Analytical and Bioanalytical Chemistry

, Volume 408, Issue 19, pp 5179–5188 | Cite as

An internal standard approach for homogeneous TR–FRET immunoassays facilitates the detection of bacteria, biomarkers, and toxins in complex matrices

  • Noam Cohen
  • Eran Zahavy
  • Ran Zichel
  • Morly FisherEmail author
Research Paper


The recent development of a homogeneous time-resolved Förster resonance energy transfer (TR–FRET) immunoassay enables one-step, rapid (minutes), and direct detection compared to the multistep, time-consuming (hours), heterogeneous ELISA-type immunoassays. The use of the time-resolved effect of a donor lanthanide complex with a delay time of microseconds and large Stokes shift enables the separation of positive signals from the background autofluorescence of the sample. However, this study shows that the sample matrices directly interfere with donor fluorescence and that interference cannot be eliminated by time-resolved settings alone. Moreover, the reduction in donor emission did not appear to be equivalent to the reduction in acceptor emission, resulting in incorrect FRET signal measurements. To overcome this limitation, an internal standard approach was developed using an isotype control antibody. This new approach was used to develop TR–FRET assays for rapid detection (15–30 min) of Bacillus anthracis spores and botulinum toxin (type E) in beverages, which are major concerns in bioterrorism involving deliberate food contamination. Additionally, we demonstrate the detection of B. anthracis-secreted protective antigen (PA) and the Yersinia pestis-secreted markers F1 and LcrV in blood cultures, which are early markers of bacteremia in infected hosts following a possible bioterror attack. The use of an internal standard enables the calculation of correct ΔF values without the need for an external standard. Thus, the use of the internal standard approach in homogeneous immunoassays facilitates the examination of any sample regardless of its origin, and therefore expands the applicability of TR–FRET assays for complex matrices.


Time-resolved fluorescence FRET Yersinia pestis Bacillus anthracis Botulinum toxin 


Compliance with ethical standards

All animal experiments were performed in accordance with Israeli law and were approved by the Ethics Committee for Animal Experiments at the Israel Institute for Biological Research.

Conflict of interest

The authors state that there are no conflicts of interest.


  1. 1.
    Lee JS, Joung HA, Kim MG, Park CB. Graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay. ACS Nano. 2012;6(4):2978–83. doi: 10.1021/nn300684d.CrossRefGoogle Scholar
  2. 2.
    Watanabea J, Ishihara K. Single step diagnosis system using the FRET phenomenon induced by antibody-immobilized phosphorylcholine group-covered polymer nanoparticles. Sensors Actuators B Chem. 2008;129(1):87–93. doi: 10.1016/j.snb.2007.07.100.CrossRefGoogle Scholar
  3. 3.
    Wei Q, Lee M, Yu X, Lee EK, Seong GH, Choo J, et al. Development of an open sandwich fluoroimmunoassay based on fluorescence resonance energy transfer. Anal Biochem. 2006;358(1):31–7. doi: 10.1016/j.ab.2006.08.019.CrossRefGoogle Scholar
  4. 4.
    Boeneman K, Delehanty JB, Susumu K, Stewart MH, Deschamps JR, Medintz IL. Quantum dots and fluorescenct protein FRET-based biosensors. In: Zahavy E, Ordentlich A, Yitzhaki S, Shafferman A, editors. Nano-biotechnology for biomedical and diagnostic research. Advances in experimental medicine and biology. Dordrecht: Springer; 2012.Google Scholar
  5. 5.
    Hildebrandt N, Geissler D. Semiconductor Quntum dots as FRET acceptors for multiplexed diagnostic ruler application. In: Zahavy E, Ordentlich A, Yitzhaki S, Shafferman A, editors. Nano-biotechnology for biomedical and diagnostic research. Advances in experimental medicine and biology. Dordrecht: Springer; 2012.Google Scholar
  6. 6.
    Spindel S, Granek J, Sapsford KE. In vitro FRET sensing, diagnostic and personlized medicine. In: Medintz IL, Hildebrandt N, editors. FRET - from theory to application. Weinheim: Wiley-VCH; 2014.Google Scholar
  7. 7.
    Harma H, Dahne L, Pihlasalo S, Suojanen J, Peltonen J, Hanninen P. Sensitive quantitative protein concentration method using luminescent resonance energy transfer on a layer-by-layer europium(III) chelate particle sensor. Anal Chem. 2008;80(24):9781–6. doi: 10.1021/ac801960c.CrossRefGoogle Scholar
  8. 8.
    Kokko T, Liljenback T, Peltola MT, Kokko L, Soukka T. Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer. Anal Chem. 2008;80(24):9763–8. doi: 10.1021/ac801875a.CrossRefGoogle Scholar
  9. 9.
    Kupstat A, Kumke MU, Hildebrandt N. Toward sensitive, quantitative point-of-care testing (POCT) of protein markers: miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection. Analyst. 2011;136(5):1029–35. doi: 10.1039/c0an00684j.CrossRefGoogle Scholar
  10. 10.
    Medintz I, Hildebrandt N, editors. FRET - Förster resonance energy transfer: from theory to applications. Wiley Online Library; 2013.Google Scholar
  11. 11.
    Cohen N, Mechaly A, Mazor O, Fisher M, Zahavy E. Rapid homogenous time-resolved fluorescence (HTRF) immunoassay for anthrax detection. J Fluoresc. 2014;24(3):795–801. doi: 10.1007/s10895-014-1354-7.CrossRefGoogle Scholar
  12. 12.
    Geissler D, Stufler S, Lohmannsroben HG, Hildebrandt N. Six-color time-resolved Forster resonance energy transfer for ultrasensitive multiplexed biosensing. J Am Chem Soc. 2013;135(3):1102–9. doi: 10.1021/ja310317n.CrossRefGoogle Scholar
  13. 13.
    Mechaly A, Cohen N, Weiss S, Zahavy E. A novel homogeneous immunoassay for anthrax detection based on the AlphaLISA method: detection of B. anthracis spores and protective antigen (PA) in complex samples. Anal Bioanal Chem. 2013;405(12):3965–72. doi: 10.1007/s00216-013-6752-1.CrossRefGoogle Scholar
  14. 14.
    Qin QP, Peltola O, Pettersson K. Time-resolved fluorescence resonance energy transfer assay for point-of-care testing of urinary albumin. Clin Chem. 2003;49(7):1105–13.CrossRefGoogle Scholar
  15. 15.
    Anisimov AP, Lindler LE, Pier GB. Intraspecific diversity of Yersinia pestis. Clin Microbiol Rev. 2004;17(2):434–64.CrossRefGoogle Scholar
  16. 16.
    Skrzypek E, Straley SC. Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis. J Bacteriol. 1995;177(9):2530–42.Google Scholar
  17. 17.
    Kobiler D, Weiss S, Levy H, Fisher M, Mechaly A, Pass A, et al. Protective antigen as a correlative marker for anthrax in animal models. Infect Immun. 2006;74(10):5871–6. doi: 10.1128/IAI.00792-06.CrossRefGoogle Scholar
  18. 18.
    Jernigan D, Raghunathan P, Bell B, Brechner R, Bresnitz E, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis. 2002;8(10):1019–28. doi: 10.3201/eid0810.020353.CrossRefGoogle Scholar
  19. 19.
    Wein LM, Liu Y. Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk. Proc Natl Acad Sci U S A. 2005;102(28):9984–9. doi: 10.1073/pnas.0408526102.CrossRefGoogle Scholar
  20. 20.
    Cohen S, Mendelson I, Altboum Z, Kobiler D, Elhanany E, Bino T, et al. Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax. Infect Immun. 2000;68(8):4549–58.CrossRefGoogle Scholar
  21. 21.
    Ben-Gurion R, Shafferman A. Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid. 1981;5(2):183–7.CrossRefGoogle Scholar
  22. 22.
    Reuveny S, White MD, Adar YY, Kafri Y, Altboum Z, Gozes Y, et al. Search for correlates of protective immunity conferred by anthrax vaccine. Infect Immun. 2001;69(5):2888–93. doi: 10.1128/IAI.69.5.2888-2893.2001.CrossRefGoogle Scholar
  23. 23.
    Diamant E, Lachmi BE, Keren A, Barnea A, Marcus H, Cohen S, et al. Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations. PLoS One. 2014;9(1):e87089. doi: 10.1371/journal.pone.0087089.CrossRefGoogle Scholar
  24. 24.
    Levy Y, Flashner Y, Tidhar A, Zauberman A, Aftalion M, Lazar S, et al. T cells play an essential role in anti-F1 mediated rapid protection against bubonic plague. Vaccine. 2011;29(40):6866–73. doi: 10.1016/j.vaccine.2011.07.059.CrossRefGoogle Scholar
  25. 25.
    Zahavy E, Fisher M, Bromberg A, Olshevsky U. Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry. Appl Environ Microbiol. 2003;69(4):2330–9.CrossRefGoogle Scholar
  26. 26.
    Flashner Y, Fisher M, Tidhar A, Mechaly A, Gur D, Halperin G, et al. The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease. FEMS Immunol Med Microbiol. 2010;59(2):197–206. doi: 10.1111/j.1574-695X.2010.00687.x.CrossRefGoogle Scholar
  27. 27.
    Knepp AM, Grunbeck A, Banerjee S, Sakmar TP, Huber T. Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry. 2011;50(4):502–11. doi: 10.1021/bi101059w.CrossRefGoogle Scholar
  28. 28.
    Vagima Y, Levy Y, Gur D, Tidhar A, Aftalion M, Abramovich H, et al. Early sensing of Yersinia pestis airway infection by bone marrow cells. Front Cell Infect Microbiol. 2012;2:143. doi: 10.3389/fcimb.2012.00143.CrossRefGoogle Scholar
  29. 29.
    Turro N, Ramamurthy V, Scaiano J. Modern molecular photochemistry of organic molecules. New York: Wiley Online Library; 2012.Google Scholar
  30. 30.
    Janzen TW, Thomas MC, Goji N, Shields MJ, Hahn KR, Amoako KK. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense. J Food Prot. 2015;78(2):355–61. doi: 10.4315/0362-028X.JFP-14-216.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Noam Cohen
    • 1
  • Eran Zahavy
    • 2
  • Ran Zichel
    • 1
  • Morly Fisher
    • 2
    Email author
  1. 1.Department of BiotechnologyIsrael Institute for Biological ResearchNess-ZionaIsrael
  2. 2.Department of Infectious DiseasesIsrael Institute for Biological ResearchNess-ZionaIsrael

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