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.
Highlights
To rapidly screen the biomarkers before routine clinical examination, the magnetic beads-based chemiluminescence immunoassay (MPs-Chemiluminescent enzyme immunoassay (CLEIA)) technique has been used because of its high sensitivity and wide dynamic range, without the requirement for radioactive reagents [1,2,3]
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
A chemiluminescence in vitro diagnostic reagent for NT-proBNP was synthesised by using Cobetia marina ALP (CmAP) with high specific activity and an improved coupling method
Summary
To rapidly screen the biomarkers before routine clinical examination, the magnetic beads-based chemiluminescence immunoassay (MPs-CLEIA) technique has been used because of its high sensitivity and wide dynamic range, without the requirement for radioactive reagents [1,2,3]. The test sensitivity is closely related to the performance of the instrument and the paired antibody, and especially to the specific activity of the enzyme. The reaction of the enzyme-catalysed substrate is continuous and mild. When the amount of a chemiluminescence substrate is sufficient, the chemiluminescence signal increases with time under a certain time range. This process requires sufficient time for the reaction solution to mix evenly. Alkaline phosphatase (ALP), often used to label antibodies, can catalyse luminescent regents to produce an enhanced chemiluminescence after the competitive immunoreaction [4,5,6]
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