Improved Sensitivity and Wide Range Detection of Small Analytes Using a Two-Antigen-Combined Competitive Immunoassay

[Image: see text] Competitive immunoassays have unique advantages in the detection of small molecules and are widely used in clinical practice. However, the concentrations of some analytes usually vary greatly among different populations, which makes it difficult to balance the sensitivity and detection range of competitive immunoassays. Studies have shown that using haptens with weaker affinity for specific antibodies as competitive antigens can help improve the sensitivity of the method. Here, we developed a competitive light initiated chemiluminescence assay based on the combination of antigens with different affinities, which has high sensitivity and wide detection range. As a proof of concept, estradiol was used as the analyte. After the mixing ratio was optimized, the two labeled haptens played different competitive roles due to the different concentrations of estradiol to be tested, which improved the sensitivity of estradiol detection, while ensuring a certain detection range. The limit of detection of this method was 5.30 pg/mL, which is lower than most current estradiol immunoassay kits. Good linearity (R(2) = 0.9902) was obtained between estradiol concentrations of 17.07–2376.22 pg/mL. This study provides a new solution for the detection of small molecule biomarkers with a large concentration span, which also has considerable potential in other immunological detection methods.