UAC Pathology Services tests for numerous infectious diseases which may be detrimental to the animal research studies performed at the University of Arizona. It is very important to detect diseases early and accurately to prevent disease transmission and to expedite appropriate disease management procedures. We utilize the Multiplexed Fluorometric Immunoassay (MFI) for serologic detection of a comprehensive panel of viral and bacterial agents with a very small amount of sera (20 microliters).
MFI technology is a solid-phase immunoassay based upon flow cytometry principles. It is also known as xMAP technology, originally developed by the Luminex Corporation. Each purified antigen is covalently linked or "labeled" onto one of 100 different types of polystyrene beads, which vary slightly in the intensity of their internal dye. If IgG antibody to a particular antigen is present, it will bind to the antigen on a specific bead and will then be detected by subsequent binding of species-specific antibody conjugated to a fluorochrome, R-phycoerythrin. The reader channels single beads through a dual laser detector which simultaneously determines both the bead type by the internal dye combination and the fluorescent intensity associated with each individual bead. The fluorescent intensity associated with 50 individual beads of each type is used to determine the median fluorescent intensity value.
The antigens used for the immunoassay are produced in our laboratory. Each viral or bacterial organism is grown to high titers in culture and taken through various purification procedures in order to remove any cellular or contaminating proteins. Purification protocols are based upon the physical properties of each antigen. For example, the purification protocol for viruses takes into account the density of the virus, whether it is enveloped or non-enveloped, and if it is secreted or membrane associated. After purification, the antigens are inactivated by chemical and/or mechanical means to ensure they are non-infectious before use. Each preparation produces enough purified antigen to meet our demand for multiple years.
For a few antigens that grow poorly in culture, we produce recombinant protein using the Invitrogen Bac-to-Bac Baculovirus Expression System. The advantage of this approach is that milligram quantities of viral protein can be produced with no contaminating mammalian proteins. In some cases viral capsid proteins also self-assemble into virus-like particles (VLPs), thereby providing conformational epitopes for improved immunogenicity. With the expression system, the DNA sequence of the protein to be produced is cloned into a bacterial plasmid. This plasmid is used to transpose the DNA sequence into a bacmid that can transfect insect cells. The insect cells produce recombinant baculovirus particles which are used to infect insect cells to make large amounts of recombinant protein. Protein is then purified similarly to non-enveloped viral antigens and capsid formation is verified by electron microscopy. VLP's are non-infectious so inactivation steps are unnecessary. Once the purified antigens are produced, they are quantitated by a bicinchoninic acid protein assay and labeled onto beads in different buffers and protein concentrations for signal (noise optimization). After optimization, every antigen preparation goes through a validation process using known positive and negative serum samples. Baseline, borderline, and positive ranges are determined for each antigen on the basis of statistical analysis.
This lab uses the Qiagen LiquiChip system to perform MFI. The antigen-bound beads are processed in a similar way to the measurement of fluorescently labeled cells in a fluorescence-activated cell sorter (FACS). Several thousand beads are read in 30 seconds. A computer algorithm processes the signals from each bead and calculates the signal average and standard deviation for each bead set. Sera that generate positive, borderline, and unexpected results are retested by an extramural reference laboratory to confirm results. Multiple controls are employed in every assay to ensure accuracy and reliability.
The MFI requires a small sample volume and is highly sensitive, specific, and fast. In combination with intramural antigen production, this technology is helping our laboratory efficiently meet the increasing demand for rodent health monitoring as the rodent populations within our facilities continue to expand.
View the Pathology Services Price List for information on available serology testing.