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Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera

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Abstract

This paper described a homogeneous method, light-initiated chemiluminescent assay (LICA), for quantitation of total testosterone in human sera. The assay was bead based and built on a competitive-binding reaction format, in which 5-α-dihydrotestosterone (5-α-DHT) competed with the testosterone in serum samples in binding with biotinylated anti-testosterone antibody. The more testosterone in the serum sample, the less 5-α-DHT that bonded with biotinylated anti-testosterone antibodies. 5-α-DHT was coupled with emission beads (doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter) via bovine serum albumin as a linker. Once streptavidin-coated sensitizer beads (modified with phthalocyanine as a photosensitizer) were added, the streptavidin/biotin reaction between 5-α-DHT-bound anti-testosterone antibody and sensitizer beads could bring emission and sensitizer beads together, which allowed energy transfer from sensitizer bead to emission bead. As such, an exciting light (680 nm) impinging on the sensitizer beads led to light emission at 520–620 nm by emission beads. The strength of the emitted light was inversely proportional to the testosterone in serum sample. The detection range of this assay was from 13.3 to 1200 ng/dL. The coefficient variation for intra- and inter-assay was lower than 15%. The recovery of this method ranged from 95.5 to 105.9% for different samples. Moreover, the LICA assay was highly specific with low cross-reactivity and interference. The concentration of testosterone from 58 serum samples analyzed by the LICA method significantly correlated (y = 0.97x + 1.87, R2 = 0.970, p < 0.001) with those obtained with the SIEMENS Centaur Xp System.

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Acknowledgements

We are grateful for the technical help from Chemclin Biotech (Beijing, China).

Funding

This work was supported by the National Natural Science Foundation of China (# 81772259).

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Authors and Affiliations

  1. Department of Pharmacology, School of Basic Medicine, Tianjin Medical University, No. 22 Qixiangtai Road, Heping District, Tianjin, 300203, China

    Yaqiong Cui & Weizhen Gao

  2. Department of Clinical Immunology, School of Medical Laboratory, Tianjin Medical University, No. 1 Guangdong Road, Hexi District, Tianjin, 300203, China

    Tiantian She, Hong Zhao, Liuxu Li & Huiqiang Li

  3. Department of Clinical Laboratory, Tianjin Central Hospital of Gynecology Obstetrics, 156 Sanlu Road, Nankai District, Tianjin, 300052, China

    Jiuzhi Li

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  1. Yaqiong Cui
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  2. Tiantian She
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  5. Liuxu Li
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Correspondence to Huiqiang Li.

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The study was approved by the ethics committee of Tianjin Medical University (TMUHMEC2017008). The sera involved in our research were from healthy individuals. Informed consent was obtained from all human participants.

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The authors declare that they have no conflicts of interest.

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Cui, Y., She, T., Zhao, H. et al. Competitive light-initiated chemiluminescent assay: using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera. Anal Bioanal Chem 411, 745–754 (2019). https://doi.org/10.1007/s00216-018-1496-6

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  • DOI: https://doi.org/10.1007/s00216-018-1496-6

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