An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus

Abstract

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Novel Foot-and-Mouth Disease Vaccine Platform: Formulations for Safe and DIVA-Compatible FMD Vaccines With Improved Potency

“…Serum samples at indicated time points were tested for the presence of antibodies against FMDV non-structural proteins (NSPs) using three commercially available competitive 3ABC Enzyme-Linked Imunosorbent Assay (cELISA) kits following the corresponding manufacturer's protocol. The three test kits were the PrioCHECK FMDV NS Antibody ELISA (Thermo Fisher Scientific) ( 47 ), VMRD FMDV Antibody Detection Kit ( 48 ), and the SERELISA FMDV NSP Antibody Competition ELISA (Zoetis Inc.). Values are cited as means ± Standard Deviations.…”

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus

“…Serum samples from vaccinated cattle were evaluated 0, 14, and 21 days post-challenge for the presence of non-structural protein antibodies by commercial ELISA. None of the cattle were seropositive on the day of challenge (doc), as presented in Table 2 , confirming the DIVA compatibility of this platform using licensed diagnostic tests [ 27 , 28 ]. After challenge, only a single calf, D21-06, became seropositive, as Table 2 shows.…”

Development of an ELISA to distinguish between foot-and-mouth disease virus infected and vaccinated animals utilising the viral non-structural protein 3ABC

“…Moreover, relative sensitivity may be affected by different geographical location. It has previously been reported that the commercial ID Screen FMD NSP competition kit gave lower sensitivity (76.9 %) but 100 % specificity upon comparison with sera identified after virus neutralization test (VNT) and 3B ELISA [59]. Analytical specificity is also an important parameter for newly developed assays, which is determined by using sera from animals infected with clinically similar diseases.…”

“…The detection of false positives was increased for both the in-house ELISA and the ID Screen FMD NSP competition kit in animals that had been vaccinated when compared with naïve animals. These false positive results may be associated with insufficient specificity of the assay or to the presence of 3ABC during some vaccine preparations, which should have been depleted during the manufacturing process [10,59]. It has previously been reported that animals receiving repeated local vaccination may elicit immune responses to 3ABC, which probably contributes to this observation [60].…”