Development and Evaluation of a Rapid Antigen Detection and Serotyping Lateral Flow Antigen Detection System for Foot-and-Mouth Disease Virus

We developed a lateral flow strip using monoclonal antibodies (MAbs) which allows for rapid antigen detection and serotyping of foot-and-mouth disease virus (FMDV). This FMDV serotyping strip was able to detect all 7 serotypes and distinguish serotypes O, A, C and Asia1. Its sensitivities ranged from 103 to 104 of a 50% tissue culture infectious dose of each FMDV stain; this is equal to those of the commercial product Svanodip (Boehringer Ingelheim Svanova, Uppsala, Sweden), which can detect all seven serotypes of FMDV, but does not distinguish them. Our evaluation of the FMDV serotyping strip using a total of 118 clinical samples (vesicular fluids, vesicular epithelial emulsions and oral and/or nasal swabs) showed highly sensitive antigen detection and accuracy in serotyping in accordance with ELISA or RT-PCR. To the best of our knowledge, this is the first report on any FMDV serotyping strip that provides both rapid antigen detection and serotyping of FMDV at the same time on one strip without extra devices. This method will be useful in both FMD-free countries and FMD-infected countries, especially where laboratory diagnosis cannot be carried out.


Introduction
Foot-and-mouth disease (FMD) is one of the most highly contagious viral diseases, and causes devastating economic damage in the countries affected by it. FMD is caused by foot-andmouth disease virus (FMDV), which belongs to the Aphthovirus genus of the family Picornaviridae, and consists of 7 immunologically distinct serotypes: O, A, C, Asia1, and South African Territories (SAT) 1, 2 and 3. Within a single serotype, there may be many different topotypes [1]; serotype A in particular displays a large number of topotypes [2]. In Asia, type O, A and Asia1 viruses have prevalently existed and the disease is endemic or sporadic in main countries [3]. Additionally, there have been some cases of FMD whose serotype is unknown in South East Asia [3]. Although outbreaks of the SAT serotypes have been largely limited to the African continent, it has been reported that the SAT2 serotype invaded Middle Eastern countries in 2000 and 2012 [4].
In an FMD outbreak, antigen serotyping should be conducted immediately in order to implement the appropriate plan for disease control (e.g., preparing vaccine and immunological diagnosis kits for the serotype in question). To serotype FMDV, indirect sandwich enzymelinked immunosorbent assay (ELISA), which is mentioned in the World Organization for Animal Health (OIE) Terrestrial Manual 2012 [5], may be used, however it lacks sufficient sensitivity and specificity [6][7][8][9]. Although some studies have reported serotyping by reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR in some regional endemic FMD strains, the mutability of viral RNA makes it difficult to apply either of these as a universal method [10]. Moreover, the recent activation of world trade and human transportation might change the epidemic styles of trans-boundary emerging diseases. The possibility of new outbreaks of the different topotypes in places where they have never previously occurred is increasing. We have developed a monoclonal antibody (MAb)-based direct sandwich ELISA (MSD-ELISA), which demonstrates higher sensitivity than those of current indirect sandwich ELISA methods reported in previous studies [8,9]. ELISA is a useful tool for antigen detection, however this should be carried out in a laboratory with the appropriate apparatus.
In this report, we developed a lateral flow assay using MAbs (FMDV serotyping strip), which allows for rapid FMD antigen detection for all 7 serotypes and FMD serotyping for types O, A, C and Asia1, specifically in the field, and especially in countries where laboratory diagnosis cannot be carried out, in vast countries in which it would take a long time to transport clinical samples to a laboratory, and/or in countries lacking transportation facilities.

Conjugation of MAb 1H5 with colloidal gold
A mixture of 0.1 ml of purified MAb 1H5, which reacts with all 7 serotypes of FMDV (200 μg/ml) and 0.8 ml of colloidal gold (pH 8.0) (Winered Chemical Corporation, Tokyo, Japan) was stabilized for 8 min at room temperature. Next, 0.1 ml of 10% bovine serum albumin (BSA) and 0.01 ml of 5% polyethylene glycol (PEG) were added, and the mixture was centrifuged at 10,000 rpm at 20°C for 30 min. After removal of the supernatant, the colloidal gold-labeled MAb 1H5 was resuspended with 1.5 ml of phosphate buffered saline (PBS) containing 0.5% BSA and 0.05% PEG, centrifuged again under the same conditions mentioned above, and resuspended with 20 mM Tris-HCl (pH 7.5) containing 10% trehalose, 10% BSA and 5% PEG. After the density was adjusted to 2.0 of OD 520 , 50 μl of the colloidal gold labeled MAb 1H5 (G-MAb) was freeze-dried in a 2-ml tube.
The cellulose fiber membrane (Cellulose Absorbent Sample Pads; Millipore), which was cut to a width of 1 cm, dipped in 100 mM Tris-HCl (pH 8.0) with 1.6 mM ethylenediaminetetraacetic acid (EDTA) for blocking against non-specific reactions due to contamination of saliva or hemolytic fluid and dried overnight at room temperature, was used as an absorption pad, and stuck with adhesive tape on the downstream side of the nitrocellulose membrane, overlapping by 5 mm (ARcare 7815; Adhesives Research, Glen Rock, PA, USA). A cotton membrane (GE Healthcare Life Sciences, Little Chalfont, UK) 2 cm in width was used as an absorption pad and stuck with adhesive tape on the upstream side of the nitrocellulose membrane, overlapping by 5 mm. Constructed membranes were then cut at a width of 0.5 cm, and the strips were stored at room temperature under low humidity.
Monoclonal antibody-based sandwich direct ELISA MSD-ELISA was established and described in our previous reports [8,9], and the protocol is described in detail [8]. In the present study, OD values over 0.1 were deemed positive.

Indirect sandwich ELISA
Indirect sandwich (IS) ELISA [23], which is a method described in the OIE manual [5], was carried out according to the relevant instruction manuals. For the purposes of the present study, OD values over 0.1 were considered positive.

Statistics
The Pearson's chi-square test was used to analyze the statistical significance of the differences in virus detection rates between IS-ELISA and the FMDV serotyping strip. To analyze correlations between the OD values of MSD-ELISA and the mABS values of the FMDV serotyping strip, Pearson's correlation coefficient was used.

RT-PCR
For the RT-PCR for the detection of FMDV nucleic acid, the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Waltham, MA, USA) and primers for the 3D region were used [15].

Lateral flow antigen detection commercial kit
A Svanodip FMDV-Ag kit (Boehringer Ingelheim Svanova, Uppsala, Sweden) was used following the manufacturer's instructions. A total of 170 μl of samples diluted with sample buffer were added to the device and, after 10 min, mABS was measured using the Immunochromato-Reader C10066.  [14] were used for comparative studies.

Clinical samples
Field samples (vesicular epithelium and vesicular lesion's swab) were submitted from Miyazaki Prefecture, Japan, for diagnosis of the 2010 FMD in Japan.

Ethics statement
The animal experiments carried out in the present study were approved by the Ethics Committee of the National Institute of Animal Health, Japan (approvals #09-029, 14-060, and 14-080). The field samples used in this study were submitted from Miyazaki Prefecture, Japan, for diagnosis of the 2010 FMD in Japan. These vesicular epithelial tissues were collected by veterinarians in accordance with the guidelines specified in the Act on Domestic Animal Infectious Diseases Control.

The FMD serotyping strip
One hundred μL of samples diluted with PBS were added to the tube containing the freezedried G-MAb, and the FMDV serotyping strip was then dipped into the tube. After 12 min, the lines were interpreted by visual observation and/or the milli absorbance (mABS) of the lines was measured using an Immunochromato-Reader C10066 (Hamamatsu Photonics, Shizuoka, Japan). With respect to visual interpretation, mABS "from 10 to 15" and "over 15" were deemed equivalent to "weak positive" and "positive," respectively.
Antigen detection for all seven serotypes and serotyping (O, A, C and Asia1). A total of 17 viruses, 16 strains of FMDV and the SVDV J1 strain, were adjusted to almost 10 6 of 50% tissue culture infectious dose (TCID 50 )/100 μL with PBS, and those of 100 μL were applied for the FMD serotyping strips.
Detection limits of the FMD serotyping strip. Ten-hold serial dilutions were prepared with PBS or running buffer for Svanodip against the 16  In this evaluation, the positions of the serotype-specific antibodies were rearranged from O, A, Asia1, C (from upstream to downstream) to C, O, A, Asia1 to inspect non-specific reactions due to contamination of hemolytic fluid or other contamination.

Antigen detection for all seven serotypes and serotyping (O, A, C and Asia1)
The FMDV serotyping strips were able to detect all 16 strains of the 7 serotypes of FMDVs including recent epidemic strains, and were able to distinguish serotypes O, A, C and Asia1, but did not react to SVDV. However, when a high titer of SAT2 was applied, a weak non-specific line was observed on the serotype A line (Fig 1).

Sensitivities of the strip
The antigen detection limits of each serotyping line of the FMDV serotyping strip were found to be almost equal to 10 3 to 10 4 TCID 50 of each virus stain (Fig 2). The FMDV serotyping strip showed high sensitivity to the serotype O ME-SA topotype compared to Svanodip. On the other hand, the FMDV serotyping strip was not sensitive to SAT3, at a detection limit of 10 5 TCID 50 , while Svanodip were able to detect 10 4 TCID 50 .

Evaluation of the FMD serotyping strip using clinical samples
As shown in Table 1

Discussion
In the present study, we developed an FMD serotyping strip that enables rapid antigen detection for all 7 serotypes and serotyping for O, A, C and Asia1, including recent epidemic FMDVs. This FMD serotyping strip allows for rapid antigen detection and 4 types of serotyping of FMDV without extra devices.
Although antigen detection ELISA requires that samples of each serotype are added to detecting wells, this FMD serotyping strip is able to perform simultaneous detection and serotyping of FMDV on a single strip. Furthermore, our lateral flow system detects not only FMDV but also type-specific FMDV. This not only increases sensitivity but also counteracts the shortcomings of MAb, which recognizes a single epitope that depends on broad intra-type reactivity.
In Southeast Asia, there have been many cases in which the causative FMDV serotype was unknown [3]. The present method will be useful not only in FMD-free countries but also in FMD-infected countries, especially where laboratory diagnosis is not possible. Thus, the FMD serotyping strip will prevent delay of serotyping reports to the OIE and we will be able to share more precise information about FMD epidemics.
The advantage of using MAbs is that it is possible to select highly efficient MAbs for high specificity and sensitivity. However, a MAb recognizes a single epitope, and thus the broad intra-and intertype reactivity of the MAbs should be evaluated.  Rapid FMDV Antigen Detection and Serotyping by Lateral-Flow Assay detection line is able to be warranty for SAT1, 2 and 3, and in view of mismatch of each serotype specific MAbs. In our previous study, we used MAb 16C6 as a detecting antibody for MSD-ELISA for type A FMDV, however it did not react to A/IRN/01/2011, although it react to A/TAI/10/2011. The strain A/IRN/01/2011 belongs to the A-Iran-05 lineage, which appeared in 2005 as a new lineage of FMDV serotype A [24,25]. That strains spread throughout Iran and moved westward into Saudi Arabia and Turkey, and were also reported in 2006 in Pakistan, and in 2007 in Jordan [25]. To solve this problem, we produced MAb 2A1 by A/IRN/01/ 2011 as the antigen, and this MAb was able to react to A/IRN/01/2011 as well as to A22 IRQ 24/64. Therefore, in the present study, we used both MAb 16C6 and 2A1 as cocktails of the antigen detection MAb on the membrane.
We used oral swabs for samples for MSD-ELISA in our previous study [9], however, we found that some oral and/or nasal vesicular lesion swabs from 2010 inhibited chromatography flow in the present study (data not shown). Saliva is expected to be a useful sample for detecting antigens, especially from preclinical infected animals [26] and subclinical infected animals such as those that are vaccinated-infected or have low pathogenic FMDV. For instance, the cattle in the 2000 FMD outbreak in Japan did not show any distinct vesicles on the feet, or in or around the oral and nasal cavities. [11][12][13]. In addition, even in typical clinical cases such as those in 2010, almost all samples were oral and/or nasal vesicular lesion swabs from the outbreak area because the vesicles readily ruptured and it was difficult to collect epithelial tissues.
On the other hand, using vesicular fluids and/or epithelial emulsions as a sample has the advantage that the sample would include a high titer of virus, and antigens can be detected a long time after the appearance of lesions. The present FMDV serotyping strip was able to detect antigens from 4 to 13 days (the end of the experiment) post-inoculation when the lesions were almost healed (Table 1; sampling date not shown).
In this study, the correlation coefficient between mABS of the FMDV serotyping strip and the OD of MSD-ELISA was significantly high (R = 0.67) and this strip was found to be more sensitive than IS-ELISA (p<0.01). As described in our previous study [9], the difference of sensitivity was probably due to the ability of MAb that we used in this system, much more than those of rabbit and guinea pig anti-sera in IS-ELISA. In addition, concerning specificity, the FMD serotyping strip showed negative results against a total of 21 of RT-PCR negative samples (14 of oral swabs or saliva and 7 of epithelial emulsions) (data not shown).
Further study is ongoing, and we are trying to establish serotype-specific MAbs against SAT1, 2 and 3, and apply them in the lateral flow assay for the detection and serotyping of all 7 serotypes of FMDVs.