochimie, Montpellier, France.
aAddress correspondence to this author at: CNRS UMR 5160, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France. Fax 33-467-548-610; e-mail sylvie.villard{at}cpbs.univ-montp1.fr.
Background: B-Type natriuretic peptide (BNP132) as well as the N-terminal fragment of the prohormone containing residues 176 (NT-proBNP176), both cleavage products of the precursor proBNP1108, are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to develop an immunoassay that specifically measures solely proBNP1108 in plasma.
Methods: After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP1108, an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP1108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP176 or synthetic BNP132. By combining mAb Hinge76 with a polyclonal antibody directed against BNP132, we were able to set up a proBNP1108-specific sandwich immunoassay able to confirm the presence of proBNP1108 in blood samples.
Results: From a cohort of 50 healthy persons and 170 patients with congestive heart failure (CHF), our assay was able to differentiate healthy individuals from CHF patients (P <0.005). Interestingly, plasma proBNP1108 concentrations were correlated with New York Heart Association classification. Moreover, a close relationship between proBNP1108 and BNP132 concentrations may exist, as a good correlation (r2 = 0.89) was obtained when their respective concentrations were compared.
Conclusion: mAb Hinge76 is the first proBNP1108-specific mAb produced that allows accurate estimation of proBNP1108 concentrations in plasma.
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