Detection Of B-type Natriuretic Peptide Research Articles

The role of brain natriuretic peptide and serum triiodothyronine in the diagnosis and prognosis of chronic heart failure

Objective The objective of this paper was to investigate the diagnostic and prognostic value of plasma B type natriuretic peptide (BNP) and serum triiodothyronine (T3) in chronic congestive heart failure (CHF).Methods 156 cases of CHF patients and 75 cases of cardiac function I patients hospitalized over the same period were utilized in this study. On admission, the patient’s BNP and T3 plasma concentrations were measured. The correlation analysis of plasma BNP and T3 in CHF patients with cardiac function classification was conducted.Results According to the NYHA grading systems, the plasma BNP levels in patients with II, III, and IV grade CHF were significantly higher than those with cardiac function I (P < 0.05); BNP levels and NYHA grading of cardiac function correlated positively. The BNP concentrations increased with CHF progression (P < 0.01). The T3 level and NYHA grading of cardiac function correlated negatively. The T3 level decreased as the degree of heart failure increased. Using CHF in combination with BNP to predict the occurrence of CHF had a sensitivity value of 90.8% with 95.5% specificity, 86.3% accuracy, and a negative predictive value of 87.7%.Conclusions Plasma BNP was more sensitive than T3 in the diagnosis of CHF. The T3 was more meaningful than the BNP in the prognosis of CHF. The BNP and T3 combination detection was more valuable in determining the severity of CHF and prognosis.

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide.

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of L-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0mM and relative binding activity was 3.5 times higher compared with that of no induction with L-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4μg/ml with optimized inducer concentration (1.0mM L-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.

Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5‐13 (C10A)

B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.

Kinetics and Thermodynamics of Antibody Binding to B-Type Natriuretic Peptide

B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using high-affinity monoclonal antibodies. Temperature dependence of the equilibrium binding constants and the kinetic rates were studied for anti-BNP mAbs 106.3 and 3-631 by means of fluorescence spectroscopy. Thermodynamic parameters including changes in the free energy, enthalpy and entropy measured at equilibrium are in a good agreement with the parameters calculated from kinetics data. The differences in thermodynamic parameters measured for the two antibodies under study support structural data obtained by NMR and X-ray crystallography.

Detection of Endogenous B-Type Natriuretic Peptide at Very Low Concentrations in Patients With Heart Failure

The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic paradox."

Biology of the Natriuretic Peptides

The biology of the natriuretic peptide (NP) system is complex, yet highly phylogenetically preserved. It regulates salt and water handling, promotes vasodilatation, and exerts favorable effects on the heart in the context of processes such as heart failure. Prior assumptions about the production of B-type NP (BNP) and its amino-terminal precursor fragment (NT-proBNP) have recently been refuted. It is now recognized that rather than a 1:1 secretion of these 2 NPs, a mixture of cleaved and uncleaved NPs is released by the cardiomyocyte. It is also recognized that BNP is rapidly modified into a mixture of various fragments. Commercial assays for the detection of BNP and NT-proBNP measure a mixture of cleaved and uncleaved NPs as well as varying amounts of degraded BNP. BNP and NT-proBNP are cleared differentially: BNP is actively removed from the bloodstream and also has passive clearance mechanisms, including renal clearance; NT-proBNP is cleared more passively by organs with high rates of blood flow, including the kidney.