2014. of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected contamination in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections. KEYWORDS: 2C, 3ABC, chemiluminescence immunoassay, Tmprss11d diagnosis, foot-and-mouth disease virus INTRODUCTION Foot-and-mouth disease (FMD) is usually a severe infectious illness that afflicts cloven-hoofed animals. The disease is usually caused by foot-and-mouth disease virus (FMDV), which belongs to the genus of the family (1). FMDV contains a positive-sense, single-stranded RNA genome that is translated into a polyprotein and is then cleaved by proteases into 4 structural proteins (SPs; VP1, VP2, VP3, and VP4) that form the viral capsid and 10 nonstructural proteins (NSPs; L, 2A, 2B, 2C, 3A, 3B, 3C, 3D, 3AB, and 3ABC) that play key roles in viral replication and host cell interactions (2,C4). Several diagnostic methods, such as virus isolation, reverse transcriptase PCR, a mucosal IgA enzyme-linked immunosorbent assay (ELISA), and NSP-based ELISAs, have been used to differentiate FMDV-infected animals from vaccinated animals (5). Among these methods, NSP-based ELISAs are the most widely used technique because they can detect seven different serotypes and discriminate infected from vaccinated animals (2, 6). Antibodies against NSPs 3ABC and 2C are the most important indicators of an FMDV contamination. The 2C protein remains membrane bound and is not present in purified vaccines (7); furthermore, antibodies against NSP 2C are induced early after FMDV contamination (8). Therefore, the ability to detect antibodies against 2C is helpful for diagnosing early infections (9). The presence of the 3ABC protein is the most reliable single indicator of infection because it produces abundant antibodies and persists in the body for a longer time than the other NSPs (10, 11). The presence of antibodies against the 2C protein and, to a lesser extent, the 3ABC protein has been used to distinguish potential carrier animals in the convalescent phase from vaccinated animals (7, 12). Diagnosis would be more accurate if researchers could simultaneously detect antibodies against both proteins. The chemiluminescence immunoassay (CLIA) was developed in the late 1970s (13). In recent years, CLIA has been shown to perform extremely well in diagnosing human diseases, such as human immunodeficiency virus (HIV) contamination (14, 15), cancer (based on the detection of tumor markers) (16), and hepatitis B (based on AT7519 HCl the detection of hepatitis B virus surface antigen [HBsAg]) (17, 18), due to its high AT7519 HCl sensitivity, high signal-to-noise ratio, and wide linear range. To date, no reports of studies that used CLIA to detect FMDV infections have been published. We developed a CLIA platform for the simultaneous detection of antibodies against NSPs 2C and 3ABC to evaluate the performance of CLIAs in distinguishing FMDV-infected pigs from vaccinated pigs. The results showed that this CLIAs have a higher sensitivity and a shorter detection time than currently available ELISAs and may serve as a basis for the development of accurate and rapid penside FMD diagnostics. RESULTS Expression and characterization of the rmu3ABC and r2C proteins. The His-tagged recombinant mutated 3ABC (rmu3ABC) protein was expressed in insoluble inclusion bodies and had an apparent molecular mass of 55 kDa on SDS-polyacrylamide gels (Fig. 1A). The His-tagged recombinant r2C (r2C) protein was also expressed in inclusion bodies and had an apparent molecular mass of 55 AT7519 HCl kDa (Fig. 1B). After washing and nickel affinity chromatography purification, we obtained highly pure rmu3ABC and r2C proteins (Fig. 1C and ?andD).D). According to the results of the Western blot analysis, the FMDV-infected positive serum sample produced a strong signal for both purified proteins (Fig. 1E). In addition, the purified 3ABC and 2C proteins showed a good power to discriminate serum samples that were positive and weakly positive in an indirect ELISA, whereas the unfavorable serum sample displayed almost no reactivity. On the basis of these results, the rmu3ABC and r2C proteins are suitable for use for detecting antibodies against NSPs from FMDV. Open in a separate window FIG 1 (A) SDS-PAGE profile of.