Determination and stability of N-terminal pro-brain natriuretic peptide in saliva samples for monitoring heart failure.

Heart failure (HF) is the main cause of mortality worldwide, particularly in the elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) is the gold standard biomarker for HF diagnosis and therapy monitoring. It is determined in blood samples by the immunochemical methods generally adopted by most laboratories. Saliva analysis is a powerful tool for clinical applications, mainly due to its non-invasive and less risky sampling. This study describes a validated analytical procedure for NT-proBNP determination in saliva samples using a commercial Enzyme-Linked Immuno-Sorbent Assay. Linearity, matrix effect, sensitivity, recovery and assay-precision were evaluated. The analytical approach showed a linear behaviour of the signal throughout the concentrations tested, with a minimum detectable dose of 1 pg/mL, a satisfactory NT-proBNP recovery (95-110%), and acceptable precision (coefficient of variation ≤ 10%). Short-term (3 weeks) and long-term (5 months) stability of NT-proBNP in saliva samples under the storage conditions most frequently used in clinical laboratories (4, - 20, and - 80 °C) was also investigated and showed that the optimal storage conditions were at - 20 °C for up to 2.5 months. Finally, the method was tested for the determination of NT-proBNP in saliva samples collected from ten hospitalized acute HF patients. Preliminary results indicate a decrease in NT-proBNP in saliva from admission to discharge, thus suggesting that this procedure is an effective saliva-based point-of-care device for HF monitoring.

Zeolite-iron oxide nanocomposite from fly ash formed a 'clubbell' structure: integration of cardiac biocapture macromolecules in serum on microelectrodes.

A new zeolite-iron oxide nanocomposite (ZEO-IO) was extracted from waste fly ash of a thermal power plant and utilized for capturing aptamers used to quantify the myocardial infarction (MI) biomarker N-terminal prohormone B-type natriuretic peptide (NT-ProBNP); this was used in a probe with an integrated microelectrode sensor. High-resolution microscopy revealed that ZEO-IO displayed a clubbell structure and a particle size range of 100-200 nm. Energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy confirmed the presence of Si, Al, Fe, and O in the synthesized ZEO-IO. The limit of detection for NT-ProBNP was 1-2 pg/mL (0.1-0.2 pM) when the aptamer was sandwiched with antibody and showed the doubled current response even at a low NT-ProBNP abundance. A dose-dependent interaction was identified for this sandwich with a linear plot in the concentration range 1 to 32 pg/mL (0.1-3.2 pM) with a determination coefficient R2 = 0.9884; y = 0.8425x-0.5771. Without  sandwich, the detection limit was 2-4 pg/mL (0.2-0.4 pM) and the determination coefficient was R2 = 0.9854; y = 1.0996x-1.4729. Stability and nonfouling assays in the presence of bovine serum albumin, cardiac troponin I, and myoglobin revealed that the aptamer-modified surface is stable and specific for NT-Pro-BNP. Moreover, NT-ProBNP-spiked human serum exhibited selective detection. This new nanocomposite-modified surface helps in detecting NT-Pro-BNP and diagnosing MI at stages of low expression.

Functionalized silicon dioxide self-referenced plasmonic chip as point-of-care biosensor for stroke biomarkers NT-proBNP and S100ß.

Surface plasmon resonance (SPR) biosensors are often used in the detection of solid, liquid or gaseous samples in diagnostics, pharmaceutics and military defense. Plasmon waveguide resonance (PWR) mode is obtained when a dielectric waveguide layer is added to the metal film. In this study, a self-referenced PWR (SRPWR) silicon dioxide (SiO2) chip was examined. The self-referenced measurement is important to compensate for temperature fluctuations, other instabilities and allows RI signal measurement without an additional reference sample, thus minimising the sample volume needed. The chip was fabricated with a multi-layer of metals and dielectrics, consisting of a 420 nm SiO2 layer, a 40 nm Ag layer and another 480 nm SiO2 layer. This chip was shown to give one internal plasmon excited on the bottom interface SiO2/Ag, which is used as self-reference in the detection. The top layer acts as a waveguide layer and can be designed to give modes with ultrahigh penetration depth. A direct assay was developed, where the recognition molecule (specific antibody) was immobilized onto the SiO2 plasmonic chip surface, via a covalent coupling protocol based on 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde. The SRPWR biosensor was developed for the sensing of two chosen stroke biomarkers: NT-proBNP and S100ß, which are sensitive and specific for stroke diagnostics. For both biomarkers, a linear decreasing pattern in the RI signal was recognized with the increasing biomarkers concentrations. Biomarkers detection was conducted in deionized water and validation was done in spiked porcine plasma. The SiO2 based plasmonic chip demonstrates a limit-of-detection of less than 1 ng/mL that is clinically relevant for both stroke biomarkers.

Electrochemical Detection of NT-proBNP Using a Metalloimmunoassay on a Paper Electrode Platform.

In this paper, we demonstrate an electrochemical method for detection of the heart failure biomarker, N-terminal prohormone brain natriuretic peptide (NT-proBNP). The approach is based on a paper electrode assembly and a metalloimmunoassay; it is intended for eventual integration into a home-use sensor. Sensing of NT-proBNP relies on the formation of a sandwich immunoassay and electrochemical quantification of silver nanoparticle (AgNP) labels attached to the detection antibodies (Abs). There are four important outcomes reported in this article. First, compared to physisorption of the detection Abs on the AgNP labels, a 27-fold increase in signal is observed when a heterobifunctional cross-linker is used to facilitate this labeling. Second, the assay is selective in that it does not cross-react with other cardiac natriuretic peptides. Third, the assay forms in undiluted human serum (though the electrochemical analysis is carried out in buffer). Finally, and most important, the assay is able to detect NT-proBNP at concentrations between 0.58 and 2.33 nM. This performance approaches the critical NT-proBNP concentration threshold often used by physicians for risk stratification purposes: ∼0.116 nM.

Original signal amplification assay for N-Terminal pro-brain natriuretic peptide detection based on Bi2MoO6 photosensitive matrix.

An original dual signal amplification immunoassay for N-Terminal pro-brain natriuretic peptide (NT-proBNP) detection is developed based on Au NPs and Zn doped CdS nanoparticles (Zn0.02Cd0.98S) co-sensitized Bi2MoO6 nanosheet photoelectrochemical (PEC) platform, the target is a good myocardial marker for diagnosing heart failure (HF). Bi2MoO6 as an outstanding photocatalysis material is successfully used in PEC analysis in this work, and the nanosheet structure provide a preponderance to capture superior Zn0.02Cd0.98S in order to achieve anticipant PEC response. The doping of Zn makes the energy band of CdS matched more compatible with Bi2MoO6, and improves the narrow band gap of CdS, making the surface plasma resonance (SPR) effect of Au NPs more significant, thus further improving the PEC response, as well as elevated the detection sensitivity of biological targets. The constructed PEC platform for NT-proBNP provides a wide detection range of 0.0001-1000 ng mL-1 and gives the minimum detection value of 0.037 pg mL-1. Great stability, high selectivity, and good reproducibility are also achieved. The proposed PEC immunoassay provides more possibilities for other protein ultra-sensitivity detection.

Electrochemiluminescence immunoassay for the N-terminal pro-B-type natriuretic peptide based on resonance energy transfer between a self-enhanced luminophore composed of silver nanocubes on gold nanoparticles and a metal-organic framework of type MIL-125.

The N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a marker of heart failure. A novel sandwich type electrochemiluminescence (ECL) immunoassay is described for the NT-proBNP. The method is based on ECL resonance energy transfer (RET) between silver nanocubes that were covered with semicarbazide-modified gold nanoparticles (AgNC-sem@AuNPs) as the donor, and a Ti(IV)-based metal-organic framework of type MIL-125 as the acceptor. The ECL signal was strongly amplified by increasing the luminous efficiency. ECL-RET occurs due to the partial overlap between the ECL emission of the AgNC-sem@AuNPs (emission wavelength at 470 nm to 900 nm) and the visible absorption spectrum of MIL-125 (absorption wavelength at 406 nm to 900 nm). This results in the quenching of ECL. The AgNC-sem@AuNPs were placed on the electrode. The antibody was immobilized on AgNC-sem@AuNPs via Au-NH2 bond, and MIL-125 was utilized as a label for the secondary antibody. The assay works in the 0.25 pg mL-1 to 100 ng mL-1 concentration range and has a 0.11 pg mL-1 lower detection limit (at S/N = 3). Graphical abstract Schematic representation of self-enhanced luminescence mechanism (semicarbazide (Sem) as co-reaction accelerator) and Electrochemiluminescence resonance energy transfer (ECL-RET): silver nanocubes (AgNCs) as the energy donor and MIL-125 as the energy acceptor.

Increased Complement Factor B and Bb Levels Are Associated with Mortality in Patients with Severe Aortic Stenosis.

Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease.

Detection of Neprilysin-Derived BNP Fragments in the Circulation: Possible Insights for Targeted Neprilysin Inhibition Therapy for Heart Failure.

BACKGROUND: Entresto™ is a new heart failure (HF) therapy that includes the neprilysin (NEP) inhibitor sacubitril. One of the NEP substrates is B-type natriuretic peptide (BNP); its augmentation by NEP inhibition is considered as a possible mechanism for the positive effects of Entresto. We hypothesized that the circulating products of BNP proteolysis by NEP might reflect NEP impact on the metabolism of active BNP. We suggest that NEP-based BNP cleavage at position 17-18 results in BNP ring opening and formation of a novel epitope with C-terminal Arg-17 (BNP-neo17 form). In this study, we use a specific immunoassay to explore BNP-neo17 in a rat model and HF patient plasma. METHODS: We injected BNP into rats, with or without NEP inhibition with sacubitril. BNP-neo17 in plasma samples at different time points was measured with a specific immunoassay with neglectable cross-reactivity to intact forms. BNP-neo17 and total BNP were measured in EDTA plasma samples of HF patients. RESULTS: BNP-neo17 generation in rat circulation was prevented by NEP inhibition. The maximum 13.2-fold difference in BNP-neo17 concentrations with and without sacubitril was observed at 2 min after injection. BNP-neo17 concentrations in 32 HF patient EDTA plasma samples ranged from 0 to 37 pg/mL (median, 5.4; interquartile range, 0-9.1). BNP-neo17/total BNP had no correlation with total BNP concentration (with r = -0.175, P = 0.680) and showed variability among individuals. CONCLUSIONS: BNP-neo17 formation is NEP dependent. Considering that BNP-neo17 is generated from the active form of BNP by NEP, we speculate that BNP-neo17 may reflect both the NEP activity and natriuretic potential and serve for HF therapy guidance.

Plasmon Near-Field Coupling of Bimetallic Nanostars and a Hierarchical Bimetallic SERS "Hot Field": Toward Ultrasensitive Simultaneous Detection of Multiple Cardiorenal Syndrome Biomarkers.

Cardiorenal syndrome (CRS) has posed tremendous challenges in patient management, and the detection of serum biomarkers may provide opportunities for early diagnosis and effective treatment. Herein, we introduce a novel surface-enhanced Raman scattering (SERS)-based sandwich immunoassay platform to simultaneously detect cardiac troponin I (cTnI), N-terminal prohormone of brain natriuretic peptide (NT-ProBNP), and neutrophil gelatinase-associated lipocalin (NGAL) for the early diagnosis of CRS by using Raman reporter-molecule-labeled Ag-Au nanostars (Ag-Au NSs) as nanotags and a three-dimensional ordered macroporous (3DOM) Au-Ag-Au plasmonic array as substrate. The Ag-Au NSs prepared by galvanic replacement feature bimetallic composition and a multibranched structure so that high SERS stability and enhancement are exhibited. Meanwhile, a 3DOM Au-Ag-Au plasmonic array was fabricated through Au-assisted electrodeposition and was further covered by a protective Au layer; it is characterized by a large specific surface area and high homogeneity, serving as a "hot field". When the nanotags and substrate were combined, "hot spots" were generated from the plasmon near-field coupling, which greatly increased the SERS enhancement. The limits of detection (LODs) were 0.76, 0.53, and 0.41 fg mL-1 for cTnI, NT-ProBNP, and NGAL, respectively, and the Raman images indicated the approximate concentration ranges of the detected proteins for visual analysis. Taking advantage of the ultrasensitivity and multiplexing capability of this approach, we further analyzed clinical blood samples with high integrality, efficiency, and accuracy. Therefore, the presented SERS immunoassay platform holds promise as an ideal test method for point-of-care detection and a powerful tool for investigations into the complex CRS-related biological process.

Cutting Edge of Brain Natriuretic Peptide (BNP) Research　- The Diversity of BNP Immunoreactivity and Its Clinical Relevance.

Brain (or B-type) natriuretic peptide (BNP) is a cardiac hormone produced in the heart and an established biochemical marker for heart failure (HF) because the level in plasma increases in proportion to disease severity. Recently, the diversity of BNP molecular forms in the peripheral circulation, which includes mature BNP (BNP1-32) and its metabolites (BNP3-32, BNP4-32, and BNP5-32), was demonstrated. Moreover, studies showed that unprocessed BNP prohormone (proBNP) is also secreted from the heart, and its secretion is increased in patients with HF. Interestingly, BNP1-32, its metabolites, and proBNP are all detected as immunoreactive BNP by the currently available BNP assay system. Current N-terminal proBNP (NT-proBNP) assay systems also can react to both NT-proBNP and proBNP. In addition, the N-terminal region of proBNP and NT-proBNP are often O-glycosylated, which may result in underestimation of total NT-proBNP level, which includes both glycosylated and non-glycosylated NT-proBNP, by the NT-proBNP assay system. More recently, we have shown that miR30-GALNT-dependent O-glycosylation in the N-terminal region of proBNP affects the processing of proBNP and contributes to its secretion from the heart. The level of proBNP relative to BNP (proBNP/BNP ratio) in the coronary sinus is higher in patients with more severe HF. The proBNP/BNP ratio and the deglycosylated NT-proBNP level may be new and clinically useful biomarkers of HF.

Sandwich-Type Electrochemiluminescence Sensor for Detection of NT-proBNP by Using High Efficiency Quench Strategy of Fe3O4@PDA toward Ru(bpy)32+ Coordinated with Silver Oxalate.

Heart failure (HF) is a burgeoning public health problem trigged by a heart circulation disorder. N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been acknowledged as a prognostic biomarker for cardiac disease. Herein, a sandwich-type electrochemiluminescence (ECL) immunosensor was introduced for sensitive detection of NT-proBNP. Gold nanoparticle modified graphene oxide-Ru(bpy)32+/Ag2C2O4 was used as a luminophore and a desirable platform for immobilization of the captured antibodies. The more stable immobilization of plentiful Ru(bpy)32+ could be implemented by direct covalent bonding chelation with Ag2C2O4. More importantly, significant quenching can be achieved by introducing polydopamine (PDA) coated Fe3O4 onto the electrode via sandwich immunoreactions. The quenching mechanism mainly showed that the excited states of Ru(bpy)32+ could be annihilated by quinone units in PDA via energy transfer. The ECL quenching efficiency was logarithmically related to the concentration of the NT-proBNP in the range from 0.0005 ng/mL to 100.0 ng/mL with a detection limit of 0.28 pg/mL. Furthermore, this specific immunosensor presented good stability and repeatability as well as selectivity, which offers a guiding significance in both fundamental and clinical diagnosis of NT-proBNP.

A sensitive immunosensor via in situ enzymatically generating efficient quencher for electrochemiluminescence of iridium complexes doped SiO2 nanoparticles.

A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy)3] doped silica nanoparticles (SiO2@Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H2O2 for efficiently quenching the ECL emission of SiO2@Ir. Typically, the SiO2@Ir not only increased the loading amount of Ir(ppy)3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H2O2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO2@Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells.

Adding Biomolecular Recognition Capability to 3D Printed Objects.

Three-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems.

Randomized placebo controlled blinded study to assess valsartan efficacy in preventing left ventricle remodeling in patients with dual chamber pacemaker--Rationale and design of the trial.

BACKGROUND: Dual chamber pacing is known to have detrimental effect on cardiac performance and heart failure occurring eventually is associated with increased mortality. Experimental studies of pacing in dogs have shown contractile dyssynchrony leading to diffuse alterations in extracellular matrix. In parallel, studies on experimental ischemia/reperfusion injury have shown efficacy of valsartan to inhibit activity of matrix metalloproteinase-9, to increase the activity of tissue inhibitor of matrix metalloproteinase-3 and preserve global contractility and left ventricle ejection fraction. PURPOSE: To present rationale and design of randomized blinded trial aimed to assess whether 12 month long administration of valsartan will prevent left ventricle remodeling in patients with preserved left ventricle ejection fraction (LVEF ≥ 40%) and first implantation of dual chamber pacemaker. METHODS: A total of 100 eligible patients will be randomized into three parallel arms: placebo, valsartan 80 mg/daily and valsartan 160 mg/daily added to previously used drugs. The primary endpoint will be assessment of valsartan efficacy to prevent left ventricle remodeling during 12 month follow-up. We assess patients' functional capacity, blood plasma activity of matrix metalloproteinases and their tissue inhibitors, NT-proBNP, tumor necrosis factor alpha, and Troponin T. Left ventricle function and remodeling is assessed echocardiographically: M-mode, B-mode, tissue Doppler imaging. CONCLUSION: If valsartan proves effective, it will be an attractive measure to improve long term prognosis in aging population and increasing number of pacemaker recipients. ClinicalTrials.org (NCT01805804).

An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding.

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.

[The endocrine heart and inflammation]. / El corazón endocrino y el proceso inflamatorio.

The endocrine heart produces the polypeptide hormones Atrial Natriuretic Factor (ANF or ANP) and Brain Natriuretic Peptide (BNP). Through the peripheral actions of these hormones the heart contributes to the regulation of the cardiac preload and afterload. More recently, new functions for these hormones have been described including the modulation of the immune response. Plasma levels of BNP but not those of ANF, increase following an acute rejection episode of a cardiac allotransplant but return to levels pre-rejection with successful treatment. This observation constitutes the first observation leading to characterizing the interactions of BNP with the immune response. Several other pathologies with an inflammatory component are now known to be associated with an increase in the production of BNP. Such an increase is due to an increase in the transcriptional activity of the BNP gene induced by cytokines and related substances. In vitro investigations have shown that an increase in BNP directly modulates immunological activity. Inflammation and hemodynamic changes co-exist in several cardiovascular diseases and therefore it may be beneficial to measure circulating levels of both ANF and BNP as biomarkers of changes in intravascular volume and of changes in intravascular volume plus inflammation, respectively. Changes in plasma ANF, that are relatively larger than those of BNP, might be an indication of hemodynamic deterioration while important changes in circulating BNP could indicate a worsening of the inflammatory process.

El corazón endocrino y el proceso inflamatorio / [The endocrine heart and inflammation].

The endocrine heart produces the polypeptide hormones Atrial Natriuretic Factor (ANF or ANP) and Brain Natriuretic Peptide (BNP). Through the peripheral actions of these hormones the heart contributes to the regulation of the cardiac preload and afterload. More recently, new functions for these hormones have been described including the modulation of the immune response. Plasma levels of BNP but not those of ANF, increase following an acute rejection episode of a cardiac allotransplant but return to levels pre-rejection with successful treatment. This observation constitutes the first observation leading to characterizing the interactions of BNP with the immune response. Several other pathologies with an inflammatory component are now known to be associated with an increase in the production of BNP. Such an increase is due to an increase in the transcriptional activity of the BNP gene induced by cytokines and related substances. In vitro investigations have shown that an increase in BNP directly modulates immunological activity. Inflammation and hemodynamic changes co-exist in several cardiovascular diseases and therefore it may be beneficial to measure circulating levels of both ANF and BNP as biomarkers of changes in intravascular volume and of changes in intravascular volume plus inflammation, respectively. Changes in plasma ANF, that are relatively larger than those of BNP, might be an indication of hemodynamic deterioration while important changes in circulating BNP could indicate a worsening of the inflammatory process.

Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.

Phage display generation of a novel humanized single-chain antibody against brain natriuretic peptide with potent neutralizing activity.

Cerebral salt wasting syndrome (CSW) is defined as a renal loss of sodium and water during intracranial disease leading to hyponatremia, which is the most frequent electrolyte disorder in critically neurological patients. Abnormal brain natriuretic peptide (BNP) secretion is implicated as the main offender. Development of antagonist against BNP is therefore of potential clinical relevance. In this study, synthetic human BNP peptide (hBNP) was used as bait and a humanized single chain fragment variable (scFv) phage antibody library as the source of antagonists. After three rounds of biopanning, hBNP-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted into pET-22b and expressed in Escherichia coli BL21 (DE3) PlysS cells. After purification by nickel-affinity and refolding, this scFv antibody (Ab) was proven to recognize hBNP specifically and sensitively in ELISA and dot-blotting assay. Its binding constant to hBNP was 1.98×10(-8) M, measured by surface plasmon resonance. Thus, the humanized scFv Ab prepared with this approach has potential therapeutic value for neutralizing abnormally high level of BNP correlated well with CSW.

Diagnostic utility of a single-epitope sandwich B-type natriuretic peptide assay in stable coronary artery disease: data from the Akershus Cardiac Examination (ACE) 1 Study.

OBJECTIVES: To assess the merit of a novel single-epitope sandwich (SES) assay specific to the stable part of BNP in patients with reversible myocardial ischemia as post-translational modifications of BNP may influence assay performance. DESIGN AND METHODS: We measured BNP concentration by a conventional assay and the SES-BNP assay in 198 patients referred for myocardial perfusion imaging (MPI). BNP concentration was determined before and immediately after exercise stress testing, and 1.5 and 4.5h later. Patients were categorized according to MPI results. RESULTS: BNP concentration was higher with both assays at all time points in patients with reversible myocardial ischemia (n=19) compared to the other patients (n=179). Measuring BNP after stress testing or calculating the changes in BNP concentration did not improve diagnostic accuracy compared to baseline measurements: SES-BNP: AUC 0.71 (95% CI 0.58-0.84) vs. conventional BNP: 0.71 (0.59-0.83), p=0.96. By linear regression analysis, reversible myocardial ischemia was significantly associated with baseline SES-BNP concentration (p=0.043), but not with measurements by the conventional assay (p=0.089). In multivariate logistic regression models, only baseline measurement with the SES-BNP assay was significantly associated with reversible myocardial ischemia: odds ratio [logarithmical transformed BNP] 2.00 (95% CI 1.16-3.47), p=0.013. The SES-BNP assay, but not the conventional BNP assay, reclassified a significant proportion of the patients towards their correct category on top of the best clinical model of our data set: NRI=0.47, p=0.04. CONCLUSIONS: The SES-BNP assay was significantly associated with reversible myocardial ischemia as assessed by several statistical indices, while a conventional BNP assay was not.