Applied Biochemistry and Biotechnology

, Volume 87, Issue 1, pp 25–35 | Cite as

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

  • Mohyee E. Eldefrawi
  • Nehad L. Azer
  • Nidhi Nath
  • Nabil A. Anis
  • Madan S. Bangalore
  • Kevin P. O’Connell
  • Robert P. Schwartz
  • Jeremy Wright


An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 µg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).

Index Entries

Morphine urinalysis monoclonal antibody polymethyl methacrylate beads KinExA™ 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Soini, E. and Hemmila, I. (1979), Clin. Chem. 25, 353–361.Google Scholar
  2. 2.
    Jolley, M. E., Wang, C. J., Ekenberg, S. J., Zuelke, M. S., and Kelso, D. M. (1984), J. Immunol. Methods 67, 21–35.CrossRefGoogle Scholar
  3. 3.
    MacCrindle, C., Schwantzer, K., and Jolley, M. E. (1985), Clin. Chem. 31, 1487–1490.Google Scholar
  4. 4.
    Glass, T. R., Lackie, S., and Hirschfeld, T. (1987), Appl. Optics 26, 2181–2187.Google Scholar
  5. 5.
    Andrade, J. D., Van Wagenen, R. A., Gregonis, D. E., Newby, K., and Lin, J. N. (1985), IEEE Trans. Electron Devices 32, 1175–1179.Google Scholar
  6. 6.
    Anis, N. A., Eldefrawi, M. E., and Wong, R. B. (1993), J. Agric. Food Chem. 41, 843–848.CrossRefGoogle Scholar
  7. 7.
    Brummel, K. E., Wright, J., and Eldefrawi, M. E. (1997), J. Agric. Food Chem. 45, 3292–3298.CrossRefGoogle Scholar
  8. 8.
    Devine, P. J., Anis, N. A., Wright, J., Kim, S., Eldefrawi, A. T., and Eldefrawi, M. E. (1995), Anal. Biochem. 227, 216–224.CrossRefGoogle Scholar
  9. 9.
    Zhao, C. Q., Anis, N. A., Rogers, K. R., Kline, R. H., Jr., Wright, J., Eldefrawi, A. T., and Eldefrawi, M. E. (1995), J. Agric. Food Chem. 43, 2308–2315.CrossRefGoogle Scholar
  10. 10.
    Toppozada, A. R., Wright, J., Eldefrawi, A. T., Eldefrawi, M. E., Johnston, E. L., Emche, S. D., and Helling, C. S. (1997), Biosens. Bioelectron. 12, 113–124.CrossRefGoogle Scholar
  11. 11.
    Nath, N., Wright, J., Eldefrawi, M. E., Darwin, D., and Huestis, H. J. Analyt. Toxicol. 23, 460–467.Google Scholar
  12. 12.
    Glass, T. (1995), Biomed. Prod. 20, 122, 123.Google Scholar
  13. 13.
    O’Connell, K. P., Valdes, J. J., Azer, N. L., Schwartz, R. P., Wright, J., and Eldefrawi, M. E. (1999), J. Immunol. Methods 225, 157–169.CrossRefGoogle Scholar
  14. 14.
    Moore, F. M. and Simpson, D. (1989), Med. Lab. Sci. 46, 309–312.Google Scholar
  15. 15.
    Moore, F. M. and Simpson, D. (1990), Med. Lab. Sci. 47, 85–89.Google Scholar
  16. 16.
    Lewellen, L. J. and McCurdy, H. H. (1988), J. Analyt. Toxicol. 12, 260–264.Google Scholar
  17. 17.
    Rubenstein, K. E., Schneider, R. S., and Ullman, E. F. (1972), Biochem. Biophys. Res. Commun. 47, 846–851.CrossRefGoogle Scholar
  18. 18.
    Cheng, Y. and Prusoff, W. H. (1973), Biochem. Pharmacol. 22, 3099–3108.CrossRefGoogle Scholar
  19. 19.
    Braithwaite, R. A., Jarvie, D. R., Minty, P. S. B., Simpson, D., and Widdop, B. (1995), Ann. Clin. Biochem. 32, 123–153.Google Scholar
  20. 20.
    Allen, L. V. and Stiles, M. L. (1981), Clin. Toxicol. 18, 1043–1065.CrossRefGoogle Scholar
  21. 21.
    Moore, F. M. and Simpson, D. (1993), Br. J. Biomed. Sci. 50, 57–59.Google Scholar
  22. 22.
    Rogers, K. R., Kohl, S. D., Riddick, L. A., and Glass, T. (1997), Analyst 122, 1107–1111.CrossRefGoogle Scholar
  23. 23.
    Blake, D. A., Chakrabarti, P., Khosraviani, M., Hatcher, F. M., Westhoff, C. M., Gobel, P., Wylie, D. E., and Blake, R. C. (1996), J. Biol. Chem. 271, 27,677–27,685.Google Scholar
  24. 24.
    Craig, L., Sanschagrin P. C., Rozek, A., Lackie, S., Kuhn, L. A., and Scott, J. K. (1998), J. Mol. Biol. 281, 183–201.CrossRefGoogle Scholar
  25. 25.
    Chuntharapai, A., Gibbs, V., Lu, J., Ow, A., Marsters, S., Ashkenazi, A., De Vos, A., and Jin Kim, K. (1999), J. Immunol. 163, 766–773.Google Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Mohyee E. Eldefrawi
    • 1
  • Nehad L. Azer
    • 1
  • Nidhi Nath
    • 1
  • Nabil A. Anis
    • 1
  • Madan S. Bangalore
    • 1
  • Kevin P. O’Connell
    • 1
  • Robert P. Schwartz
    • 2
  • Jeremy Wright
    • 3
  1. 1.Department of Pharmacology and Experimental TherapeuticsUniversity of Maryland School of MedicineBaltimore
  2. 2.Department of PsychiatryUniversity of Maryland School of MedicineBaltimore
  3. 3.Department of Pharmaceutical SciencesUniversity of Maryland School of PharmacyBaltimore

Personalised recommendations