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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.
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Abstract |
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Methods: After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP1–108, an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP1–108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP1–76 or synthetic BNP1–32. By combining mAb Hinge76 with a polyclonal antibody directed against BNP1–32, we were able to set up a proBNP1–108-specific sandwich immunoassay able to confirm the presence of proBNP1–108 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 proBNP1–108 concentrations were correlated with New York Heart Association classification. Moreover, a close relationship between proBNP1–108 and BNP1–32 concentrations may exist, as a good correlation (r2 = 0.89) was obtained when their respective concentrations were compared.
Conclusion: mAb Hinge76 is the first proBNP1–108-specific mAb produced that allows accurate estimation of proBNP1–108 concentrations in plasma.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human gene for BNP codes a 134–amino acid preproBNP precursor, which after removal of a 26-amino acid signal peptide gives rise to a 108-amino acid proBNP polypeptide (proBNP1–108) (6). Further processing releases a mature 32-amino acid BNP molecule (BNP1–32), which corresponds to the C-terminal sequence of the human proBNP and a 76–amino acid N-terminal fragment (NT-proBNP1–76). The former exhibits the biological activity, whereas no defined biological function has been found to be associated with the latter. The processing site in proBNP1–108 occurs immediately downstream from the Arg73-X-X-Arg76 sequence, at a cleavage site similar to that recognized by the ubiquitous endoprotease furin (7)(8). However, other endoproteases, such as corin (9)(10), or prohormone convertases (11) may be involved in the posttranslational maturation of proBNP1–108. Indeed, the cardiac processing of proBNP1–108 is still poorly characterized, as is that of the proBNP-derived peptide molecular forms circulating in the bloodstream. It is now well established that BNP1–32 is one of the major circulating forms (12)(13), and it seems likely that BNP1–32 may undergo deletion of the 2 N-terminal amino acid residues (Ser-Pro) (14). In addition to BNP1–32, other forms of proBNP-derived peptides have been reported to circulate in the bloodstream, including the intact precursor (13) and N-terminal proBNP fragments (15)(16). Using gel-filtration HPLC followed by immunoassays for BNP1–32 and proBNP1–108, Shimizu et al. (13) analyzed plasma samples from heart failure patients and reported the existence of 2 molecular forms, high–molecular-mass BNP, corresponding to the precursor proBNP1–108, and low–molecular-mass BNP, corresponding to BNP1–32.
Over the past decade, it has been demonstrated that the B-type natriuretic peptides have great pathophysiologic importance and therefore are of great significance in the diagnosis of heart failure as well as in risk stratification (17)(18). Growing interest in the clinical determination of B-type natriuretic peptides has led to the development of fully automated immunoassays for BNP1–32 and for NT-proBNP1–76. Studies have compared the clinical performance characteristics of both BNP1–32 and NT-proBNP1–76 assays (19)(20)(21). Overall, their performances are comparable, and both have become increasingly integrated into routine clinical practice. Nevertheless, the BNP1–32 and NT-proBNP1–76 assay characteristics need to be better understood for their optimal use in a clinical setting. This is why the IFCC Committee on Standardization of Markers of Cardiac Damage has prepared guidelines for analytical validation of BNP1–32 and NT-proBNP1–76 assays (22). One of these recommendations is to analyze the capacity of the BNP1–32 and the NT-proBNP1–76 assays to cross-react with proBNP1–108 and its multimers, a possible source of result disagreement. Indeed, because of the different sets of antibodies used in these different immunoassays, discrepancies between the reported concentrations of proBNP-derived peptides in healthy persons as well as in heart failure patients have been reported (23). In this situation, the availability of an alternative assay that would specifically quantify the precursor form in biological samples is desirable.
The aims of the present study were to obtain a monoclonal antibody (mAb) specific for proBNP1–108 without cross-reactivity with either BNP1–32 or NT-proBNP1–76 and to use this specific mAb in an assay for proBNP1–108 measurement in plasma samples.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antigens
Human recombinant proBNP1–108 and NT-proBNP1–76 proteins were purchased from Hytest. Both were isolated from recombinant Escherichia coli cultures. The resulting proteins had purities of 95% and 98%, respectively, according to Tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proBNP1–108 stock solution was 1 g/L in 50 mmol/L Tris acetate (pH 7), 750 mmol/L NaCl, whereas the NT-proBNP1–76 stock solution was at 1 g/L in 10 mmol/L potassium phosphate (pH 7.4), 150 mmol/L NaCl. BNP1–32 was purchased from Sigma and had been synthesized chemically, with a purity >97%, as determined by HPLC. Initially lyophilized, the BNP1–32 was dissolved in phosphate-buffered saline (PBS), pH 7.4, at a final concentration of 1 g/L. Different proBNP-derived peptides containing in their sequence the cleavage site (Arg76-Ser77) of proBNP1–108 with a variable number of flanking residues were synthesized by use of an automated peptide synthesizer (Pioneer). Coupling reactions were performed with the Fmoc/tBu strategy. The peptides were purified on a reversed-phase C18 column (Prep-Pak; particle size, 15 µm; pore size, 300 Å; Waters) in a Prep LC 4000 system (Waters) after trifluoroacetic acid cleavage. The purity of the peptides was established by analytical reversed-phase HPLC (DeltaPak C18 column; particle size, 15 µm, pore size, 300 Å; Waters). The relative molecular masses of the peptides were determined by electrospray ionization mass spectrometry (LCT; Micromass). For coupling to the keyhole limpet hemocyanin molecule (KLH), the peptides were synthesized with the addition of a Cys residue at their amino terminus. Coupling took place between the Cys-SH group and a primary amino group of the carrier protein and was performed with the heterobifunctional coupling reagent sulfo-SMCC (Pierce), according to the manufacturer’s instructions.
monoclonal antibody
Production of the mAb was carried out by immunization of 5-week-old BALB/c mice (Charles River Laboratories) with 1 subcutaneous injection followed by 2 intraperitoneal injections, at 3-week intervals, of one specific peptide coupled to the KLH. To that end, the KLH-conjugated peptide was emulsified in the same amount of Freund’s complete adjuvant for the first injection and of Freund’s incomplete adjuvant for the 2 subsequent booster injections. The mice were bled on day 78, and serum immunoreactivity was tested by ELISA against recombinant proBNP1–108 and NT-proBNP1–76, and BNP1–32. The mouse that showed the most specific immune response for proBNP1–108 was sacrificed, and its splenocytes were isolated and fused according to the procedure described by Kohler and Milstein (25). Hybridomas were screened for antibody production against proBNP1–108 as follows. Briefly, plates (Nunc Maxisorp) were coated overnight at 4 °C with recombinant proBNP protein (0.5 mg/L) or an irrelevant protein [glutathione S-transferase (GST); Sigma] as control. The plates were blocked by addition of 100 µL of 0.01 mol/L PBS (pH 7.4) containing 1 mL/L Tween 20 (PBS-T) and supplemented with 10 g/L bovine serum albumin (BSA; fraction V; Sigma), and incubated for 1 h at 37 °C. The plates were washed 3 times with PBS-T, and 75 µL of each hybridoma culture supernatant plus 25 µL of PBS-T containing 1 g/L BSA were added to each well. The plates were then incubated for 2 h at room temperature and washed 3 times with PBS-T. Peroxidase-conjugated donkey anti-mouse whole-molecule IgG (Jackson) diluted 1:2000 in PBS-T containing 1 g/L BSA was added to each well, and the plates were incubated for 1 h at room temperature. After 3 washings with PBS-T, substrate mixture (100 µL of 0.5 g/L o-phenylenediamine and 0.4 mL/L hydrogen peroxide in 0.1 mol/L citrate buffer, pH 4) was added to each well, and the plates were incubated in the dark at room temperature for 20 min. The reaction was stopped with 50 µL of 2 mol/L H2SO4, and the absorbances were measured at 490 nm with the PR2100 microplate reader (Bio-Rad Laboratories). Positive hybridomas were subcloned by limiting dilution, and those showing the highest reactivity and the best specificity for proBNP1–108 were selected for further study. They were cultured in vitro under conditions in which mAbs are naturally secreted into the culture medium and in which high mAb concentrations are expected. The mAbs were further purified on a protein G column (Amersham), and their isotypes were determined by use of the Mouse Monoclonal Antibody Isotyping Kit (Roche). To simplify the study, we focused our attention on mAb Hinge76, which showed the best affinity and specificity for the intact proBNP1–108 protein. This was evaluated by ELISA as follows: Plates were coated overnight at 4 °C with recombinant proBNP1–108, BNP1–32, and NT-proBNP1–76 proteins as well as with an irrelevant protein (GST) at a similar molar concentration (40 nmol/L). The plates were blocked by the addition of 100 µL of PBS-T containing 10 g/L BSA and incubated for 1 h at 37 °C. The plates were washed 3 times with PBS-T, and mAb Hinge76 diluted in PBS-T containing 1 g/L BSA was added to each well. mAb Hinge76 was then detected as described above.
epitope mapping
Overlapping pentadecapeptides frameshifted by 3 residues and spanning the entire proBNP1–108 sequence were prepared on cellulose membranes (Intavis) by the Spot technique (26) with an ASP222 robot (Abimed) as described previously (27). Once the epitope was identified, series of analog peptides were prepared in which each position of the reactive sequence was substituted by an alanine. The set of membrane-bound peptides was probed by incubation for 90 min at 37 °C with 1 mg/L mAb Hinge76 in Tris-buffered saline containing 1 mL/L Tween 20, 0.1 g/L saccharose, and Blocking Buffer (Roche) diluted 1:50 in Tris-buffered saline–Tween. Binding of mAb Hinge76 to peptides was detected with an alkaline phosphatase–conjugated anti-mouse IgG antibody (Sigma) followed by the phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate disodium salt–thiazolyl blue tetrazolium bromide (BCIP/MTT). Membranes were scanned with a CanonScan N650U (Canon), and spot intensity was registered by the Scion Image software. Membranes were regenerated as described previously (27).
digestion by furin
Recombinant proBNP1–108 at 100 µg/L was dialyzed against 150 mmol/L NaCl–5 mmol/L CaCl2–50 mmol/L Tris-HCl, pH 7.5. Digestion was performed in the same buffer in the presence of 10 U of recombinant furin (Sigma). An aliquot was removed after 1, 2, and 3 h of digestion at 37 °C. The reaction was stopped by freezing the sample to –80 °C. The samples were then loaded in duplicate on 2 sodium dodecyl sulfate–polyacrylamide gels (16%), subjected to electrophoresis, transferred to nitrocellulose, and incubated with either the rabbit polyclonal antibody raised against BNP1–32 (Strategic Biosolutions; 1 mg/L) or mAb Hinge76 (1 mg/L), followed by incubation with peroxidase-conjugated anti-rabbit immunoglobulin (1:8000 dilution; Jackson ImmunoResearch) or by peroxidase-conjugated anti-mouse immunoglobulin (1:3000 dilution; Jackson ImmunoResearch), respectively. Antibody binding was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
intact PROBNP immunoassay
mAb Hinge76 was coated at 5 mg/L on microplates. The wells were blocked by the addition of 250 µL of PBS-T containing 10 g/L nonfat dried milk and incubated for 1 h at 37 °C. The plates were washed 3 times with PBS-T, and 50 µL of patient sample diluted in 50 µL of PBS-T containing 1 g/L nonfat dried milk was added to each well. The plates were incubated for 2 h at room temperature under gentle shaking and washed 3 times with PBS-T. Bound proBNP1–108 molecule was then revealed by a rabbit polyclonal antibody directed against the BNP part (Strategic Biosolutions), used at 1 mg/L diluted in PBS-T containing 0.1 g/L BSA and incubated for 1 h at room temperature. Peroxidase-conjugated goat anti-rabbit whole-molecule IgG (Sigma) diluted 1:40 000 in PBS-T containing 1 g/L nonfat dried milk as well as irrelevant murine IgG at 150 mg/L was added to each well, and the plates were incubated for 1 h at room temperature. After 3 washings with PBS-T, substrate mixture (100 µL of 0.5 g/L o-phenylenediamine and 0.4 mL/L hydrogen peroxide in 0.1 mol/L citrate buffer, pH 4) was added to each well, and the plates were incubated in the dark at room temperature for 20 min. The reaction was stopped by addition of 50 µL of 2 mol/L H2SO4, and the absorbances at 490 nm were measured. Human recombinant proBNP1–108 from Hytest was used as calibrator because it has the advantages of having no His tag at its extremities and having the same sequence as the natural protein. We constructed the calibration curve by diluting the highest calibrator, human recombinant proBNP1–108 at a concentration of 20 000 ng/L in 500 mL/L horse plasma, in PBS-T containing 1 g/L nonfat dried milk and then diluting in the same buffer mixture to prepare calibrators with final concentrations of 10 000, 7500, 5000, 2500, 1250, 625, 313, and 156 ng/L. In preliminary experiments, we verified that the horse plasma BNP1–32 concentration was <100 ng/L, which is the decision threshold in the Access BNP assay. The within-run (n = 12) and between-day imprecision (n = 10 days) was determined at 3 calibrator concentrations (5000, 1000, and 500 ng/L), prepared by diluting calibrator in 500 mL/L horse plasma in PBS-T containing 1 g/L nonfat dried milk.
stability
We tested analyte stability in 4 different plasma samples, obtained from CHF patients classified as NYHA class IV, stored at 4 °C and at room temperature (22 °C). Immediately after blood centrifugation, an aliquot (control) was removed, and other aliquots were left standing for 10 and 24 h at the above temperatures. The different aliquots were stored at –80 °C until determination of proBNP1–108 concentrations at each time point, in duplicate.
statistical analysis
Plasma concentrations of proBNP1–108 and BNP1–32 did not follow a gaussian distribution. Thus, their concentrations are reported as the median and interquartile range (25th–75th percentiles). An independent Student t-test was carried out to compare proBNP1–108 and BNP1–32 concentrations within each NYHA class as well as within the different LVEF subgroups. The results were regarded as statistically significant for a P value <0.05. Moreover, to assess the relationship between these 2 analytes, we log-transformed their concentrations before statistical analysis and performed a linear regression analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, designated mAb Hinge76, was able to recognize recombinant human proBNP1–108 in a concentration-dependent manner, without any cross-reaction with the BNP1–32 peptide (Fig. 1A
). A minor cross-reactivity with NT-proBNP1–76 was observed at high mAb concentrations (
0.5 mg/L), but this was not considered significant. To identify the precise epitope recognized by mAb Hinge76, we synthesized overlapping pentadecapeptides frameshifted by 3 residues, spanning the entire sequence of human proBNP1–108. mAb Hinge76 interacted with 6 peptides that shared the motif 75PRSPKM80, corresponding to the hinge region of intact proBNP1–108 (Fig. 1B
). Western blotting experiments confirmed that the integrity of the proBNP1–108 cleavage site (Arg76-Ser77) is crucial for the binding of mAb Hinge76 to proBNP1–108. mAb Hinge76 was unable to recognize the recombinant proBNP1–108 digested by furin (Fig. 1C
, gel a, lane 2), an endoprotease having cleavage specificity for the Arg76-Ser77 sequence, whereas a BNP1–32 polyclonal antibody was able to recognize BNP1–32 after furin digestion (Fig. 1C
, gel b, lane 4).
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A proBNP1–108-specific immunoassay, combining mAb Hinge76 as the capture antibody with a polyclonal anti-BNP antibody as the secondary antibody, was set up to investigate the presence and the amount of the actual circulating form of proBNP1–108 in blood samples. A typical calibration curve as well as the data for diluted plasma samples are shown in Fig. 2
. The assay showed good linearity with the recombinant proBNP1–108 in the range from 150 to 10 000 ng/L. Plasma samples diluted in the same buffer as the calibrator gave a concentration–response curve parallel to the proBNP1–108 calibration curve, suggesting that an entity immunologically indistinguishable from recombinant proBNP1–108 was present in the human plasma samples. To determine the assay detection limit, defined as the lowest proBNP1–108 concentration that could be differentiated from zero, we measured the calibrator diluent 20 times in a single run. The detection limit, calculated as mean + 3 SD of these measurements, was 10 ng/L. The intraassay CVs were 5%, 3.4%, and 3% at 500, 1000, and 5000 ng/L, respectively. At the same calibrator concentrations, the interassay CVs were 6.1%, 6.1%, and 3.5%, respectively. Dilution of plasma samples with high concentrations of endogenous proBNP1–108, as well as mixing of plasma samples with high and low concentrations of recombinant proBNP1–108, gave results within 5% of the theoretical concentrations (data not shown). There was no significant cross-reactivity of the assay against human recombinant NT-proBNP1–76 or human BNP1–32 peptide introduced in a large molar excess (Table 1A
). Preliminary stability experiments indicated that, at room temperature, the measured concentration of proBNP1–108 at 10 h was 76% of the initial concentration, whereas at 24 h, the measured concentration was 59% of the original. At 4 °C, plasma appeared relatively more stable with measured concentrations that were 89% and 79%, respectively, of the original concentration (Table 1B
).
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EDTA-plasma samples from 50 healthy individuals and 171 CHF patients were assayed with the proposed proBNP1–108 assay. The mean proBNP1–108 concentration in samples from healthy individuals was 57 ng/L (median, 54 ng/L; range, 42–99 ng/L). The concentration in the NYHA class I CHF patients was significantly increased (median, 73 ng/L) compared with the concentration in the healthy individuals (median, 54 ng/L; P <0.0005). Mean increases of 2- to 23.5-fold were observed for the samples from patients in NYHA classes I and IV, respectively (Fig. 3A
). Increases in intact proBNP1–108 concentrations paralleled the disease severity, as judged by the NYHA classification. Moreover, patients for whom the LVEF value was available were grouped into 4 classes: LVEF <30% (n = 34), LVEF 31%–40% (n = 25), LVEF 41%–50% (n = 22), and LVEF >50% (n = 34). The proBNP1–108 concentrations were increased in patients with decreased LVEF. When we considered all of the groups, mean proBNP1–108 concentrations and LVEF were negatively correlated (Fig. 3B
). For the correlation study between BNP1–32 and proBNP1–108 concentrations, we studied 250 samples from CHF patients. The coefficient of determination (r2) between intact proBNP1–108 and BNP1–32 concentrations was 0.89, showing the close relationship between the 2 analytes (Fig. 4
). Concerning the relative ratio of proBNP1–108 to BNP1–32, for some of the patients, proBNP1–108 was the predominant form; for the others, the concentration of the 2 analytes was equivalent.
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Discussion |
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Using this mAb specific for the intact form, and a commercial anti-BNP132 antibody, we developed an immunoassay for proBNP1–108. For the detection antibody, an NT-proBNP1–76 or a BNP1–32 antibody could be used. We favored the second choice for several reasons. First, Seidler et al. (28) reported that proBNP1–108 might circulate as a trimer. This oligomerization occurs through leucine zipper-like coiled-coil motifs present at the proBNP1–108 NH2 terminus. To date, all of the antibodies directed against the N-terminal region have been raised against small peptides or the monomeric form of NT-proBNP1–76 so that, depending on the localization of their epitopes, their ability to recognize the circulating trimer form remains to be established. Second, several studies have demonstrated that the integrity of the BNP1–32 ring structure, formed by a disulfide bridge between Cys86 and Cys102, is crucial for the biological activity of the hormone (29), so that use of an antibody that recognizes this native loop would, to some extent, guarantee that a putatively functional molecule is being measured. Shimizu et al. (13) proposed an immunoassay to measure proBNP1–108 by combining mAb BC203, which is specific for the COOH terminus of BNP1–32, and a polyclonal antibody specific for the NH2 terminus of the precursor. However, as reported in their study, mAb BC203 may capture both proBNP1–108 as well as BNP1–32; consequently, mAb BC203 could become saturated with BNP1–32, leading to possible underestimation of the quantity of circulating proBNP1–108. On the other hand, mAb BC203 is one of the mAbs used in the Shionoria BNP IRMA (Shionogi & Co, Ltd.) (30); consequently, this test measures intact proBNP1–108 in addition to the chosen analyte. Conscious of this lack of reliable region-specific antibodies, Goetze et al. (31) preferred to quantify the total amount of precursor and its N-terminal fragment in plasma. To be certain that the same epitope present in different fragments was recognized with the same affinity when bound to the same antibody, they digested all of their samples with trypsin to release a small proBNP1–21 fragment. This small peptide could come from the intact form as well as from NT-proBNP1–76, both these forms being subsequently detected by a competitive immunoassay measuring the proBNP1–108 N-terminal epitope. On the other hand, in the innovative method proposed here, the use of mAb Hinge76, which is specific for the precursor form, allows the capture of only the intact form from plasma, ensuring for the first time an accurate determination of proBNP1–108 concentrations. Our decision to focus on a mAb specific for proBNP1–108 only was motivated by the fact that this molecule may merge the advantages of both BNP1–32 and NT-proBNP1–76. It shares the ring structure with BNP1–32, which determines the hormone activity, although no natriuretic activity has yet been shown to be associated with the precursor. In addition, like NT-proBNP1–76, it would have a longer half-life than BNP1–32, which is rapidly trapped by specific receptors. In fact, from a study in sheep plasma, it appears that proBNP1–108 might have a longer half-life than BNP1–32 in circulation (32).
Our study confirms that proBNP1–108 circulates in blood and that its cleavage to yield the BNP1–32 hormone may not be linked exclusively to the myocyte secretion process. Furthermore, it demonstrates that measurement of proBNP1–108 may have clinical value. Our assay allows the differentiation of healthy individuals from patients suffering from heart failure and the stratification of patient samples based on the severity of the heart failure. We observed some overlap between the different NYHA classes for proBNP1–108 as well as for BNP1–32 concentrations, but because of the subjective nature of the NHYA classification, this result was expected. Furthermore, proBNP1–108 concentrations and LVEF were inversely correlated in the CHF patients in our study. However, this trend should be confirmed in more homogeneous cohorts, as some individual values were far from the regression line. This could be explained by the facts that (a) the LVEF was determined by different techniques (electrocardiography, scintigraphy, and angiography) and (b) we did not have the data to divide the patients with abnormal left ventricular dysfunction into those with purely systolic or purely diastolic dysfunction.
Additional investigations are needed to improve our understanding of the physiologic meaning of the differences observed on a molar basis between proBNP1–108 and BNP1–32. Indeed, it appears that the relative ratio of proBNP1–108 to BNP1–32 varies from one patient to another, as already observed by Tateyama et al. (12). Thus, further analyses of different clinical situations appear to be necessary to determine whether the differences in the relative proportions of the various forms of natriuretic peptides reflect different stages of heart failure. Another aspect to clarify is the mechanism of proBNP1–108 clearance, which may facilitate interpretation of plasma concentrations. For example, comparisons of the quantities of NT-proBNP1–76, BNP1–32, and proBNP1–108 in the same patient with impaired renal function could be informative.
In conclusion, intact proBNP1–108, like its cleavage products, could be used as a biomarker in the diagnosis of patients with suspected CHF; however, large-scale investigations are needed to definitively clarify its possible additive clinical contribution.
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Acknowledgments |
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Footnotes |
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); 0.1 mg/L (
); 0.05 mg/L (
); 0.01 mg/L (
); 0.005 mg/L (
); 0.001 mg/L (
), and 0 mg/L (
). (B), Spot method identification of peptides recognized by mAb Hinge76: panel 1, reactivity of mAb Hinge76 with 6 peptides within the proBNP1–108 sequence; panel 2, sequences of reactive pentadecapeptides and identification of the minimal epitope (boxed). (C), loss of reactivity of the proBNP1–108 after digestion by furin: Western blotting of recombinant proBNP1–108 digested by furin (lanes 2 and 4) or undigested (lanes 1 and 3) detected by mAb Hinge76 in gel a or by a polyclonal BNP antibody in gel b.
and 
