Serum amyloid A (SAA), discovered and named over 50 years ago, is a protein with a length of 150 kb and a size of 12,000 (Sack Jr., 2018). It exhibits remarkable immune activity and undergoes substantial changes in plasma concentration during the acute phase reaction (Zhu et al., 2021). Furthermore, SAA has been implicated in the progression of rheumatoid arthritis (Zhou et al., 2022), tumor development (Lee and Beatty, 2021), Crohn's disease, cardiovascular disease (Shridas et al., 2021), transplant rejection, and other pathological conditions. Lactoferrin (LTF) is an iron-bound glycoprotein present in various bodily fluids. LTF in stool, saliva, tears, and cerebrospinal fluid helps diagnose inflammatory bowel disease, Alzheimer's disease, dry eye disease, and bacterial meningitis, respectively (Maffei et al., 1999; Versura et al., 2010; Dastych et al., 2015; Njunge et al., 2017; Rubio et al., 2019; Gonzalez-Sanchez et al., 2020). In blood, its concentration typically remains low at approximately 0.2–0.6 mg/L (Kucia et al., 2020). However, when the body develops an infection and sepsis, LTF is rapidly released from neutrophils, leading to a significant increase in its concentration in the blood. This makes it a promising biomarker for early diagnosis (Brock, 2012; Decembrino et al., 2017; Ahmed et al., 2019; Kell et al., 2020). The clinical significance of both inflammatory markers has garnered remarkable attention in recent years, resulting in the growing importance of their quantitative detection.

Currently, a range of techniques have been developed for the detection of SAA, including radioimmunoassay (RIA) (Marhaug, 1983), enzyme⁃linked immunosorbent assay (ELISA) (Carbone et al., 2021), turbidimetric inhibition immunoassay (Hansen et al., 2006), chemiluminescence immunoassay (Hulten et al., 1999) etc. The aforementioned methods, however, possess inherent drawbacks including radioactive contamination, intricate operational procedures, and limited sensitivity. Moreover, The clinical application of LTF detection in body fluids is significantly limited by its reliance on ELISA, thus impeding its potential for widespread adoption (Dastych et al., 2015). While these traditional immunoassays allow for the detection of only one marker per sample at a time, it is worth exploring multiple detection techniques that enable the simultaneous detection of multiple markers in a single reaction, thus achieving cost, time and labor reduction objectives (Rojas et al., 2022). Our team has previously developed a multiple immunodetection system utilizing stable element labeling technique and ICP-MS, which exhibits remarkable advantages including exceptional sensitivity, specificity, throughput, and detection range. Furthermore, its reliability has been extensively validated through clinical laboratory applications (Jiang et al., 2020a; Jiang et al., 2020c). Therefore, the objective of this study is to establish a duplex immunodetection system for the two inflammatory markers, conduct method validation and performance verification in accordance with CLSI standard protocols, and evaluate its reliability through analysis of clinical samples.