Aldosterone (ALD), a hormone that regulates sodium balance and blood pressure in humans and a steroid hormone, also has a variety of pathophysiological effects (Leopold and Ingelfinger, 2023). ALD makes a major contribution to the pathogenesis of arterial hypertension, and primary aldosteronism (PA) is the most common form of secondary hypertension accounting for up to 10 % of all cases (Birukov et al., 2019; Quencer et al., 2023). Accurate and precise quantization of ALD is therefore essential for the screening, diagnosis, and subtype evaluation of PA (Brown, 2024; Carvajal et al., 2022). Increasing evidence also points to ALD as a major cardiovascular risk factor even in the absence of PA (Turcu et al., 2022). Furthermore, ALD measurements are also used to investigate patients with adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular channelopathies (Reincke et al., 2021; Buffolo et al., 2022).

The current mainstay for measuring aldosterone involves antibody-based methods, which are rapid and easy to perform (Hanquez et al., 1987; Barnes, 2013). However, these methods have the disadvantages of cross-reactivity and poor inter-laboratory reproducibility (Tetti et al., 2024; Tan et al., 2020; Gibbons et al., 2021; Hubl et al., 1990). Kit performance such as sensitivity and specificity highly depends on the choice of monoclonal antibodies (mAbs) (Hanquez et al., 1988). Competitive enzyme-linked immunoassay (ELISA)-based methods have been developed using serotype-specific MAb (anti-ALD 601#) in our laboratory. In addition, the current ELISA kit with anti-ALD 601# available in the market is licensed for diagnostic use. However, anti-ALD 601# has a low affinity for the antigens because somatic hypermutations did not occur. To use antibodies with low affinity for the antigens as research tools for diagnostics, it is necessary to enhance the affinity for the antigens.

Phage display technology has been extensively used since its development for the isolation of antibodies for research purposes and well suited for the selection and evolution of high-affinity antibodies (Saw and Song, 2019). In this approach, gene libraries are constructed with mutations in the antigen-binding regions such as variable light chain (VL) and variable heavy chain (VH), the libraries were displayed on the phages, and the antibodies with high affinity for the antigens were then isolated by biopanning (Li et al., 2015; Zheng et al., 2024; Kim et al., 2024; Hayrapetyan et al., 2023). In order to construct gene libraries with mutations, site-specific complementarity determining regions-3 (CDR3) mutagenesis in both the VL and VH domains were developed. For antibody affinity maturation, libraries of mutated wild-type single-chain variable fragments (scFvs) positive clones were constructed using an overlap extension PCR-based approach (Hutchings and Sato, 2024; Peissert et al., 2023; Sompunga et al., 2019; Lim et al., 2019; Ye et al., 2022). Finally, to obtain stably expressed recombinant monoclonal antibody against ALD, the anti-ALD-scFv-3302# with high affinity obtained by competitive ELISA screening was sequenced and primers were designed for transfection into Chinese hamster ovary-S (CHO-S) cells (Fig. 1).