Development of an NT-ProBNP Assay Reagent Based on High Specific Activity Alkaline Phosphatase CmAP and an Improved Coupling Method

Abstract

(1) Background: Chemiluminescent enzyme immunoassay (CLEIA) is an efficient analytical method. Alkaline phosphatase (ALP) with high specific activity is the basis for CLEIA to achieve high sensitivity. In this study, a high specific activity Cobetia marina ALP (CmAP) and an improved coupling method were used to develop an N-terminal pro-B-type natriuretic peptide (NT-proBNP) diagnostic reagent. (2) Methods: The purification method of CmAP was improved and the related enzyme activities were assessed. The enzyme and magnetic beads were coupled only to the Fc region of the detection antibody and the capture antibody, respectively, by using a specially improved method. The NT-proBNP in human serum was assessed. (3) Results: The specific activity of the purified CmAP was found to be 13,133 U/mg. No loss in the enzyme activity was observed after its storage at room temperature for 4 months. The sensitivity of the in vitro diagnostic reagents was found to be 0.58 ng/L. (4) Conclusions: CmAP can be applied as a substitute for the commercial ALP. Analytical parameters indicated that the chemiluminescence diagnostic reagent for NT-proBNP is adequately sensitive and reliable for detecting the serum NT-proBNP, which suggests that both the enzyme and coupling method are suitable for the CLEIA.

Ultrasensitive and Rapid Detection of N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP) Using Fiber Optic Nanogold-Linked Immunosorbent Assay

“…Recently, a wide range of optical biosensing techniques have been developed for determination of NT-proBNP, including colorimetry [ 12 ], fluorescence [ 13 ], chemiluminescent immunoassay [ 14 ], dynamic light scattering (DLS) [ 15 ], surface-enhanced Raman scattering (SERS) [ 16 ], and fluorescent immunochromatographic assay (F-ICA) [ 25 ]. Herein, we present a method of detecting and quantifying NT-proBNP concentrations via an immunoassay that employs an antibody sandwich model.…”

“…Thus far, a number of analytical methods in the literature have been reported for the analysis of NT-proBNP, including radioimmunoassay (RIA) [ 11 ], colorimetry [ 12 ], fluorescent bead-based immunoassay [ 13 ], chemiluminescent immunoassay [ 14 ], dynamic light scattering [ 15 , 16 ], surface-enhanced Raman scattering [ 17 ], amperometric immunosensors based on enzyme label [ 18 ], silver nanoparticle label method [ 19 ], silver nanodisk label method [ 20 ], photoelectrochemical immunosensor method [ 21 ], electrochemiluminescent (ECL) immunosensor method [ 22 ], field-effect transistor biosensor method [ 23 ], giant magnetoresistance assay [ 24 ], and fluorescence-based lateral flow immunoassay [ 25 ]. While very sensitive electrochemical biosensors for the detection of NT-proBNP have been reported [ 18 , 19 , 20 , 21 , 22 , 23 ], the requirement of a reference electrode in electrochemical biosensors may be a weak point, as liquid junction error often exists in practical situations [ 26 ].…”