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.
Similar content being viewed by others
References
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.
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.
Macadams MR, White RH, Chipps BE. Reduction of serum testosterone levels during chronic glucocorticoid therapy. Ann Intern Med. 1986;104:648–51.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Laulua SL, Kalpa KJ, Straseskib JA. How low can you go? Analytical performance of five automated testosterone immunoassays. Clin Chem. 2018;58:64–71.
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.
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.
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.
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.
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.
Anderson DC. Sex hormone-binding globulin. Clin Endocrinol. 1974;3:69–96.
Zettner A. Principles of competitive binding assays (saturation analyses). Equilibrium Techniques. Clin Chem. 1973;19:699–705.
Zettner A, Duly PE. Principles of competitive binding assays(saturation analyses). Sequential Saturation. Clin Chem. 1974;20:15–4.
Uraipong C, Wong V, Lee NA. A testosterone specific competitive enzyme immunoassay for monitoring water quality. Bull Environ Contam Toxicol. 2013;90:585–90.
Boscato LM, Stuart MC. Heterophilic antibodies: a problem for all immunoassays. Clin Chem. 1988;34:27–33.
Glick MR, Ryder KW, Jackson SA. Graphical comparisons of interferences in clinical chemistry instrumentation. Clin Chem. 1986;32:470–5.
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.
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.
Hammond GL, Bocchinfuso WP. Sex hormone-binding globulin: gene organization and structure/function analyses. Horm Res. 1996;45:197–201.
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.
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.
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.
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.
Bielefeld Sevigny M. AlphaLISA immunoassay platform —the “no-wash” high-throughput alternative to ELISA. Assay Drug Dev Technol. 2009;7:90–2.
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).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
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.
Electronic supplementary material
ESM 1
(PDF 297 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-1496-6