This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, M. Articles by Stilwell, K. Search for Related Content PubMed PubMed Citation Articles by Lin, M. Articles by Stilwell, K.

 Previous Article  |  Next Article 

Clinical and Diagnostic Laboratory Immunology, Jul 1996, 438-443, Vol 3, No. 4
Copyright © 1996 by the American Society for Microbiology. All rights reserved.

Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization

M Lin, EA Sugden, ME Jolley and K Stilwell
Immunology Section, Animal Diseases Research Institute, Nepean, Ontario, Canada.

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli- polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.

This article has been cited by other articles:

• Lin, M., Trottier, E., Pasick, J., Sabara, M. (2004). Identification of Antigenic Regions of the Erns Protein for Pig Antibodies Elicited during Classical Swine Fever Virus Infection. J Biochem 136: 795-804 [Abstract] [Full Text]  
• Lin, M., Lin, F., Mallory, M., Clavijo, A. (2000). Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum. J. Virol. 74: 11619-11625 [Abstract] [Full Text]  
• Tencza, S. B., Islam, K. R., Kalia, V., Nasir, M. S., Jolley, M. E., Montelaro, R. C. (2000). Development of a Fluorescence Polarization-Based Diagnostic Assay for Equine Infectious Anemia Virus. J. Clin. Microbiol. 38: 1854-1859 [Abstract] [Full Text]