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

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.

Development in competitive immunoassay of a point-of-care testing for cotinine (COT) detection in urine

“…Zhang et al (2022) demonstrated that a lower concentration of antibodies can greatly influence the signal produced between low, moderate, and high amounts of serum. 35 A lower concentration of antibodies results in fewer binding sites for antigens in the sample, which fully block the binding site on the antigen at the TL. Excessive immobilization of antibodies on AuNPs can lead to false negative results, as the antibodies can still bind with the antigen at the TL.…”

“…Therefore, it is necessary to use the minimal concentration of antibody-immobilized AuNPs that can produce a band when tested with the negative sample to improve the sensitivity in competitive assay. 35…”

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers

“…However, the homologous combination does not provide satisfactory sensitivity, because the binding affinity of the labeled antigen to the antibody is higher than that of the antigen to be measured (18,19). Few sensitive homologous assays for steroids have been developed (1)(2)(3)(4)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”