## 4 Discussion  
CLEIA is one of the most commonly used in vitro diagnostic methods that possesses the longest chemiluminescence time. However, CLEIA also has some limitations. For example, the sensitivity of the test is over dependent on the specific activity of the enzyme, however, enzymes are generally proteins and are easily inactivated. Therefore, specific activity and stability of the marker enzyme are highly required in the CLEIA. CmAP is derived from a deep-sea bacterium that can adapt to extreme environments and possesses the highest specific activity of up to 15,000 U/mg. According to the method of [PERSON] et al. [9], we expressed CmAP in _E. coli_ and improved the purification method. Finally, we obtained the purified enzyme with specific activity of more than 13,000 U/mg. Its specific activity is much higher than that of all commercial ALPs. The analysis of enzyme properties indicated that the enzyme possesses extremely strong stability, and almost no loss in its activity was observed when it was stored at room temperature for 4 months. The aforementioned advantages indicate that CmAP can be used as an alternative to commercial ALP as a marker enzyme. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Antigens**} & **Tested Concentration** & **NT-proBNP Concentration** & **CR** \\  & **(ng/mL)** & **Determined (ng/L)** & **(\%)** \\ \hline cTn complex & 100 & NA & 0 \\ CK-MB & 100 & NA & 0 \\ Myoglobin & 500 & NA & 0 \\ BNP & 100 & 8.8 & 0.0088 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Cross-reactivity of MPs-CLEIA with other cardiac biomarkers. Figure 5: Correlation between results measured using the commercially available Roche NT-proBNP diagnostic kit (c\({}_{0}\)) and the proposed chemiluminescencec diagnostic reagent (c\({}_{1}\)). Y = 0.9322X + 182.2 (r = 0.985, \(p\) < 0.001). Normally, the Fc region of most antibodies has a conserved oligosaccharidesacharide chain, but the Fab region lacks this feature [21]. In this study, this characteristic feature of an antibody was exploited. In the preparation of enzyme-labelled antibody, the oligosaccharidesacharide chain in the Fc region was oxidised to the aldehyde group through a reaction with sodium periodate, and then the reactive thiol group was produced in the Fc region through the aldehyde amine condensation reaction. The thiol group was coupled with SMCC-treated CmAP, so that CmAP could specifically bind to the Fc region of the antibody without affecting the variable region of the antibody. At present, [PERSON]'s reagent is being used in most studies to introduce sulfhydryl groups onto the antibody surface [19; 22], but this reaction is random. Similar to the detection antibody coupling method, we developed a new method by coupling magnetic beads to the capture antibody. We used biotin-LC-hydrazide to react with the aldehyde group formed by the oligosaccharidesacharide chain oxidation to form a stable hydrazone bond, and then connected it with streptavidin magnetic beads through biotin at the other end, which also did not affect the variable region of the capture antibody. However, until now, most studies have used flourescein isothiamate to modify the capture antibody, which could also randomly react with amino groups on the surface of the antibody [19; 23; 24; 25]. The modified group may occupy the variable region of the capture antibody. The NT-proBNP chemiluminescence diagnostic reagent was prepared using the improved method described in this paper. Compared with the traditional coupling method, the improved coupling method leads to better performance of the diagnostic reagent (data not shown). The sensitivity of CLEIA to NT-proBNP was 0.58 ng/L in this work. This is the lowest sensitivity value of CLEIA in NT-proBNP detection until now, which can be used for the diagnosis of acute HF. However, the sensitivity of the electrochemiluminescence immunoassay (ECLIA) to NT-proBNP can reach or be even lower than 0.1 ng/L [20; 26], which is much lower than that of CLEIA.