Cloning, Expression and Functionality Evaluation of Recombinant Monoclonal Antibody Against VP1 Capsid Protein of FMD Virus

1. Introduction
oot-and-mouth disease (FMD) is a highly contagious viral disease of mammals and causes significant economic losses in susceptible cloven-hoofed animals. The virus belongs to the genus Aphtho virus in the family Picornaviridae. Its genome is a positive single stranded RNA, 8.5 kbp length, with a single open reading frame [1].
There are seven different serotypes, A, O, C, Asia1, and Southern African Territories (SAT) 1, 2, and 3. Infection with one serotype does not confer immunity against another. FMD cannot be differentiated clinically from other vesicular diseases; therefore,its laboratory diagnosis is important [1, 2]. The intact virion included an icosahedral capsid structure containing 60 copies of structural proteins (VP1-VP4) and 7 non-structural proteins. The VP1 capsid protein has the highest copy number among all FMDV proteins and consist of 213 amino acids. The G±H loop (residues 141-160) of VP1 capsid protein is highly variable, yet the main antigenic region contains a highly conserved triplet of amino acids, Arg±Gly±Asp (RGD), across FMDV types causing the production of protective antibodies against FMDV types. Studies show that 40% of secreted antibodies against FMDV are stimulated by this loop [2-4].
In this study, a cross-reactive recombinant mAb against conserved RGD region of the G±H loop was designed as an scFv with a flexible linkers for detection of FMDV serotype O. Our goal was to assess the possibility of recombinant scFv-mAb production using an Escherichia coli expression system at a low cost and in a short time [2, 3, 5].

2. Materials and Methods

2.1 Engineering and prediction of physicochemical properties of recombinant scFv-mAb
For engineering of the mAb against the G±H loop region of FMD virus VP1 capsid protein (Arg±Gly±Asp, RGD motif), the PDB ID “1ejo” was retrieved and subjected to the required truncations to achieve the desired scFv. The advantages of this antibody included low folding complexities, a well- annotated sequence and 3D structure, and low post-translational modifications, especially glycosylations [3].
The truncation was performed at ARG 2112 (arginine amino acid) in light chain and Lys 2621 in the heavy chain. The antibody domain were fused together using a poly Gly-Ser linker (GSGGGGS). The physicochemical properties of the final engineered FMD virus mAb were analyzed using the ProtParam tool on the ExPasy web server [6]. Through this web server, various parameters were calculated, including: Molecular weight (MW [kDa]), theoretical isoelectric point (pI), estimated half-life under in vitro and in vivo conditions, stability index, aliphatic index, and grand average of hydrophobicity (GRAVY) [6].

2.2. Bacterial strains and culture media
E. coli DH5α (Novagen Co.) was used as a cloning host for the production and maintenance of the expression vector, and E. coli BL21(DE3) Rosetta strain (Novagen Co.) was used as the expression host of eukaryotic proteins containing codons rarely used in E. coli. Luria Bertani (LB) was used as bacterial culture media [7].

2.3. Plasmid preparation
The recombinant scFv-mAb antibody (50.306 kD) gene cassette was designed using bioinformatic tools and was inserted between NcoI and BamHI restriction. It was then chemically synthesized by Shine Gene Molecular Biotech Co. (Shanghai, China) into the pET28a(+) expression vector. Chloramphenicol and kanamycin antibiotics employed as selectable markers for the Rosetta strain and the pET28a(+) expression vector, respectively.

2.4. Plasmid cloning and extraction 
Competent E. coli DH5a strain cells (200 µL) were prepared using the CaCl2 method and transformed with pET28a(+) vector (5 µL) via the Novagen heat -shock transformation method [6]. Subsequently, selection for transformants was accomplished by plating on LB agar plates containing kanamycin (35 µg/mL) and incubation at 37 °C for 18-24 hours.
Plasmid DNA was extracted and purified from the transformants using the Favor Prep™ Plasmid DNA Extraction Mini Kit, based on kit instructions [8]. Briefly 3 mL of well-grown transformant culture was centrifuged at 11000×g for 1 minute, and the supernatant was discarded completely. The cell pellet was resuspended in 200 μL of FAPD1 buffer (RNase A added) by pipetting. The lysate was clarified by centrifugation at 18,000×g for 5 minutes. During centrifugation, a FAPD column placed in a collection tube. The resulting supernatant was carefully transferred to an FAPD column placed in a collection tube and centrifuged at 11000×g for 30 seconds. After discarding the flow-through, the column was washed with 400 μL of WP buffer (11000×g for 30 seconds) followed by 700 μL of wash buffer (11,000×g for 30 s). Then, the flow-through was discarded, and column was placed back into the collection tube. Next, 700 μL of wash buffer added to the FAPD column and centrifuged at 11000×g for 30 seconds. This process continued by discarding the flow-through and replacing the column back into the collection tube. The column was then centrifuged at full speed (18000×g) for an additional 3 minutes to dry the FAPD column. Then, the FAPD column was placed into a new 1.5 mL microcentrifuge tube. Following this, added 50 μL ~ 100 μL of Elution buffer or ddH2O was added to the center of the FAPD column membrane. The column was allowed to stand for 1 minute and then centrifuged at full speed (18000×g) for 1 minute to elute the plasmid DNA. Finally the DNA was stored at -20 °C. Also, 5 microliters of the extracted plasmid were run on a 0.8% agarose gel at 85 V for 90 minutes, and its quality was checked [8].

2.5. Cloning and protein expression
E. coli BL21 (DE3) Rosetta strain (Novagen) competent cells (200 µL) were prepared using the CaCl2 method and transformed with 5 µL of pET28a(+) vector using the Novagen heat shock transformation method. Subsequently, selection for transformants accomplished by plating on LB agar plates containing chloramphenicol (70 µg/mL), kanamycin (35 µg/mL), and incubated at 37 °C for 18-24 hours [7].
Then, transformants were cultured in LB broth containing chloramphenicol (70 µg/mL) and kanamycin (35 µg/mL) and incubated overnight at 37 °C with stirring at 210 rpm. Sub-culturing was done at 1:50 (v/v) ratio in 100 mL of fresh LB broth containing chloramphenicol (70 µg/mL) and kanamycin (35 µg/ml) and incubated under the above mentioned conditions. As soon as optical density (OD) at 600 nm reached ~0.8, 1 mL of culture was withdrawn as an expression negative control. The expression of the target protein was then induced by the addition of isopropyl-β-d-galactopyranoside (IPTG; Sigma-Aldrich, St. Louis, USA) at a final concentration of 0.5, 0.75 and 1.0 mM. The culture was incubated at 30 °C, 210 rpm. Expression time-course studies were performed at 0, 4, 8 and 12 hours after induction. Finally, pellets were harvested by centrifugation of each sample at 7000×g, 15 minutes at 20 °C and stored at -80 °C for further processing [9].

2.6. Protein expression analysis and extraction
Collected pellets were resuspended in protein sample buffer (5X) plus 2-mercapto ethanol (2 ME) according to the Laemmli method [7]. Based on the hypothetical MW of scFv (50.306 kDa), the resolving and stacking sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel concentration were selected as 12% and 4%, respectively. Electrophoresis was done in running buffer (25 mM Tris-base, 192 mM glycine, 1% SDS, pH 8.3; CinnaGen Co., Tehran, Iran) at 90 V for 2–3 hours. The gel was stained with a staining solution of 1% Coomassie blue R-250 (Merck, Darmstadt, Germany) and de-stained using a solution of 7% acetic acid (Merck, Darmstadt, Germany), 5% methanol (Merck, Darmstadt, Germany) and 88% water. A standard molecular marker (CinnaGen Co., Tehran, Iran) was run in parallel with the samples to estimate the molecular weights of the proteins. Moreover, for periplasmic proteins extraction, a sonication method (Sonicator; Hielscher Ultra-sound Technology, Brandenburg, Germany) consisting of 5 times ×1 minute sonication and 1 minute interval on ice was followed. Also, 1.0 mM phenyl methyl sulfonyl fluoride (PMSF), (Merck, Darmstadt, Germany) was added to each sample to inhibit potential proteases in extracted samples [7].

2.7. Western blotting (WB) for confirmations of specificity
The specificity of recombinant scFv-mAb was confirmed by WB method. Briefly, SDS-PAGE was followed as described above without gel staining. The sandwich was arranged in a cathode to anode direction as follows: Support pad, Whatman NO.1 filter paper, SDS-PAGE gel, nitrocellulose membrane, Whatman NO.1 filter paper and support pad. The Blotting was implemented at 20 V for 2–3 hours using transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol for 1L double-distilled water, pH 8.3). Then, the nitrocellulose membrane was blocked with 5% (w/v) skim milk powder in PBS-T buffer (PBS with 0.05% tween 20 (v/v) overnight at 4 °C. Subsequently, washing was done three times with PBST, and the membrane was then incubated with an anti-poly histidine monoclonal antibody at 1:1000 dilutions for 2 hours, at room temperature. After three additional washes, the membrane was soaked in a horseradish peroxidase (HRP)- conjugated anti-His tag antibody (Sigma-Aldrich) solution. DAB solution (Merck, Darmstadt, Germany) was then added as the enzyme chromogen substrate. After incubation in dark place at room temperature and the appearance of thescFv-mAb band color, the reaction stopped with distilled water [7].

2.8. Protein purification and concentration measurement
The production of recombinant scFv-mAb, in either solubilized or insolubilized form, was analyzed by 12% SDS-PAGE gel electrophoresis (Hercules, USA). Then, Ni2+-NTA affinity chromatography (nickel-nitrilotriacetic acid) resin (QIAGEN, USA) was used for protein purification based on affinity to 6×His-tagg at the C-terminus of scFv-mAb. The purification was performed under native conditions with equilibration, washing (plus 20 mM imidazole) and elution (plus 250 mM imidazole) buffers according to the manufacturer’s protocol. Collected samples from various purification runs were analyzed using SDS-PAGE as previously described. 
The concentration of purified scFv-mAb measured at 595 nm (25 ˚C) using the colorimetric Bradford assay method in comparison with bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, USA) as a standard  (0-20 mg/mL). A standard curve was constructed with a serial dilution from 0.00 to 20.00 mg/mL using Graph Pad online server regression tools [10] via a linear regression calculator and plotted against BSA [7, 10, 11].

2.9. Chequerboards and evaluation of scFv-mAb function by using sandwich (capture) enzyme-linked immunosorbent assay (ELISA) 
The diagnostic value analysis of purified recombinant scFv-mAb was the most important part of the present study, as it shows its proper folding and functionality. It is performed by indirect sandwich ELISA (S-ELISA, Capture antibody ELISA), which reflects the affinity, folding, function, and specificity of the scFv-mAb. A 96-well flat bottom polystyrene high -bind microplate (Corning Co., USA) was coated with 400 ng/well of scFv- mAb (as a capture antibody) in coating buffer (1.50 g/L Na2CO3, 2.93 g/L Na2HCO3 in 1000 mL distilled water, pH 9), and incubated overnight at 4 °C. Also, BRS (bovine reference serum against FMDV serotype O [BRS-O]) was used as a standard positive antibody control for the FMD virus (virus neutralization test (VNT) Log10≥1/8, 1:10 dilution/well).

The supernatant was then removed, and the wells were washed three times with PBS-T buffer. Subsequently 150 μL of blocking buffer (PBST plus 2% sodium caseinate) was added for 2 hours and incubated at room temperature. The supernatant was removed, and the wells were washed three times with PBS-T. In the chequerboard assay, different concentrations (μg/well) of rmAb (1.5, 1, 0.5, 0.25, 0.125, 0.061) were used. Also, 2, 1.5, 1, 0.5 μg/well of ultracentrifuged concentrated viral antigen FMDV serotype O2016 (previously prepared by Razi Vaccine & Serum Research Institute, Department of FMD) mixed with 0.03 μg/well of PEG+NACL were added (data not shown). After 45 minutes of incubation at 37 ° C and three washes with PBS-T, 100 μL/well of secondary antibody (BRS with VNT 50%≥Log10 1.8) was added in 1:10 dilution. 4 negative controls (nonvaccinated calf serum, age below 6 month) and 2 positive controls (BRS) were also used. The plate was then incubated at 37 ° C for 45 minutes and washed three times with PBS-T. Next, 100 μL/well of goat anti -bovine HRP- conjugated antibody was added at a 1:10000 dilution with PBS-T. After 30 minutes of incubation at 37 ° C, reactions were developed by adding 100 μL/well of TMB substrate (3, 3′, 5, 5′- tetra methyl benzidine) (IDvet Co., Grabels, France). Finally, 50 μL/well of 1 M H2 So4 (Merck, Darmstadt, Germany) was added to stop the reaction. Absorbance at 450 nm was determined by an ELISA microtitre plate reader (Denly, well Scan Co.). The OD of highest dilution of each sera that was 2.5 times bigger than OD of the negative control serum (Mean of negative ±2 SD) was considered as the endpoint titer [7, 12, 13].

3. Results

3.1. scFv-mAb structure analysis 
The final scFv-mAb with 470 amino acid lengths was designed and linked together using a peptide linker (GSGGGGS). These nucleotide sequence of the engineered recombinant anti-FMD virus antibody was as follows:
CCATGGGCaaatacctattgcctacggcagccgctggattgttattactcgcggcccagccggccatgGAAGTTATGCTGGTTGAATCTGGTGGTGGTCTGGTTAAACCGGGTGGTTCTCTGAAACTGTCTTGCACCGCTTCTGGTTTCATCTTCAACCGTTGCGCTATGTCTTGGGTTCGTCAGACCCCGGAAAAACGTCTGGAATGGGTTGCTACCATCTCTTCTGGTGGTACCTACACCTACTACCCGGACTCTGTTAAAGGTCGTTTCACCATCTCTCGTGACAACGCTAAAAACACCCTGTACCTGCAGATGTCTTCTCTGCGTTCTGCTGACACCGCTATGTACTACTGCGTTCGTCGTGAAGACGGTGGTGACGAAGGTTTCGCTTACTGGGGTCAGGGTACTGTTGTTACCGTAAGCGCTGCTAAAACCACCCCGCCGTCTGTTTACCCGCTGGCTCCGGGTTCTGCTGCTGCTGCTGCTTCTATGGTTACCCTGGGTTGCCTGGTTAAAGGTTACTTCCCGGAACCGGTTACCGTTACCTGGAACTCTGGTTCTCTGTCTTCTGGTGTTCACACCTTCCCGGCTGTTCTGCAGTCTGACCTGTACACCCTGTCTTCTTCTGTTACCGTTCCGTCTTCTACCTGGCCGTCTGAAACCGTTACCTGCAACGTTGCTCACCCGGCTTCTTCTACCAAAGTTGACAAAAAAATCGTTCCGCGTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTTCTGACATCGTTCTGACCCAGTCTCCGGCTTCGCTCGCTGTAAGCCTGGGTCAGCGTGCTACCATCTCTTGCCGTGCTTCTGAATCTGTTGACTCTTCTGGTCACTCTTTCATGCACTGGTACCAGCAGAAACCGGGTCAGCCGCCGAAACTGCTGATCTACCGTGCTTCTAACCTGGAATCGGGTATCCCAGACAGGTTCTCTGGTTCTGGCTCTCGTACCGACTTCACCCTGACCATCGACCCGGTTGAAGCTGACGACGTTGCTACCTACTACTGCCAGCAGTCTAACGAAGTTCCGCTGACCTTCGGTGCTGGTACCAAACTGGACCTGAAACGTGCTGACGCTGCTCCGACCGTTTCTATCTTCCCGCCGTCTTCTGAACAGCTGACCTCTGGTGGTGCTTCTGTTGTTTGCTTCCTGAACAACTTCTACCCGAAAGACATCAACGTTAAATGGAAAATCGACGGTTCTGAACGTCAGAACGGTGTTCTGAACTCTTGGACCGACCAGGACTCTAAAGACTCTACCTACTCTATGTCTTCTACCCTGACCCTGACCAAAGACGAATACGAACGTCACAACTCTTACACCTGCGAAGCTACCCACAAAACCTCTACCTCTCCGATCGTTAAATCTTTCAACCGTAACGAATGCGGTCCGGGTGGTCAGCACCACCACCACCACCACCACCACTGCTAAGGATCC
As shown in the sequence, the signal peptide (PelB signal peptide) at the beginning is showed in lowercase at the 5’ terminal, before NcoI restriction enzyme (RE) site (C↓CATGG). A dash is drawn below the start and end codons. At the 3’ terminal of the ordered sequence, the His-tag, stop codon, and BamHI RE site (G↓GATCC) have been located.
After initial analysis, the final confirmation of the suitable physicochemical characteristics of the construct’s structure was reported (Table 1) [3].

3.2. Plasmid dilution, amplifying and extraction
Plasmid dilution implemented based one manufacturer’s instruction with some modifications. The lyophilized vector was dissolved in 100 μL of distilled water to obtain a final concentration of 2 μg/100 μL. From this, 75 μL was stored at -20 °C as a stock solution, and 25 μL was used as working solution. Also, pET28a(+) was successfully cloned in E. coli DH5α, screened using antibiotic- rich media, and extracted using the FavorPrep™ Plasmid DNA Extraction Mini Kit. At the end of this process, 90 μL was stored at -20 °C as extracted plasmid stock, while 10 μL was used as working solution. The concentration of the extracted plasmid solution, measured using a Nanodrop device, was 100 ng/mL. For analysis, 8 μL of the extracted plasmid solution was used for 0.8% (w/v) agarose gel electrophoresis. The results revealed typical, distinct high- purity band corresponding to circular and linear plasmids (~6958 bp) (Figure 1) [14, 15]. 

3.3. Transformation 
The selection of transformants was evidenced by the growth of E. coli DH5a on LB agar medium containing (30 μg/mL kanamycin) and E. coli BL21(DE3) Rosetta on LB agar medium containing kanamycin (35 µg/mL) and chloramphenicol (70 µg/mL) after 18-24 hours of incubation at 37 °C, in compared to the negative control. This confirmed the successful transformation of the pET28a(+) vector into its hosts (data not shown) [16].

3.4. Expression of recombinant scFv-mAb
The transformed E. coli BL21(DE3) Rosetta strain, induced with 0.5, 0.75 and 1 mM IPTG and incubated at 37 ˚C for 12 hours, expressed a significant protein fraction in both soluble and sonicated (inclusion body) (Figures 2A and 2B). All expression samples were run on 12% under reducing condition to confirm the expression of scFv. The SDS-PAGE results clearly showed a distinct band at the predicted position in the induced bacterial cell extract. 

As depicted in Figure 2 and evaluated by the presence of ~50 kDa band, the expression was successful for both (0.5 and 1 Mm) IPTG concentrations and duration time of 4 to 12 hours. Additionally, the presence of scFv bands was confirmed by WB [16, 17].

3.5. Concentration calculation of purified scFv-mAb
The optimum conditions for the purification of recombinant scFv-mAb by Ni2+-NTA agarose affinity chromatography included three-step binding process, followed by five washes in the presence of 25 mM imidazole to remove non-specific contaminants. Elution was subsequently implemented using an elution buffer (pH 7.5) containing 0.3 M imidazole to achieve maximum efficiency.
Following a single purification step, SDS-PAGE analysis showed a purity of more than 90%. The concentration of the purified scFv-mAb protein under these optimum condition was calculated to be approximately 2.00 mg/ml using the Bradford assay [14].
3.6. Efficiency evaluation of purified scFv by sandwich (capture) ELISA
By analyzing the chequerboard results, and based on curve break pattern, optical absorption jump, and optical absorption preferences between 0.8 and 1, the optimum concentration for the virus (0.5 μg/well of O2016 serotype) and scFv-mAb (0.4 μg/well) were determined with the highest signal to noise ratio for capture ELISA (Figure 3).

By assessing mean OD of negative serum at a 1:10 dilution against 0.4 μg/well of coated scFv, an OD of 0.3 was eventually selected as the cut-off. ELISA results showed that the scFv-mAb fragment successfully detected FMDV serotype O2016 with signals above 0.3 (optimum concentration of antigen was 0.5 μg/well). Additionally, the mean OD of the 4 negative controls was 0.17. Based on obtained results, when the recombinant scFv-mAb was coated on the plate, it exhibited an OD of 0.3≤X≤1.5 at 450 nm wavelength across various positive control treatments 

There was also at least 0.4 OD difference between the purified protein and the crude or lysate protein. Altogether, the present results indicated good sensitivity of the synthetic recombinant scFv-mAb for the detection of FMDV serotype O whole particles [16, 17].

4. Discussion 
This study describes the development of scFv-mAb against the VP1 capsid of FMDV in prokaryotic host for the detection of FMDV serotype O. Although primarily designed for serotype O, it may also be used for the detection of another FMDV serotypes, such as A, C, Asia 1, SAT 1, 2 and 3. Furthermore, it has potential applications in the preparation of antigenic panel (especially in OIE reference countries) and for use in ELISA, diagnostic kits, lateral flow test, virus neutralization test, and as a positive serum control.
Recombinant antibodies have important roles in treatment, research, and diagnosis. While full-length antibodies require mammalian expression systems due to their complex folding and post-translational modification, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli based expression system. Some commercial recombinant antibodies produced in E. coli are currently used in clinical treatment, including Certolizumabpegol (CIMZIA®), Ranibizumab, Brolucizumab, Caplacizumab (trade name Cablivi®) Moxetumabpasudotox (MxP) [18].
 In research settings, 1F10 (O UK) has been used as Pan-FMDV mAb [13, 19]. Also, Ochao (2000) reported crystallographic determinations for complexes of 4C4 or SD6 mAb with AnSA peptides, revealing important structural characteristics of the conserved RGD motif [15, 20]. Both studies showed that after binding of the viral peptide to both 4C4 and SD6 mAbs a similar pattern of interactions occurred. It was further established that Asp143 and Leu144 residues were structurally conserved and constituted part of the cell receptor recognition motif [21-23].
In 2000, Ochoa et al. registered the neutralizing monoclonal antibody 4C4 in the PDB database [3].
Its corresponding peptide adopted a compact fold with a nearly cyclic conformation and a specific disposition of the receptor-recognition motif Arg-Gly-Asp (RGD). It was complicated with the Fab fragment of the neutralizing monoclonal antibody 4C4. Although various studies have been conducted on antibody response against FMDV, determination and registration of antibody crystallographic structure remain rare and are limited to this structure.
To date, there are three hundred and twenty-four mAbs introduced for the O, A, C and Asia 1 serotypes of FMDV. Specifically; these include 130 mAbs for serotype O, 108 mAbs for serotype A, 53 mAbs for serotype Asia 1, and 33 mAbs for serotype C [11, 12, 19, 24].
The initial approach involved using an indirect sandwich ELISA with relatively low specificity and sensitivity, employing polyclonal immune sera as both capture (rabbit) and detecting conjugate antibody (guinea pig). Then, it was updated by using polyclonal and mAb as a capture and conjugate antibody, respectively [25, 26]. 
Grazioli et al. (2020) developed and validated a simplified serotyping ELISA based on monoclonal antibodies for the detection of FMDV serotypes such as O, A, C and Asia 1 [12]. They employed a Pan-FMDV mAb (1F10) as a detector conjugate in a multiplex ELISA, achieving 79% sensitivity compared to the 72% sensitivity of the polyclonal ELISA. This multiplex ELISA is simple, rapid, and stable. So, it could replace existing polyclonal ELISAs for FMDV detection and serotyping. These Pan-FMD antibodies showed valuable results in LFIAs, also a sandwich-type immunoassay combined with a set of well-characterized mAbs. One LFIA antibody work as detecting and identifying O, A and Asia-1 serotypes, while the second antibody enables the detection and differentiation of SAT1 and SAT2 serotypes [2].
Also, another mAb, which is actually a nanobody (M170 Nab), has been produced in Lama glama (PDB ID:7DST), a member of the Camelid family. These antibodies are produced specifically against VP3 protein of FMDV serotype O [15]. 
The production of recombinant antibodies in E. coli was first described in 1988 for Fv and scFv fragments. In the present study, we successfully expressed recombinant scFv with relatively high efficiency and performance in the E. coli BL21(DE3) Rosetta strain. Numerous studies are in line with our study [16, 27, 28]. The expression of scFv for the detection of different serotypes of FMDV has yielded very effective results. scFv-mAb against 3ABC was effectively produced in E. coli in 2014 [17, 29]. In another study in 2003, a known mAb against FMDV serotype O expressed in E. coli [30]. Commercial recombinant antibodies produced in prokaryotic and eukaryotic hosts for human and animal diseases include some products from Creative biolabs, Biocompare, Sinobiological and etc.
Further studies could further demonstrate the value of this recombinant antibody in the future, potentially expanding its commercial applications significantly.
It is noteworthy that the cost of scFv production after purification is estimated at approximately 1 USD per 2 mg/mL, which is significantly lower than other expressing platforms such as hybridoma technology and mammalian expression systems.

 
5. Conclusion 
The ELISA assay showed the reliable detection of FMDV serotype O using the recombinant scFv‑mAb fragment, indicating its high specificity and sensitivity. Also it support the diagnostic potential of this recombinant antibody and warrant further validation studies to confirm its efficacy and expand its possible commercial applications in both diagnostic and therapeutic contexts for FMDV.

Acknowledgements
The authors express their gratitude to Majid Esmaeilizad (Department of Biotechnology) and the staff of the Foot-and-Mouth Disease Departments at Razi Vaccine & Serum Research Institute for their assistance and for providing the research environment. Also, the authors wish to express their sincerest gratitude Davoud Mohammadbagher for his invaluable support during this study.

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Data availability
The data that support the findings of this study are available upon request from the corresponding author.

 
Funding
This study was supported by Razi Vaccine & Serum Research Institute, Karaj, Iran.

Authors' contributions
Data acquisition, data assessment and elucidation: Mohammad Mahdi Ranjbar; Writing the original draft: Mozhgan Helalinasab; Review and editing: Mohammad Kazem Shahkarami.
Conflict of interest

The authors declared no conflict of interest. 

References

1. World Organization for Animal Health (OIE). Chapter 3.1. 8. Foot and mouth disease (infection with foot and mouth disease virus). Manual of diagnostic tests and vaccines for terrestrial animals 2018. 2019 [Updated 2026 Jan 27]. Available from: [Link]
2. Cavalera S, Russo A, Foglia EA, Grazioli S, Colitti B, Rosati S, et al. Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect. Talanta. 2022; 240:123155. [DOI:10.1016/j.talanta.2021.123155] [PMID]
3. Ochoa WF, Kalko SG, Mateu MG, Gomes P, Andreu D, Domingo E, et al. A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: A role for water molecules. J Gen Virol. 2000; 81(Pt 6):1495-505. [DOI:10.1099/0022-1317-81-6-1495] [PMID]
4. Marrero R, Limardo RR, Carrillo E, König GA, Turjanski AG. A computational study of the interaction of the foot and mouth disease virus VP1 with monoclonal antibodies. J Immunol Methods. 2015; 425:51-7. [DOI:10.1016/j.jim.2015.06.008] [PMID]
5. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. ScFv antibody: Principles and clinical application. Clin Dev Immunol. 2012; 2012:980250. [DOI:10.1155/2012/980250][PMID]
6. Gasteiger E, Hoogland C, Gattiker A, Duvaud SE, Wilkins MR, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The Proteomics Protocols Handbook. Springer Protocols Handbooks. New Jersey: Humana Press. [DOI:10.1385/1-59259-890-0:571]
7. Sambrook J. Plasmid and their usefulness in molecular cloning. Mol Cloning Lab Man. 2001; 1:1-31. [Link]
8. No Author. Plasmid DNA Extraction Mini Kit, based on kit instructions [Internet]. 2026 [8 April 2026]. Available from: [Link]
9. Chan CE, Chan AH, Lim AP, Hanson BJ. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays. J Immunol Methods. 2011; 373(1-2):137-146. [DOI:10.1016/j.jim.2011.08.005][PMID]
10. Graph Pad. Graph Pad online server regression tools [Internet]. 2026 [Updated 8 April 2026]. Available from: [Link]
11. Brocchi E, Crosatti ML, Grazioli S, Bugnetti M. Preliminary evaluation of panels of monoclonal antibodies developed against four vaccine strains of FMDVes of serotype A for antigenic profiling and vaccine matching of field isolates. Abstract book of the Open Session of the EuFMD Standing Technical Committee, Erice, Italy, 14-17 October, 2008; Appendix 5, pp. 85. [Link]
12. Grazioli S, Ferris NP, Dho G, Pezzoni G, Morris AS, Mioulet V, et al. Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1. Transbound Emerg Dis. 2020; 67(6):3005-15. [DOI:10.1111/tbed.13677] [PMID]
13. Ferris NP, Grazioli S, Hutchings GH, Brocchi E. Validation of a recombinant integrin αvβ6/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease. J Virol Methods. 2011; 175(2):253-60. [DOI:10.1016/j.jviromet.2011.05.026] [PMID]
14. Yaghoobizadeh F, Ardakani MR, Ranjbar MM, Galehdari H, Khosravi M. Expression, purification, and study on the efficiency of a new potent recombinant scFv antibody against the SARS-CoV-2 spike RBD in E. coli BL21. Protein Expr Purif. 2023; 203:106210. [DOI:10.1016/j.pep.2022.106210][PMID]
15. Dong H, Liu P, Bai M, Wang K, Feng R, Zhu D, et al. Structural and molecular basis for foot-and-mouth disease virus neutralization by two potent protective antibodies. Protein Cell. 2022; 13(6):446-53. [DOI:10.1007/s13238-021-00828-9][PMID]
16. Baumgarten T, Ytterberg AJ, Zubarev RA, de Gier JW. Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress. Appl Environ Microbiol. 2018; 84(12):e00270-18.  [DOI:10.1128/AEM.00270-18][PMID]
17. Chitray M, Opperman PA, Rotherham L, Fehrsen J, Van Wyngaardt W, Frischmuth J, et al. Diagnostic and epitope mapping potential of single-chain antibody fragments against foot-and-mouth disease virus serotypes A, SAT1, and SAT3. Front Vet Sci. 2020; 7:475. [DOI:10.3389/fvets.2020.00475][PMID]
18. Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli expression system in producing antibody recombinant fragments. Int J Mol Sci. 2020; 21(17):6324. [DOI:10.3390/ijms21176324][PMID]
19. Ferris NP, Nordengrahn A, Hutchings GH, Reid SM, King DP, Ebert K, et al. Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples. J Virol Methods. 2009; 155(1):10-7. [DOI:10.1016/j.jviromet.2008.09.009] [PMID]
20. Tami C, Taboga O, Berinstein A, Núñez JI, Palma EL, Domingo E, et al. Evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo. J Virol. 2003; 77(2):1219-26. [DOI:10.1128/JVI.77.2.1219-1226.2003][PMID]
21. Verdaguer N, Sevilla N, Valero ML, Stuart D, Brocchi E, Andreu D, et al. A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: Implications for intratypic antigenic variation.J Virol. 1998; 72(1):739-48. [DOI:10.1128/JVI.72.1.739-748.1998][PMID]
22. Wang G, Liu Y, Feng H, Chen Y, Yang S, Wei Q, et al. Immunogenicity evaluation of MS2 phage-mediated chimeric nanoparticle displaying an immunodominant B cell epitope of foot-and-mouth disease virus. PeerJ. 2018; 6:e4823. [DOI:10.7717/peerj.4823][PMID]
23. JacksonT, King AM, Stuart DI, Fry E. Structure and receptor binding. Virus Res. 2003; 91(1):33-46. [DOI:10.1016/S0168-1702(02)00258-7] [PMID]
24. Brocchi E, Gamba D, Poumarat F, Martel JL, De Simone F. Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies. Rev Sci Tech. 1993; 12(2):559-70.  [DOI:10.20506/rst.12.2.702.] [PMID]
25. Ferris NP, Dawson M. Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet Microbiol. 1988; 16(3):201-9. [DOI:10.1016/0378-1135(88)90024-7] [PMID]
26. Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: A sensitive, rapid and reliable technique for primary diagnosis. Res Vet Sci. 1987; 43(2):225-32. [DOI:10.1016/S0034-5288(18)30778-1] [PMID]
27. Golchin M. Recombinant antibodies against the K99 colonisation factor of E. coli [PhD dissertation]. University of Glasgow; 2004. [Link]
28. Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J. Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Fact. 2018; 17(1):50.[DOI:10.1186/s12934-018-0894-y][PMID]
29. Sharma GK, Mahajan S, Matura R, Subramaniam S, Mohapatra JK, Pattnaik B. Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus. Biologicals. 2014; 42(6):339-45. [DOI:10.1016/j.biologicals.2014.08.005] [PMID]
30. ShengFeng C, Ping L, Tao S, Xin W, GuoFeng W. Construction, expression, purification, refold and activity assay of a specific scFv fragment against foot and mouth disease virus. Vet Res Commun. 2003; 27(3):243-56. [DOI:10.1023/A:1023300825438] [PMID]