Detection of Antibodies to EAV by Microsphere Immunoassay

Abstract Research Plan In Scientific Terms: Scientific Importance: The serologic diagnosis of equine viral arteritis (EVA) is currently based on a virus microneutralization (VN) assay as described in the OlE Manual. Several enzyme-linked immunosorbent assays (ELISAs) recently have been described that detect antibodies to equine arteritis virus (EAV), however none has yet gained widespread acceptance and the VN assay remains the gold standard for detection of serum antibodies to EAV. The VN assay is time consuming (3-4 days), labor intensive and expensive. Furthermore, it has recently been shown that some commercial inactivated equine herpesvirus 1 and 4 vaccines induce serum cytotoxicity that can interfere with accurate interpretation of the virus neutralization test. Therefore, our principal objective is to develop a rapid, sensitive and reliable immunological assay to detect antibodies to equine arteritis virus (EAV) in horses using a novel solution phase microsphere assay based on a flow cytometric platform. We hypothesize that the microsphere immunoassay (MIA) can be used to detect antibodies to EAV in equine sera and the specificity and sensitivity of this assay would be equivalent to that of the standard VN assay. The most well established microsphere assay system is the Luminex xMap system (Luminex Corp., Austin, TX) and this system uses a proprietary precision process to internally label polystyrene microspheres (5.6 microns in diameter microbeads) with two spectrally distinct fluorochromes. Using precise ratios of these two fluorochromes, a spectral array is created encompassing 100 different microspheres sets with unique spectral signature. Therefore, each microsphere set can be distinguished based on its spectral signature and each microsphere set can process a different reactant specific for each of 100 different analytes (e.g. antigens). Hence, this technology allows multiplexing of up to 100 unique assays within a single sample, both rapidly and precisely. In this proposed study, we will covalently coat the microspheres with recombinant envelope (GP5 and M) and nucleocapsid (N) proteins of EAV (full-length or their truncated antigenic regions) generated in our laboratory. The test equine serum will then be added to the reaction tube containing the mixture of microspheres coated with the these recombinant EA V proteins. If EAV protein specific IgG antibodies are present in the test equine serum, they will bind to the microspheres conjugated with individual proteins. Subsequently, polyvalent goat anti-equine IgG conjugated with phycoerythrin (PE) will be added to provide a signal for detection. Because the reporter signal (pE-label) is also a color, there are two sources of signal: the color-code inside the microsphere and the PE reporter on the surface of the microsphere. The reaction mixture will be then injected into the Luminex flow cytometer (Luminex 100), where two lasers illuminate the colors inside the bead (i.e. detection of red to orange ratio to identify specific bead sets at 635 nm) and on the surface of each bead (PE label at 523 nm). Advanced optics in the Luminex instrument will capture the color signals, and digital signal processing will translate the signals into real-time, quantitative data for each reaction. We will use known positive and negative equine sera to EAV to develop the assay, and it will be further validated by screening of a large number of equine sera (up to 2000 samples) collected from experimentally and naturally EAV infected horses. The data from this assay will be compared to results obtained with the standard VN assay for its specificity and sensitivity. The MIA offers many distinct advantages over traditional ELISA and VN assays. These include accuracy, reproducibility, flexibility, versatility, and high throughput screening and reduced reagent consumption and sample usage. Importance to the Equine Industry: EVA is an economically important disease of horses that occurs in many parts of the world. The EA V carrier stallion is the essential natural reservoir of the virus and has a pivotal role in the transmission and maintenance of EA V infection in horse populations; thus outbreaks of EVA can be prevented by the identification of persistently infected stallions. Many countries require certification of horses as free of anti-EAV antibodies for international movement. For example the OlE EVA Reference Laboratory at the Gluck Equine Research Center annually processes approximately 800 equine serum samples from around the world. An additional 11 ,000 (approximately) serum samples are submitted to the Kentucky Livestock Disease Diagnostic Center for testing for antiEAV antibodies in respect of the thoroughbred yearlings and breeding stock sales each year. Therefore, development of rapid, sensitive and specific microsphere immunoassays that can detect EAV-specific antibodies in horse sera will dramatically enhance current immunodiagnostic capabilities. Furthermore, the MIA has the important advantage over other conventional serologic assays in that it can be modified and used for the detection of antibodies to other equine viral infections in a multiplex assay thus, we will utilize the same approach to detect antibodies to other equine viruses after developing the EAV-specific assay. Abstract of Research Plan in Lay Language: Scientific Importance: Clinically, equine viral arteritis (EVA) resembles a number of other infectious and non-infectious diseases of horses and, therefore, a presumptive diagnosis based solely on the clinical signs of EVA must be confirmed by laboratory diagnosis. Equine arteritis virus (EAV) infection is frequently confirmed serologically by demonstration of virus specific antibodies in paired serum samples (seroconversion) or a significant (four-fold or greater) rise in antibody titer to the virus. A number of serological procedures are employed for antibody detection; the virus neutralization (VN) test has been used successfully for many years for the diagnosis of acute EAV infection, for import-export testing, and in seroprevalence studies. Several enzyme-linked immunosorbent assays (ELISAs) recently have been described that detect antibodies to EAV, however none has yet gained widespread acceptance and the VN test remains the gold standard for detection of serum antibodies to EAV. The VN test is time consuming (3-4 days), labor intensive and expensive. Furthermore, it has recently been shown that the use of some inactivated equine herpesvirus 1 and 4 vaccines induce serum cytotoxicity that can interfere with accurate interpretation of the VN test. Recent development of a novel solution phase microsphere assay system has revolutionized serological diagnostic testing of many infectious diseases of humans and animals, and there is an enormous opportunity to do the same for the horse. Therefore, our main objective is to develop a rapid, sensitive and reliable immunological assay to detect antibodies to EAV in horses using this novel solution phase microsphere (5.6 microns in diameter microbeads) assay based on a flow cytometrlc platform. We hypothesize that microsphere immunoassay (MIA) can be used to detect antibodies to EAV in equine sera and that the specificity and sensitivity of this assay would be equivalent to that of the standard VN assay. Briefly, we will coat color (fluorochromes) coded microbeads with three recombinant EAV proteins (GP5, M and N) developed in our laboratory. The test equine serum will then be added to the reaction tube containing the mixture of beads coated with the three recombinant proteins. If EAV protein specific antibodies are present in the test equine serum, they will bind to the beads conjugated with individual viral proteins. Subsequently, the bound antibodies will be detected using a special instrument (Luminex flow cytometer, Luminex Corp., Austin, TX). Advanced optics in the Luminex instrument will capture the color signals, and digital signal processing will translate the signals into real-time, quantitative data for each reaction. We will use known positive and negative equine sera to EAV to develop the assay, and it will be further validated by screening of a large number of equine sera (up to 2000 samples) collected from experimentally, and naturally EAV infected horses. The data from this assay will be compared with the results from standard VN test for specificity and sensitivity. The proposed microsphere immunoassay (MIA) offers many distinct advantages over traditional ELISA and VN assays. These include accuracy, reproducibility, flexibility, versatility, and high throughput screening and reduced reagent consumption and sample usage. Importance to the Equine Industry: The EA V carrier stallion is the essential natural reservoir of the virus and has a pivotal role in the transmission and maintenance of EA V infection in horse populations; thus outbreaks of EVA can be prevented by the identification of persistently infected stallions. The EA V carrier status of stallions is first evaluated by serological assay to detect the presence or absence of antibodies to EAV. For example, the OlE reference laboratory at the Gluck Equine Center annually processes approximately 800 serum samples from around the world, and approximately 11 ,000 addnional samples are submitted to the Kentucky Uvestock Disease Diagnostic Center for testing for anti-EAV antibodies in respect of annual yearling and breeding stock sales. Therefore, development of a rapid, sensitive and specific MIA that can detect antibodies to EAV will dramatically enhance current immunodiagnostic capabilities. Furthermore, the MIA has the important advantage over other conventional serologic assays in that it can be modified and used for the detection of antibodies to other equine viral infections in a multiplex assay.