Mohamed Ibrahim’s research while affiliated with The University of Texas at Dallas and other places

Foot-and-mouth disease virus (FMDV) is one of the most devastating animal viruses that affect livestock worldwide. The 1B capsid of FMDV has been widely used to detect and confirm the infection. In the present study, the sequence coding for 1B subunit of FMDV capsid was expressed in insect cells using the baculovirus expression system under the polyhedrin (polh) promoter. The expression of 1B capsid protein was validated in the culture filtrate of insect cells using SDS-PAGE and western blotting. The culture filtrate containing recombinant 1B capsid (r1B) was used as a coated antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The antigenicity and specificity of r1B against SAT 2 serotype-specific antibodies were assessed. Our results revealed that a protein concentration as low as 25 ng could detect SAT 2-specific antibodies in ELISA. The results highlight the application of insect cells developed r1B protein in the detection of FMDV. Further studies are required to determine the ability of r1B to detect other FMDV serotypes.

Foot-and-mouth disease virus (FMDV) is one of the most devastating viral pathogens of cloven-hoofed animals. The detection of antibodies (Ab) against FMDV structural proteins (SP) using virus neutralization test (VNT) and liquid-phase blocking ELISA (LPBE) is the standard procedure in use for monitoring seroconversion in animals post vaccination, the prevalence of infection-surveillance, proving clinical cases and seronegative status of FMDV-free/naïve-animals prior transportation. However, due to variations within SP of FMDV serotypes, each serotype-specific Ab should be detected separately which is laborious and time-consuming. Accordingly, it is crucial to develop a sensitive, rapid, and accurate test capable of detecting FMDV-specific Ab, regardless its serotype. This study describes the heterologous expression of VP2 protein in E. coli, and its evaluation as a capture antigen in a simple indirect ELISA for serotype-independent detection of anti-FMDV Ab. Sequence analysis revealed that the VP2-coding sequence is considerably conserved among FMDV serotypes. The recombinant VP2 (rVP2), a 22 kDa polypeptide, was purified to near homogeneity by affinity chromatography under native conditions. Immunoreactivity of the rVP2 was confirmed by using a panel of positive sera including sera from animals vaccinated with the local trivalent vaccine and guinea pig FMDV antiserum, which is routinely used as tracing/detecting Ab in LPBE testing. The results obtained from the VP2-based ELISA were comparable to those determined by VNT and LPBE standard diagnostic assays. Specificity and sensitivity of rVP2 in capturing anti-FMDV Ab were 98.3% and 100%, respectively. The developed VP2-ELISA is proved reliable and time-efficient assay for detection of FMDV seropositive animals, regardless the FMDV serotype that can be implemented in a combination with VNT and/or LPBE for rapid diagnosis of an ongoing FMDV infection.

Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic diversity among its serotypes, requiring several anti-FMDV antibodies for its laboratory diagnosis, which complicated the used techniques. To conquer this cumbersome, we developed a new panel of different single-chain fragment variable (scFv) for serotype-independent detection of FMDV. The recombinant VP2 capsid protein, as a relatively conserved protein among FMDV serotypes, was expressed in E. Coli, and injected in mice. Spleen's RNA was extracted for isolating the coding sequences of IgG variable domains that were assembled into repertoires of scFv. Phage library displaying scFv was constructed with ∼1.9 × 108plaque forming units. Characterization of the library showed eight of unique scFvs, which were expressed as bacterial periplasmic proteins with apparent molecular weight of ∼27 kDa. Our data revealed the broad-spectrum binding affinity of the eight scFvs as both coating and tracing antibodies to FMDV serotypes A, O, and SAT 2.