Development of a novel homogeneous nanoparticle-based assay for rapid and high-throughput quantitation of the sst2 protein in human serum

Abstract

Purpose: The suppression of tumorigenicity 2 (ST2) protein is a member of the interleukin-1 receptor family with the transmembrane (ST2L) and soluble (sST2) subtypes and plays an important role in several diseases. Therefore, the present study aimed to establish and validate a novel amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the detection of sST2 in human serum. Materials and Methods: Based on a sandwich-type immunoassay format, sST2 was captured using two different anti-sST2 antibodies. One of the antibodies was biotinylated while the other one was coated with AlphaLISA chemibeads. Thereafter, multiple tests were conducted to optimize the working conditions and validate analytical performance. Results: The optimum concentration of the biotinylated antibodies was 1 μg/mL while the optimal dilution ratio for the anti-sST2 antibodies and conjugated chemibeads was 1:500. In addition, the optimal antigen-antibody reaction time was 15 minutes (min). Notably, the developed method showed a short turnaround time of about 25 min. Moreover, the assay exhibited high sensitivity with a limit of detection (LOD) of 0.176 ng/mL and a limit of quantification (LOQ) of 0.8 ng/mL. Furthermore, the intra-assay precision and inter-assay precision values were 5.29–7.10% and 9.41%–13.66%, respectively. It is also noteworthy that the test results deviated by less than ±10% when samples had ≤10.0 ng/mL of triglycer-ides, ≤0.5 mmol/L of bilirubin, ≤5.0 g/L of triglyceride, and ≤250 μg/L of biotin. Additionally, the developed assay was almost consistent with the commercially available PresageTM ST2 assay kit, with a Spearman correlation coefficient of 0.916 and an R2 of 0.963 as well as a slope of 0.957 from linear regression analysis. Conclusion: The present study showed that the sandwich AlphaLISA is a rapid, high-throughput, and reliable test for studying the levels of sST2 in a variety of diseases.