Validation of a fluorescence polarization assay (FPA) performed in microplates and comparison with other tests used for diagnosing B. melitensis infection in sheep and goats

A. Minas, A. Stournara, M. Minas, J. Stack, E. Petridou, G. Christodoulopoulos, V. Krikelis

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

Fluorescence polarization assay (FPA) is a relatively new test for the serological diagnosis of Brucella spp. infection in animals. FPA, carried out in 96-well microplate format, was validated here for diagnosing B. melitensis infection in sheep and goats. This study included sera from 1933 sheep and goats, from animals reared in naturally infected flocks (verified by culture) and showing a positive reaction to two different tests conducted in parallel. In addition, 2154 sera originating from healthy sheep and goats, reared in areas where B. melitensis had never been isolated, were assayed. The optimum cut-off value offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 15 mP over the mean value of the buffer control used in each microplate as determined by receiver operating characteristic analysis. The DSn and DSp of the FPA for small ruminants carried out in microplates at this cut-off value were calculated to be 95.9% and 97.9% with 95% confidence intervals (95% CI) of 94.9-97.7% and 97.2-98.4%, respectively. The accuracy of the FPA, as expressed by determination of the area under the curve, was 0.991. Indirect ELISA and FPA tests offered the highest DSn when compared with the Rose Bengal test, the complement fixation test, the modified Rose Bengal test and competitive ELISA. The parallel or serial combination of FPA with indirect ELISA offers the highest DSn and DSp. As temperature can affect the results of the FPA, all reagents must be at the same temperature and the standard for comparison must always be read under the same conditions as the sera under test. FPA performed in microplates is a promising assay; the DSn and accuracy are better than those of the tests currently approved for diagnosing B. melitensis in small ruminants. Because of its simplicity, speed, and accuracy, this test can improve capacity for laboratory testing and the efficacy of an eradication program based on a test-and-slaughter policy.

Original languageEnglish
Pages (from-to)94-103
Number of pages10
JournalJournal of Immunological Methods
Volume320
Issue number1-2
DOIs
Publication statusPublished - Mar 30 2007
Externally publishedYes

Keywords

  • B. melitensis
  • Brucellosis in small ruminants
  • Fluorescence polarization assay
  • Serology
  • Validation

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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