## 1 Introduction  
To rapidly screen the biomarkers before routine clinical examination, the magnetic beads-based chemiluminescence immunoassay 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 chemiluminescencence 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 reagents to produce an enhanced chemiluminescencence after the competitive immunoreaction [4; 5; 6].  
ALP (EC 3.1.3.1) catalyses the hydrolysis of phosphate compounds under alkaline conditions [7]. ALP exists in a wide range of organisms, from bacteria to humans. _Cobetia marina_, isolated from the coelomic liquid of a mussel, produces intracellular ALP (CmAP) that possesses high activity. The native protein at the final purification stage has a specific activity of approximately 15,000 U/mg, which makes it one of the most active ALPs studied to date [8]. The _CmAP_ gene was first cloned into _Escherichia coli_, and then the active protein was expressed successfully by [PERSON] et al. The purified recombinant CmAP also possesses a high specific activity of 12,700 U/mg [9].  
Heart failure (HF) is considered a complex syndrome, and it is the final stage in the development of a coronary heart disease [10]. Early diagnosis and timely treatment are of great significance in improving the prognosis of patients with HF [11; 12]. The N-terminal pro-B-type natriuretic peptide (NT-proBNP) can sensitively and specifically reflect changes in the left ventricular function [13; 14; 15]. B-type natriuretic peptide (BNP) is a preproBNP precursor, and it contains 134 amino acids. The precursor can be cleaved to a 108 amino acid polypeptide, which is called proBNP (precursor of BNP). The stimulation of proBNP through cardiomyocytes (e.g., cardiomyocyte stretching), results in the hydrolysis of proBNP into an NT-proBNP (1-76 amino acids) and a bioactive hormone BNP (77-108 amino acids) [16; 17]. Both the peptides are released into the circulation. In plasma, the NT-proBNP and BNP molecules exist in a ratio of 1:1; the former is more stable than the latter, and can reflect changes in the ventricular function. The exclusion cut-off point of the NT-proBNP in acute HF is less than 250 ng/L [18].  
On the basis of the available reports, we speculated that CmAP is suitable for antibody labelling. The Fc region of most antibodies contains an oligosaccharide chain, which is not present in the variable region. Based on this feature, we developed an improved antibody coupling method (coupling of enzymes to the detection antibody and that of magnetic bead to the capture antibody). In the present work, we developed a chemiluminescence in vitro diagnostic reagent by using CmAP and an improved coupling method. As already stated, the NT-proBNP is an important cardiac biomarker, and its structure is very simple. Therefore, in this study, we used NT-proBNP as a tool to develop a high sensitivity in vitro diagnostic reagent. Figure 1 illustrates the principle of CmAP-based MPs-CLEIA. The CmAP-labelled detection antibody and the magnetic bead-coupled capture antibody prepared in this study possess high antigen-binding efficiency. The analytical parameters were assessed to explore whether this CmAP-based chemiluminescence diagnostic reagent is sensitive and reliable. We hope that this research can promote the development of the CLEIA.  
Figure 1: Schematic illustration of the magnetic beads-based chemiluminescence immunoassay (MPs-CLEIA).