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Foot-and-mouth disease (FMD) is a highly contagious and the most important animal disease caused by foot-and-mouth disease virus (FMDV). FMDV is an RNA virus belonging to the Aphthovirus genus within the family Picornaviridae and has seven distinct serotypes (O, A, C, SAT1-3 and Asia 1) and numerous subtypes[10]. Once outbreak of the disease takes hold it can severely impact livestock industry and cause huge economic losses in affected countries or regions. Therefore, vaccination of susceptible animals is one of the major strategies and is extensively used to control and prevent occurrence of FMD in developing countries where the disease most frequently occurs. However, this has raised a new problem of how to distinguish between infected and vaccinated animals in herds after immunization. The identification of infected animals is important to facilitate monitoring animal health in a herd, and to help the control and eradication of the disease in countries where FMD is endemic and controlled by preventive vaccination. Vaccinated animals principally produce antibodies against structural proteins (SPs) as the vaccines are based on partially purified killed virus, while a true FMDV infection induces antibodies to both the structural proteins (SPs) and non-structural proteins (NSPs)[8, 11]. Thus, a serological survey for antibodies to NSPs of FMDV can be used to differentiate infected from vaccinated animals in a herd[1, 11]. Based on this premise, several ELISA tests based on regions of the NSP 3ABC have been developed to differentiate infected and vaccinated animals in a herd[2, 3, 9].
There are nine FMDV non-structural proteins which are primarily involved in replication and assembly functions. In this work we investigated the ability of monoclonal antibodies (mAbs) to the non-structural protein 3AB to distinguish natural infection and immune response to vaccination. In this study, an E.coli system was used to express the 3AB epitope and specific mAbs targeting the 3AB protein were generated. The mAbs showed high specificity and sensitivity, and have potential use in future laboratory diagnostics.
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The 3AB was purified by one-step affinity purification using glutathione Sepharose 4B as described elsewhere[4, 10, 12]. The presence of recombinant protein in the eluted fractions was confirmed by SDS-PAGE. The recombinant 3AB protein weight was 33 kDa, which accounted for 30 % of total protein in E. coli lysates (Fig. 1).
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In this study, two mAbs (C6 and E7) were obtained from the weights of the heavy chain and light chain were about 45.0 mice abdominal cavity. SDS-PAGE showed that the molecular and 25.0 kDa (Fig. 2), which was consistent with the predicted molecular weight, and reacted to the 3AB protein specifically with Western blot (Fig. 2).
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In this test, FMDV O, A, Asia1 and C Type and swine vesicular disease (SVD) were used to detect the specificity of prepared mAbs. The results indicated that no cross reaction was found with SVDv. The mAbs reaction abilities with FMDV O, Asia1, A and C Type antigens were shown in Table 1.
Table 1. Specific test results of selected mAbs
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In the isotype test, C6 and E7 were associated with IgG2b. As shown in Table 2, the ascites titers of mAbs were between 1-2×106. In the stability test, the titers of prepared mAbs were consistently maintained when passaged to thirty generations (as shown in Table 2). All of these results showed that the developed mAbs possessed good specificity and high titers.
Table 2. Isotype identification and titer