ABSTRACT
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.
ABSTRACT
BACKGROUND: Light-initiated chemiluminescent assays (LICA) are homogeneous assays that are sensitive, specific, and free of separation and washing steps and have high throughput and high precision. METHODS: In this research, we developed a competitive method by LICA to achieve accurate quantification of estradiol (E2) in human serum. E2 competed with estriol (E3) for binding to anti-human E2 antibodies. E3 was linked to biotin via bovine serum albumin as a linker. As this assay used competition between the labeled tracer and the analyte, an increase in E2 concentration will cause a signal decrease. RESULTS: The expected detection range of E2 was 20-5000 pg/mL. The analytical and functional sensitivities were 7.16 and 13.7 pg/mL, respectively. The intra- and inter-assay coefficients of variation were both below 15%, and the recovery rate ranged from 97.5% to 106.8%. The interference rates ranged from -3.6% to 5.4% and met detection requirements for E2 in hyperbilirubinemia, hemolysis, and lipemia in clinical samples. In addition, the cross-reactivity rates between E2 and structural analogs and some reproductive hormones varied from 1.9% to 10.6% which showed that LICA is highly specific for E2. Moreover, our results showed high accordance with the IMMULITE 2000 (y = 0.6695x + 47.92, r2 = .843) and VIDAS systems (y = 1.099x - 821.5, r2 = .9392). CONCLUSION: Our data show that the LICA, which is easy to automate, is a promising technique for quantification of E2 in human serum and could be used for clinical detection.
