Advertisement

Archives of Pharmacal Research

, Volume 20, Issue 1, pp 46–52 | Cite as

The optimization of ELISA for methamphetamine determination: the effect of immunogen, tracer and antibody purification method on the sensitivity

  • Jeongeun Choi
  • Myung Ja Choi
  • Choonmi Kim
  • Young Shik Cho
  • Jaeho Chin
  • Young-Ah Jo
Research Articles

Abstract

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

Key words

Methamphetamine ELISA Antibody purification method Coating tracer Optimization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References Cited

  1. Beckett, A. H. and Rowland, M. Urinary excretion kinetics of methylamphetamine in man.J. Pharm. Pharmac. 17, 109s-114s (1965).Google Scholar
  2. Choi, M. J., Choi, J., Park, J. and Eremin, S. A. Localization of the epitope in methamphetamine and its antibody use for the detection of methamphetamine and benzphetamine by polarization fluoroimmunoassay.,J. Immunoassay, 16, 263–278 (1995).PubMedCrossRefGoogle Scholar
  3. Choi, M. J., Gorovits, B., Choi, J., Song, E. Y., Nam, K. S. and Park, J. A visual membrane immunoassay for the detection of methamphetamine using an enzyme-labeled tracer derived from methamphetamine and amphetamine.Biol. Pharm. Bull., 17, 875–880 (1994).PubMedGoogle Scholar
  4. Colbert, D.L., Eremin, S.A. and Landon, J. The effect of fluorescein labels on the affinity of antisera to small haptens.J. Immunological Methods, 140, 227–233 (1991).CrossRefGoogle Scholar
  5. Eremin, S.A., Gallacher, G., Lotey, H., Smith, D.S. and Landon, J. Single-reagent polarization fluoroimmunoassay of methamphetamine in urine.Clin. Chem. 33, 1903–1906 (1987).PubMedGoogle Scholar
  6. Eremin, S.A., Schiavetta, D.E., Lotey, H., Smith, D.S. and Landon, J. Design and development of a single-reagent polarization fluoroimmunoassay for methamphetamine.Ther. Drug Monit. 10, 327–332 (1988).PubMedCrossRefGoogle Scholar
  7. Goodrow, M.H., Harrison, R.O. and Hammock, B.D. Hapten synthesis, antibody development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.J. Agric Food Chem. 38, 990–996 (1990).CrossRefGoogle Scholar
  8. Lebish, P., Finkle, B.S. and Brackett, J.W. Detection of amphetamine, methamphetamine and related amines in blood and urine by gas chromatography with hydrogen flame ionisation detector.Clin. Chem., 16, 195–200 (1970).PubMedGoogle Scholar
  9. Mason, P.A., Bal, T.S. and Moffat, A.C. Development and evaluation of a radioimmunoassay for the detection of amphetamine and related compounds in biological fluids.Analyst., 108, 603–607 (1983).PubMedCrossRefGoogle Scholar
  10. Nam, K.S., Kim, J.W., Choi, M.J., Han, M.Y., Choe, I.S. and Chung, T.W. Production and characterization of monoclonal antibody that simultaneously recognizes methamphetamine and its major metabolite.Biol. Pharm. Bull., 16, 490–492 (1993).PubMedGoogle Scholar
  11. Tamaki, Y., Fukuda, M., Kishida, T. and Takahashi, N. Solid-phase micro ELISA for methamphetamine.,Jpn. J. Legal Med., 37, 417–420 (1983).Google Scholar
  12. Terada, M., Yamamoto, T., Yoshida, T., Kuroiwa, Y. and Yoshimura, S. Rapid and highly sensitive method for determination of methamphetamine and amphetamine in urine by electron-capture gas chromatography.J. Chromat., 237, 285–292 (1982).CrossRefGoogle Scholar

Copyright information

© The Pharmaceutical Society of Korea 1997

Authors and Affiliations

  • Jeongeun Choi
    • 1
  • Myung Ja Choi
    • 1
  • Choonmi Kim
    • 2
  • Young Shik Cho
    • 3
  • Jaeho Chin
    • 1
  • Young-Ah Jo
    • 1
  1. 1.Doping Control CenterKorea Institute of Science and TechnologySeoulKorea
  2. 2.College of PharmacyEwha Womans UniversitySeoulKorea
  3. 3.Department of Diagnostic ProductsKorea Green Cross CorporationYongin, Kyunggi-doKorea

Personalised recommendations