Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms into the body, with bacteria being the most common causative agent. Sepsis often leads to organ dysfunction, especially in patients with septic shock, which can be high in mortality if left untreated (Jarczak et al., 2021). At present, the international diagnosis of sepsis is mainly based on infection or suspected infection + Sequential Organ Failure Assessment (SOFA) score ≥ 2, and the diagnosis of patients with septic shock mainly revolves around tissue hypoperfusion and high lactate levels that are often difficult to correct (Makic and Bridges, 2018). However, due to individual differences in patients, such as those in age, sex, underlying disease, drug use, and even different medical resources, it may affect the manifestation and reporting of symptoms in patients so that the diagnosis and classification of sepsis is often delayed. Moreover, such diagnostic criteria are often cumbersome to calculate and require more laboratory examinations. It is difficult to make a sepsis-related diagnosis in time for patients outside of the ICU ward (Font et al., 2020). As a subset of sepsis, the pathogenesis of septic shock is not yet clear, which is mainly characterized by the occurrence of uncontrolled systemic inflammatory response syndrome induced by pathogenic microorganisms, resulting in serious tissue and cell destruction, disordered metabolism, insufficient perfusion of tissues and organs, and multiple organ dysfunction (van der Poll et al., 2017). Compared with sepsis alone, septic shock has a higher mortality rate, so there is an urgent need for new clinical and laboratory tools that assist in early detection of septic shock (Seymour and Rosengart, 2015). Due to the complex pathophysiology of sepsis patients, procalcitonin (PCT) (Zhou et al., 2019), C-reactive protein (CRP) (Giannakopoulos et al., 2017), interleukin-6 (IL-6) (Song et al., 2019), interleukin-8 (IL-8) (Fu et al., 2019), Monocyte chemokine-1 (MCP-1) (Bozza et al., 2007; He et al., 2017), Myeloid cell triggering receptor-1 (TREM-1) (Sandquist and Wong, 2014; Siskind et al., 2022), and others have been investigated and evaluated for their use in the diagnostic classification and prognosis of sepsis patients. However, most clinical studies suggest a limited role for these markers.

Human neutrophil lipocalin (HNL) is mainly produced by neutrophils and renal tubular epithelial cells, and exists in multiple forms, including monomers, homodimers, and heterodimers. Among them, dimerized HNL is a marker for neutrophil activation and it is strongly associated with infection, giving it a higher efficacy in the diagnosis of bacterial infections compared to markers PCT and CRP (Venge et al., 2019; Venge and Xu, 2019). Moreover, HNL level has been shown to be significantly higher in serum than in plasma because of the additional release of HNL into the extracellular environment during the clotting process in the blood (Venge et al., 2015). This is important because higher concentrations of HNL in the serum are positively correlated with more serious infection. Therefore, detecting HNL expression in patients' serum could assist in the clinical diagnosis of infection, judgment of infection severity, and monitoring of antibiotic treatment (Fang et al., 2020).

HNL is currently detected using commercially available ELISA kits. ELISAs, however, are cumbersome and time-consuming to perform, including multiple cleaning steps to remove non-specific adsorbed reactants (Yu et al., 2015). Therefore, in this study, we developed an amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) that mainly depends on the interactions between donor beads and recipient beads. When biological reactions in the assay make the donor beads and recipient beads come into close proximity, the laser excites the cascade reaction, resulting in an amplified signal. Specifically, under the irradiation of the laser (wavelength:680 nm), the photosensitizer on the donor beads converts the oxygen in the surrounding environment into more active monomer oxygen. Monomer oxygen diffuses to the receptor beads and reacts with the chemiluminescent agent on it, further activating the fluorescent group on the same receptor beads to emit fluorescence (wavelength: 615 nm) (Liu et al., 2013; Zhang et al., 2021). Compared with an ELISA, the AlphaLISA technique has many advantages, including a short reaction time and large detection range (Yan et al., 2018).