Analytical and Bioanalytical Chemistry

, Volume 411, Issue 3, pp 745–754 | Cite as

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

  • Yaqiong Cui
  • Tiantian She
  • Hong Zhao
  • Jiuzhi Li
  • Liuxu Li
  • Weizhen Gao
  • Huiqiang LiEmail author
Research Paper


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.

Graphical abstract


Testosterone 5-α-Dihydrotestosterone Competitive immunoassay Light-initiated chemiluminescent assay (LICA) Quantitative assay 



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


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

Compliance with ethical standards

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.

Competing interests

The authors declare that they have no conflicts of interest.

Supplementary material

216_2018_1496_MOESM1_ESM.pdf (298 kb)
ESM 1 (PDF 297 kb)


  1. 1.
    Spartt DI, Bigos ST, Beitins I, Cox P, Longcope C, Orav J. Both hyper-and hypogonadotropic hypogonadism occur transiently in acute illness: bio- and immunoactive gonadotropins. J Clin Endocrinol Metab. 1992;75:1562–70.Google Scholar
  2. 2.
    Woolf PD, Hamill RW, McDonald JV, Lee LA, Kelly M. Transient hypogonadotropic hypogonadism caused by critical illness. J Clin Endocrinol Metab. 1985;60:444–50.CrossRefGoogle Scholar
  3. 3.
    Macadams MR, White RH, Chipps BE. Reduction of serum testosterone levels during chronic glucocorticoid therapy. Ann Intern Med. 1986;104:648–51.CrossRefGoogle Scholar
  4. 4.
    Kamischke A, Kemper DE, Castel MA, Lüthke M, Rolf C, Behre HM, et al. Testosterone levels in men with chronic obstructive pulmonary disease with or without glucocorticoid therapy. Eur Respir J. 1998;11:41–5.CrossRefGoogle Scholar
  5. 5.
    Anttila L, Koskinen P, Erkkola R, Irjala K, Ruutiainen K. Serum testosterone, androstenedione and luteinizing hormone levels after short-termmedroxyprogesterone acetate treatment in women with polycystic ovarian disease. Acta Obstet Gynecol Scand. 1994;73:634–6.CrossRefGoogle Scholar
  6. 6.
    Anttila L, Koskinen P, Kaihola H-L, Erkkola R, Irjalii K, Ruutiainen K. Serum androgen and gonadotropin levels decline after progestogen-induced withdrawal bleeding inoligomenorrheic women with or without polycystic ovaries. Fertil Steril. 1992;58:697–703.CrossRefGoogle Scholar
  7. 7.
    Badoud F, Boccard J, Schweizer C, Pralong F, Saugy M, Baume N. Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. J Steroid Biochem Mol Biol. 2013;138:222–2235.CrossRefGoogle Scholar
  8. 8.
    Owen LJ, Wu FC, Büttler RM, Keevil BG. A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by LC-MS/MS. Ann Clin Biochem. 2016;53:580–7.CrossRefGoogle Scholar
  9. 9.
    Tobias HJ, Zhang Y, Auchus RJ, Brenna JT. Detection of synthetic testosterone use by novel comprehensive two-dimensional gas chromatography combustion isotope ratio mass spectrometry (GC×GCC-IRMS). Anal Chem. 2011;83:7158–65.CrossRefGoogle Scholar
  10. 10.
    Levesque A, Letellier M, Swirski C, Lee C, Grant A. Analytical evaluation of the testosterone assay on the Bayer Immuno 1 system. Clin Biochem. 1998;31:23–8.CrossRefGoogle Scholar
  11. 11.
    Yockell-Lelièvre H, Bukar N, McKeating KS, Arnaud M, Cosin P, Guo Y, et al. Plasmonicsensors for the competitive detection of testosterone. Analyst. 2015;140:5105–11.CrossRefGoogle Scholar
  12. 12.
    Huang Y, Shi M, Zhao S, Liang H. A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection. Electrophoresis. 2011;32:3196–200.CrossRefGoogle Scholar
  13. 13.
    Wang H, Li J, Zhang X, Hu B, Liu Y, Zhang L, et al. A microfluidic indirect competitive immunoassay for multiple and sensitive detection of testosterone in serum and urine. Analyst. 2016;141:815–9.CrossRefGoogle Scholar
  14. 14.
    Laulua SL, Kalpa KJ, Straseskib JA. How low can you go? Analytical performance of five automated testosterone immunoassays. Clin Chem. 2018;58:64–71.Google Scholar
  15. 15.
    Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography–mass spectrometry in sera from 116 men, women and children. Clin Chem. 2003;49:1381–95.CrossRefGoogle Scholar
  16. 16.
    Ullman EF, Kirakossian H, Singh S, Wu ZP, Irvin BR, Pease JS, et al. Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A. 1994;91:5426–30.CrossRefGoogle Scholar
  17. 17.
    Ullman EF, Kirakossian H, Switchenko AC, Ishkanian J, EricsonM, Wartchow CA, et al. Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method. Clin Chem 1996;42:1518–1526.Google Scholar
  18. 18.
    Hou C, Zhao L, Geng F, Wang D, Guo LH. Donor/acceptor nanoparticle pair-based singlet oxygen channeling homogenous chemiluminescence immunoassay for quantitative determination of bisphenol A. Anal Bioanal Chem. 2016;48:8795–804.CrossRefGoogle Scholar
  19. 19.
    Bian Y, Liu C, She T, Wang M, Yan J, Wei D, et al. Development of a light-initiated chemiluminescent assayfor the quantitation of sIgE against egg white allergens based on component-resolved diagnosis. Anal Bioanal Chem. 2018;410:1501–10.CrossRefGoogle Scholar
  20. 20.
    Anderson DC. Sex hormone-binding globulin. Clin Endocrinol. 1974;3:69–96.CrossRefGoogle Scholar
  21. 21.
    Zettner A. Principles of competitive binding assays (saturation analyses). Equilibrium Techniques. Clin Chem. 1973;19:699–705.Google Scholar
  22. 22.
    Zettner A, Duly PE. Principles of competitive binding assays(saturation analyses). Sequential Saturation. Clin Chem. 1974;20:15–4.Google Scholar
  23. 23.
    Uraipong C, Wong V, Lee NA. A testosterone specific competitive enzyme immunoassay for monitoring water quality. Bull Environ Contam Toxicol. 2013;90:585–90.CrossRefGoogle Scholar
  24. 24.
    Boscato LM, Stuart MC. Heterophilic antibodies: a problem for all immunoassays. Clin Chem. 1988;34:27–33.Google Scholar
  25. 25.
    Glick MR, Ryder KW, Jackson SA. Graphical comparisons of interferences in clinical chemistry instrumentation. Clin Chem. 1986;32:470–5.Google Scholar
  26. 26.
    Dawson DA, Genco N, Bensinger HM, Guinn D, Il'giovine ZJ, Wayne Schultz T, et al. Evaluation of an asymmetry parameter for curve-fitting in single-chemical and mixture toxicity assessment. Toxicology. 2012;292:156–61.CrossRefGoogle Scholar
  27. 27.
    Dawson DA, Scott BD, Ellenberger MJ, Poch G, Rinaldi AC. Evaluation of dose-response curve analysis in delineating shared or different molecular sites of action for osteolathyrogens. Environ Toxicol Pharmacol. 2004;16:13–23.CrossRefGoogle Scholar
  28. 28.
    Hammond GL, Bocchinfuso WP. Sex hormone-binding globulin: gene organization and structure/function analyses. Horm Res. 1996;45:197–201.CrossRefGoogle Scholar
  29. 29.
    Zhao H, Lin G, Liu T, Liang J, Ren Z, Liang R, et al. Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA). J Immunol Methods. 2016;437:64–9.CrossRefGoogle Scholar
  30. 30.
    Zou LP, Liu TC, Lin GF, Dong ZN, Hou JY, Li M, et al. AlphaLISA for the determination of median levels of the free beta subunit of human chorionic gonadotropin in the serum of pregnant women. J Immunoassay Immunochem. 2013;34:134–48.CrossRefGoogle Scholar
  31. 31.
    He A, Liu TC, Dong ZN, Ren ZQ, Hou JY, Li M, et al. A novel immunoassay for the quantization of CYFRA 21–1 in human serum. J Clin Lab Anal. 2013;27:277–83.CrossRefGoogle Scholar
  32. 32.
    Harndahl M, Justesen S, Lamberth K, Røder G, Nielsen M, Buus S. Peptide binding to HLA class I molecules: homogenous, high-throughput screening, and affinity assays. J Biomol Screen. 2009;14:173–80.CrossRefGoogle Scholar
  33. 33.
    Bielefeld Sevigny M. AlphaLISA immunoassay platform —the “no-wash” high-throughput alternative to ELISA. Assay Drug Dev Technol. 2009;7:90–2.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yaqiong Cui
    • 1
  • Tiantian She
    • 2
  • Hong Zhao
    • 2
  • Jiuzhi Li
    • 3
  • Liuxu Li
    • 2
  • Weizhen Gao
    • 1
  • Huiqiang Li
    • 2
    Email author
  1. 1.Department of Pharmacology, School of Basic MedicineTianjin Medical UniversityTianjinChina
  2. 2.Department of Clinical Immunology, School of Medical LaboratoryTianjin Medical UniversityTianjinChina
  3. 3.Department of Clinical LaboratoryTianjin Central Hospital of Gynecology ObstetricsTianjinChina

Personalised recommendations