Within-run CVs ranged from 5.2%C8.0% in proBNP assay and from 7.0%C8.4% in total BNP assay, and between-run CVs ranged from 5.3C7.4% in proBNP ST271 assay and from 2.9%C9.5% in total BNP assay, respectively. good linearity (correlation coefficients?=?0.998C1.00), and analytical recovery was 90C101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.41.2 pM and 1.00.7 pM, respectively, and were 80129 pM and 4270 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects. Conclusions/Significance Our immunochemiluminescent assay is usually sufficiently quick and precise for program determination of total BNP and proBNP in human plasma. Introduction Brain (also known as B-type) natriuretic peptide (BNP) has been used as a biomarker of heart failure for more than a decade [1]. Indeed, guidelines for the treatment of heart failure recommend measurement BNP before making a diagnosis [2], [3]. During the process by which BNP is usually secreted from cardiac myocytes, its 108-amino acid precursor, proBNP, is usually cleaved to form the 32-amino acid peptide BNP and the 76-amino acid peptide N-terminal proBNP fragment (NT-proBNP) [4]. Recent studies have shown that in addition to BNP and the NT-proBNP, levels of uncleaved proBNP are also Rabbit polyclonal to PPP6C considerably increased in plasma of patients with heart failure [5], [6], [7]. This is noteworthy in part because the immunoassay system currently being used to measure BNP levels also detects proBNP, as the anti-BNP antibody cross-reacts with proBNP. Consequently, the present assay system actually steps not the active BNP level, but the total BNP (BNP+proBNP) level [8]. It is important to know the proBNP level and/or proBNP/total BNP ratio in heart failure, because proBNP has much less ability to induce cGMP production (about 13C17%) than BNP, and higher levels of the low-activity proBNP may be associated with the development of heart failure [7]. Consistent with that ST271 idea, we recently used the combination of gel-filtration and a fluorescent immunoenzyme assay with BNP extracted from plasma to show that although proBNP/total BNP ratios vary widely in heart failure, they are higher in cases of ventricular overload than in atrial overload [6]. Regrettably, the method used in that study requires a great deal of time and effort, and extraction of the peptide from plasma may cause underestimation of the proBNP levels due to its high adsorptive house [9]. To overcome those shortcomings, we developed a sensitive method to more quickly and very easily measure levels of proBNP and total BNP. Our idea was to make a sandwich immunoassay using a common capture antibody realizing the C-terminal region of both BNP and proBNP and detection antibodies that identify different epitopes: the N-terminal region of proBNP and the ring structure of BNP (Physique 1). Using this approach, we were able to develop a sensitive immunochemiluminescent assay for proBNP and total BNP in plasma. Here, we report around the assay’s overall performance and its use to compare plasma levels of total BNP and proBNP in healthy subjects and patients with heart failure. In addition, we measured NT-proBNP and compared it with total BNP and proBNP. Open in a separate window Physique 1 Schematic diagram ST271 of the total BNP and proBNP assay systems.BC203(Fab’) is usually a common capture antibody in both systems. KY-BNP-II(Fab’) is the detection antibody for the total BNP assay, and 18H5(Fab’) is the detection antibody for the proBNP assay. ALP: Alkaline phosphatase; CDP-Star EmeraldII (Chemiluminescent Substrate): Disodium 2-chloro-5-(4-methoxy-spiro1,2-dioxetane-3,2-(5-chloro)-tricyclo [3,3,1,13,7]decan-4-yl)-1-phenyl phosphate. Materials and Methods All patients provided written informed consent for all those blood sample analyses, and the protocol was approved by the Ethical Committee of Kyoto University or college Graduate School of Medicine. Sample analyses were also conducted in accordance with the guidelines and procedures of the Institutional Review Table for the use of human subjects in research at the Diagnostics Division of Shionogi & Co., Ltd. Peptides and Reagents Glycosylated proBNP and recombinant proBNP were purchased from Hytest Ltd. (Turk, Finland). The protein content was determined by amino acid analysis. BNP was.
