Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals. Foot-and-mouth disease is the most economically significant infection of domestic livestock. It causes production losses, particularly to the dairy and pig industries and is a major constraint in the international trade of live animals and their products. Its pathogen is foot-and-mouth disease virus (FMDV) which is a member of the genus Aphthovirus and of the family Picornavidae and includes seven serotypes. Serotype A is widely distributed and very active in the Middle East and Asia .
Monoclonal antibodies (mAbs) have been widely used in infectious diseases research, diagnosis and therapy. Compared to the tests based on polyclonal anti-sera, diagnostic tests with mAbs always have higher specificity, accuracy and efficiency . In this study, we report the generation of an anti-FMDV A Type mAbs. Two mAbs 7B11 and 8H4 were screened after immunization of mice with A Type FMDV. The specificity, stability, titers and neutralization activity of the mAbs were analyzed.
The A Type FMDV was purified by one-step affinity purification using glutathione Sepharose 4B as described in Refs [7, 9, 12]. The presence of recombinant protein in the eluted fractions was confirmed by SDS–PAGE. The recombinant A Type FMDV was 34 kDa, which accounted for 30 % of total protein in E. coli lysates (Fig. 1).
In this study, two mAbs (7B11 and 8H4) were obtained. All of them were produced from the mice abdominal cavity. SDS-PAGE showed that the molecular weights of the heavy chain and light chain were about 45.0 and 25.0 kDa (Fig. 2A), which was consistent with the predicted molecular weight, and reacted to VP1 protein specifically with Western blot (Fig. 2B).
In the neutralization assay, the result indicated that prepared mAbs could inhibit virus strain A/AV88 from infecting BHK-21 cells (Table 1). When the mAbs 7B11 dilution ratio reached 1:1024, 50% of the cells still survived; indicationg that the titers of mAbs were more than 1:1024. For mAbs 8H4 when the dilution ratio reached 1:512, 50% cells still survived; this showed that the titers of mAbs were more than 1:512.
Table 1. Detection of mAbs neutralization titers by virus neutralization test
In this test, FMDV O, 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 SVD and FMDV O, Asia1 and C Type antigens (Table 2).
Table 2. The results of the cross-reaction test
In the isotype test, 7B11 was found to be from IgG1 whereas 8H4 belonged to IgG2b. As shown in Table 3, the ascites titer of mAbs was between 1-2×105. In the stability test, the titers of prepared mAbs were invariably maintained when passaged to thirty generations (as shown in Table 3). All of these results showed that the developed mAbs possessed good specificity and high titers.
Table 3. The ascites titer and stability of the screened monoclonal antibodies