Issues for Assay Development

It is essential that the binding characteristics (affinities) ofthe antibodies to the antigen not be altered when one of these two antibodies is already bound to the antigen. This objective is facilitated ifthe detected epitopes are not close in space. Because NT-proBNP is a larger molecule (8.5 kDa) than BNP (3.5 kDa), it was easier to design an immunometric assay with antibodies directed against sterically remote epitopes of the analyte. In addition to targeting epitopes that are sterically distant, the antibodies of an immunometric assay optimally should target epitopes that are stable, i.e., not subject to conformational changes or degradation that might interfere with binding of the antibody. Therefore, rational assay development should be based on a comprehensive knowledge of the structure, formation, degradation, as well as circulating forms of the analyte to be detected.

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Fig. 2. Principle and antibody selection strategies of immunometric BNP assays.

The structure, formation, secretion, and degradation of BNP are well described; however, information on circulating products of degradation is limited. High-performance liquid chromatography (HPLC) ofplasma samples reveals predominantly BNP 3-32, which is formed by proteolytic cleavage of serine and proline at the amino-terminal end of BNP 1-32, and small amounts of BNP 1-32 in vivo and in vitro (4). This modification may affect the affinity of antibodies that bind to epitopes at the amino-terminal end of the peptide and, thus, be responsible for variation in the stability of BNP as measured by different commercial assays (Fig. 2). To provide optimal results, BNP assays should have 100% crossreac-tivity with BNP 3-32. To date, no additional degradation products of BNP 1-32 have been found in plasma from patients with heart failure by HPLC analysis, and the carboxyl terminal appears to be stable (4,5).

In contrast to BNP, the metabolism and degradation of proBNP are poorly understood. It is not yet clear whether proBNP is split into NT-proBNP 1-76 and BNP only within cardiomyocytes, or whether this processing also occurs in serum (Fig. 3). Although split prohormone can be found in extracts of cardiomyocytes, intact proBNP has also been found in human blood, indicating that both proBNP and BNP are secreted by cardiac myocytes (6,7). Notably, a "high molecular mass" form of BNP with a molecular mass of approximately three times that of proBNP has also been detected by HPLC analysis of plasma from patients with heart failure (6,7). This finding suggests that proBNP circulates in plasma as a trimer. Although all NT-proBNP assays should in theory crossreact with proBNP, this assertion has not been tested directly owing to difficulty in synthesizing proBNP. Split products of the N-terminal portion of proBNP have been detected in circulating plasma as well.

Description of Assays

At present, three assays for BNP (Biosite, Bayer, Abbott) are commercially available for use in the clinical laboratory. The assay manufactured by Biosite was developed as a quantitative POC test that has now also been licensed to be performed on an automated high-

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