Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus.

Two novel formats of ELISA for the detection of antibodies against swine vesicular disease (SVD) virus were developed. One of the tests described is a monoclonal antibody-based competitive ELISA (MAC-ELISA). In this test, specific antibodies in serum are detected due to their ability to compete with a neutralizing monoclonal antibody (MAb). The second is an indirect trapping ELISA which employs isotype-specific MAbs to detect swine IgG or IgM specific for SVD virus. The diagnostic sensitivity and specificity of the MAC-ELISA was studied on 5671 field sera of known origin, enabling the cut-off level to be defined. Using the MAC-ELISA, 100% of sera from infected pigs were found positive, whereas only 0.45% of negative sera gave a false-positive result. A positive correlation between MAC-ELISA and virus neutralizing titres was recorded for pig sera collected sequentially after experimental infections. The results from the isotype-specific ELISA revealed the dynamics of the antibody response to SVD virus in pigs. The first antibodies were detectable as early as 3 days after experimental infection. Up to the 10th day, demonstrable antibodies were exclusively of the IgM class. IgG developed later, between 11 and 14 days postinfection and remained at a plateaux level throughout the whole investigation period. The two tests satisfy different diagnostic requirements: the MAC-ELISA is useful as a screening test, the isotype-specific ELISA has potential application for the determination of stage of infection. Both tests benefit from the use of MAbs in terms of specificity and standardization and have advantages over the virus neutralization test.

Diagnostic potential of Mab-based ELISAs for antibodies to non-structural proteins of foot-and-mouth disease virus to differentiate infection from vaccination.

This paper summarises the development of monoclonal antibody (Mab)-based immunoassays measuring antibodies to non-structural proteins of FMDV to differentiate infection from vaccination. Of the three non-structural proteins 2C, 3C and 3ABC evaluated in this study, the polypeptide 3ABC was the most immunogenic. Three ELISAs for the detection of antibodies to 3ABC were developed. Two assays rely on the competition of test sera against either a anti-3A Mab or against antisera to 3ABC raised in rabbits and guinea-pigs. The third, 3ABC Mat-ELISA, based on the direct binding of antibodies to the 3ABC trapped by a specific Mab, provided the best combination of specificity and sensitivity. The 3ABC Mat-ELISA was extensively validated for cattle, either in experimental and in field conditions, showing specificity of 99% in vaccinated and in naive cattle and the capacity to detect silent infections in FMD-vaccinated populations. The test showed similar specificity and sensitivity in experimentally vaccinated and infected sheep.

Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA.

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.