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Saturday, February 26, 2011

[AlternativeAnswers] A fluorescent bead-based multiplex assay for the simultaneous detection of antib


Vet Immunol Immunopathol. 2010 Dec 10; [Epub ahead of print]

A fluorescent bead-based multiplex assay for the simultaneous detection of
antibodies to B. burgdorferi outer surface proteins in canine serum.

Wagner B, Freer H, Rollins A, Erb HN.

Department of Population Medicine and Diagnostic Sciences, College of
Veterinary Medicine, Cornell University, Ithaca, NY, USA; Animal Health
Diagnostic Center, College of Veterinary Medicine, Cornell University,
Ithaca, NY, USA.

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs,
horses and other species. It is caused by infection with spirochetes of the
Borrelia burgdorferi sensu lato group which are transmitted to the
host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly
diagnosed by serological testing. The gold standard for the detection of
antibodies to B.
burgdorferi is a two-step procedure of an ELISA followed by confirmatory
Western blotting (WB). Here, we developed and validated a new bead-based
multiplex assay for the detection of antibodies to B. burgdorferi in canine
serum which combined the testing by ELISA and WB in a single quantitative
test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were
expressed in E. coli. The recombinant proteins were coupled to fluorescent
beads providing the matrix of the assay. Two sets of canine sera were used
for validation of the multiplex assay. First, sera from 79 dogs with known
ELISA and WB results were used to establish the conditions of the assay.
These samples were selected to provide similar numbers of pre-tested sera
ranging from negative to high positive results and included sera from
vaccinated and/or naturally infected dogs. A high correlation was observed
for detection of antibodies to B. burgdorferi in the single and multiplex
assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for
OspA, OspC and OspF, respectively. Second, a total of 188 canine serum
samples that were not tested previously were used for further multiplex
assay validation. All samples were also blindly analyzed for antibodies to
burgdorferi antigens by WB. The WB results provided a 'relative gold
for each antigen and were used to perform a receiver operating curve
The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for
Multiplex assay interpretation ranges for antibodies to all three B.
burgdorferi antigens in canine serum were established by likelihood
analysis. The diagnostic sensitivities of the individual OspA, OspC and
bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic
specificities were 90%, 89% and 86%, respectively. The new multiplex assay
provides a sensitive and fully quantitative platform for the simultaneous
evaluation of antibodies to B.
burgdorferi OspA, OspC and OspF antigens and distinguishes between
antibodies that originated from vaccination or natural exposure to B.
Copyright A(c) 2010. Published by Elsevier B.V.

PMID: 21208663 [PubMed - as supplied by publisher]

[Non-text portions of this message have been removed]

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