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Brucella FPA

BRUCELLA FLUORESCENT POLARIZATION ASSAY

The Serology Section began offering Brucella Fluorescent Polarization Assay (FPA) testing on Friday September 9, 2011.  This test was introduced largely due to new regulations released by the Canadian Food Inspection Agency stating swine exported from the United States to Canada on or after June 9, 2011 must test negative to Brucellosis by the FPA prior to arrival in Canada.

The test is run in a 96-well plate format allowing for high daily sample throughput.  The Brucella FPA kit has been approved by several different organizations for the testing of animals. The United States Department of Agriculture (USDA) has approved its use for the testing of cattle, bison and swine sera and has listed it as an official test for both screening and confirmation. 

The data from the various studies clearly show that the FP test performs as well as or better than the conventional serological tests. In addition, it is faster to perform, and the automated plate reading process removes much of the potential for human error with subjective interpretation of standard agglutination tests.

The test principle is based on the observation that fluorescent molecules, when excited by polarized light, will emit polarized light. In solution, the polarization of the emitted light is inversely proportional to the molecule’s rotational speed, which is influenced by solution viscosity, absolute temperature, molecular volume and the gas constant. If one keeps viscosity and temperature constant, then the key variable for rotational speed differences is molecular volume or molecular weight.

Fluorescence polarization (FP) measurements are made through two different polarizing filters that are parallel and perpendicular to the plane of the polarized excitation source. Polarization values for any fluorophore complex are inversely related to the speed of molecular rotation of that complex.

 Because the speed of rotation is also inversely related to the size of the molecule, polarization values will be high with large molecule complexes, and low with small molecules. The test uses an O-polysaccharide (OPS) extracted from Brucella abortus a bacterium which is labeled with fluorescein.  A fluorescence polarization instrument is used to measure the polarization state of the light emitted by the OPS conjugate. The fluorescence intensity of the emitted light is monitored through both parallel and perpendicular filters. The degree of transfer of emission intensity from parallel to perpendicular is proportional to the rotational speed of the fluorescent molecule. If a molecule is very large (antibody bound to conjugate), there is very little movement during excitation, and thus, little transfer from the parallel signal to the perpendicular signal. However, if the fluorescent molecule is small (unbound OPS conjugate), rotational speed is faster, resulting in a greater degree of transfer from the parallel to perpendicular signal.

Please contact the VDL at 515-294-1950 with any questions you may have about this new Brucellosis testing option.

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