Development and exploratory evaluation of a quantitative immunochromatographic assay for serum ACOX1 in intrahepatic cholestasis of pregnancy

Serum biomarkers play a key role in the early diagnosis and risk stratification of intrahepatic cholestasis of pregnancy (ICP). Our previous study showed that acyl-CoA oxidase 1 (ACOX1) was elevated in the serum and placenta of ICP patients. In this study, a pair of commercially available anti-ACOX1 antibodies was purchased and used to develop a double-antibody sandwich time-resolved fluorescent nanomicrosphere immunochromatographic (TRFNI) strip for the quantitative detection of ACOX1 in serum. Methodological evaluation showed that the strip exhibited good linearity over a calibration set consisting of a blank and standards from 0.25 to 20 ng/mL, with intra-assay and inter-assay coefficients of variation (CVs) below 10% and 15%, respectively. The buffer-based LOD was 0.19 ng/mL, and the lower limit of the reportable quantitative range should not be set below the calculated LOQ of 0.29 ng/mL. In a preliminary accelerated storage assessment, the T/C ratio at 1 ng/mL remained relatively stable during 20 days at 37 °C. The TRFNI assay also showed correlation with a commercial ELISA kit and exhibited recovery rates of 85.5-100.5%. Furthermore, ACOX1 demonstrated an area under the receiver operating characteristic curve (AUC) of 0.731 for distinguishing severe from mild ICP and an AUC of 0.700 for discriminating adverse perinatal outcomes. This assay may warrant further evaluation as an exploratory adjunctive approach for measuring serum ACOX1 in ICP, pending prospective validation against disease-control cohorts and full serum-matrix analytical characterisation.