To generate antibodies with high affinity for F. verticillioides from maize ear, a representative strain, wh1-2, from a F. verticillioides population collected from maize ear rot samples in China was selected for antigen preparation. Species identity and potential fumonisin production were molecularly identified (data not shown). The cell wall-bound proteins (CWPs) were prepared from this fumonisin-producing strain and used to immunize chickens. Indirect ELISA against CWPs with polyclonal antisera from immunized chickens showed a clear robust immune response up to 1:128000, and then mRNA from the spleen cells of chickens was isolated and transcribed into cDNA for construction of phage display library.

A phagemid library with a size of 1.4 × 10⁷ clones and 95% clones containing expected inserts was constructed and a good diversity was revealed by BstN I digestion (data not shown). Solid phase panning was employed for screening potential highly reactive scFv antibodies specific to F. verticillioides. To select high-affinity scFvs, the number of washing with 0.1% (v/v) PBST and PBS was increased by five (i.e., the first round, 5 times; the second round, 10 times; and the third round, 15 times), while the concentration of coated antigens (CWPs) was decreased by 50 μg/mL from initial concentration of 200 μg/mL in each round of the succeeding panning. Under such conditions, the ratios of output and input phages increased steadily after each round of panning (Table 1), with an approximately 38-fold increase of phage recovery after the third round compared with that from the first round of selection, demonstrating an efficient enrichment of specific antibodies. Thus, the panned library was used for subsequent selection of high affinity antibodies.

To obtain high affinity scFv antibodies, 48 clones from the panned library were randomly selected and analyzed by phage ELISA. As shown in Figure 1, 23 clones had a positive reaction against CWPs of F. verticillioides with varied signal intensities. DNA sequencing of the 10 selected positive clones identified four different scFvs named as FvCA1, FvCA2, FvCA3 and FvCA4, respectively. Sequence analyses indicated that these scFv antibodies contained conserved sequences in their framework regions but had variable sequences in their CDRs, particularly in CDRH3 (Figure 2). They shared 73% sequence identity with each other at their amino acid levels. Their overall conserved sequences and domains were in accordance with chicken antibody sequences in the database of NCBI (data not shown).

The four selected scFvs expressed in soluble form in bacteria displayed a little size variation as revealed by immunoblot (Figure 3). Three antibodies, FvCA2, FvCA3 and FvCA4, had one intact band while three or four bands were seen for FvCA1 antibody, probably due to incomplete expression or protein degradation. ELISA with four soluble purified scFv antibodies revealed that the FvCA4 had the highest affinity for CWPs of F. verticillioides, followed by FvCA3. These two antibodies had a three to four-fold higher binding capacity than the remaining two antibodies, FvCA1 and FvCA2 (Figure 4). The FvCA4 antibody was the best of the four antibodies in both soluble antibody ELISA and phage ELISA. This suggested that phage displayed antibody, especially antibodies with high binding capacity, can be efficiently panned and selected for a given target. However, there were some discrepancies between two ELISAs showing inverse values for FvCA2 and FvCA3, which were antibodies with moderate binding capacity (Figures 1 and 4). FvCA2 had a higher binding value in phage ELISA than FvCA3 but a lower binding was seen in soluble antibody ELISA than for FvCA3. This discrepancy may be due to the alteration of antibody configuration when fused with pIII protein of phage particle, as observed by others [40].

To know whether the three-dimension structures of the selected antibodies have any potential impact on their binding capacity, the amino acid sequences of the four scFvs were subjected to modeling using DeepView (Swiss-PdbViewer) based on the respective crystal structure from CPHmodels-3.2 Server [50] (Figure 5). This modeling analysis indeed revealed a major structural difference among these scFvs in the central cores of protein scaffolds, consisting of CDRH3, CDRL1 and CDRL3 regions. Antibodies FvCA3 and FvCA4 formed a compact structure in this region (Figure 5c,d), while a rather loose configuration was assembled in the structures of FvCA1 and FvCA2 antibodies (Figure 5a,b). Taking into account the different affinities (Figure 4), we supposed that this region might be the centre of the binding site and directly in physical contact with antigen(s), since CDRH3 and CDRL3 were considered to lie generally in the centre of the traditional antigen binding site [51]. Therefore, we could speculate that a compact spatial configuration of paratope is more favorable for the binding of an antibody to CWPs from F. verticillioides. The other regions, such as CDRH1, CDRH2 and CDRL2, seemed to have less impact on the antigen-antibody interactions because they reside in the periphery of protein scaffolds.

Notably, most variation in structures was mainly caused by CDRH3 region (Figure 5), in which there was the most diversity of amino acid and length among the six CDRs (Figure 2). For example, FvCA1 had the longest amino acid, and FvCA2 formed a β-turn; FvCA4 exposed its CDRH3 region more closely to the surface compared to that from FvCA3. Therefore, we hypothesized that the residues of CDRH3 played a vital role in these selected antibodies. Previous studies have also confirmed that the length and heterogeneity of the CDRH3 sequence were associated to antibody binding affinity, specificity, paratope shape, and activity (such as neutralization potency) [52–56]. Properties of the selected scFvs such as FvCA1, FvCA2 or FvCA3 might be further improved by CDR shuffling [57–59], chain shuffling [60], or DNA shuffling technology [39,40].

The above analyses indicated that the FvCA4 antibody had the best binding capacity and proper overall three-dimensional structure. Thus, this antibody was selected for further characterization in its soluble form. The germinated conidiospores of different Fusarium species and other fungi were tested for this antibody by ELISA. As shown in Table 2, FvCA4 antibody had a high sensitivity to all Fusarium species but no cross-reactivity with non-Fusarium fungi, suggesting that FvCA4 is a Fusarium-specific antibody. To further reveal whether FvCA4 antibody was able to react with the components from CWPs of this fungus, an immunoblot assay was carried out with the CWPs from F. verticillioides, F. asiaticum, Aspergillus flavus, and Sclerotinia sclerotiorum. The results indicated that this antibody indeed bound to components from CWPs of Fusarium species but did not cross-react with that of A. flavus and S. sclerotiorum (Figure 6). Different sizes of CWPs from F. verticillioides and F. asiaticum detected in the blot suggested that these two Fusarium species have varied cell wall compositions as they belong to different species and have their own favorable hosts and peculiar infection mechanisms. Nevertheless, these results congruously indicated that FvCA4 is a Fusarium genus-specific antibody.

Immunofluorescence labeling was used to identify the site of FvCA4 binding. Figure 7a,c,e,g show the normal morphology of conidiospores and hyphae of F. verticillioides. Figure 7b,f show the binding of FvCA4 localized with the brightest fluorescence intensity to the cell walls of both conidiospores and hyphae, confirming the specificity of this antibody for a cell surface target. No fluorescence labeling was visible after incubation with the nonspecific scFv antibody PIPP specific to a human antigen HCG [32,40,61] (Figure 7d,h). These results confirmed that FvCA4 binds to a cell surface antigen of F. verticillioides.

To study the feasibility of FvCA4 for detection of contaminated samples, various concentrations of mycelium were used for ELISAs to determine detection limit. Twice higher optical density of samples to controls was considered as valid detection [62] and the result showed that the FvCA4 antibody was able to detect as low as approximately 10⁻² μg/mL of F. verticillioides mycelium (Figure 8a). The mycelium concentration in relation to the OD_405nm values could be expressed by logarithmic curve [y = 0.160ln(x) + 0.835, R² = 0.913]. To determine the minimum level of mycelium biomass present in maize grains, mycelium was mixed with maize grains and the extracts were used for ELISAs. Figure 8b shows that FvCA4 was able to detect approximately 10⁻² mg mycelium per gram of maize grains (i.e., 0.1 μg/mL). The detection limit of mycelium was higher in maize grains than in PBS buffer, as observed by others [24]. Logarithmic curve regularity between mycelium biomass per gram of maize and the OD_405nm values was illustrated as follows: [y = 0.142ln(x) + 0.936, R² = 0.813]. Therefore, the mycelium biomass in contaminated food/feed can be assessed conveniently by an ELISA detection using the FvCA4 antibody.

To study the stability of antigens detected by the antibody, naturally contaminated and artificially infected maize samples were heated to 100 °C for 10 min and assayed by an ELISA. The results indicated that comparable reaction signals were detected from both heated and unheated samples. These results suggested that the antigens present in F. verticillioides are thermostable and FvCA4 can efficiently detect contaminated field samples and processed food products.

A fungus strain of F. verticillioides, wh1-2, was isolated from the rot kernels of maize, collected from the seriously epidemic area of maize ear rot in Wuhan, China. This strain was identified by PCR with genus- and fumonisin-producing-specific primers [63] and cultured in Czapek-Dox Broth medium (pH 8.0) at 30 °C with shaking at 200 r/min for 5 days. The mycelia were collected and freeze-dried, and the cell wall-bound proteins (CWPs) were prepared as described [64].

For immunization, 12 week-old “White leghorn” chickens (Gallus domesticus) were injected intramuscularly with 100 μg antigen emulsified with an equal-volume complete Freund’s adjuvant (Sigma). Three additional injections were given at 2-week intervals with an equal-volume of incomplete Freund’s adjuvant (Sigma). The blood was collected after the third immunization and the antisera titer was measured by indirect ELISA [25]. Briefly, 96-well microtiter plate wells were coated with 100 μL CWPs (20 μg/mL) and blocked with 150 μL 2% (w/v) skimmed milk in PBS. Wells were washed three times with PBS, followed by incubation with 2-fold diluted immune and non-immune sera from 1:1000 initial dilution for 1.5 h at 37 °C. Then ELISA detection was performed with 1:5000 diluted alkaline phosphatase (AP)-conjugated anti-chicken IgY (Promega) and the enzyme reaction was activated by 0.2% (w/v) p-nitrophenyl phosphate (pNPP) substrate solution. The absorbance at 405 nm was measured on an ELISA Microplate Reader (Multiskan MK3, Thermo Fisher).

Total RNA was isolated from the spleen cells collected after the fourth immunization using TRNzol-A+ total RNA extraction reagent (Tiangen) and mRNA was purified using Oligotex mRNA mini kit (Qiagen) following the Manufacturer’s instructions. The first strand of V_H and V_L cDNAs were synthesized with VH-cDNA and VL-cDNA primers (Table 3) respectively, using the SuperScript III Reverse Transcriptase Kit (Invitrogen). Subsequently, V_H domains were amplified by PCR using sense primer VHF and antisense primer CHIC-Gly, as well as the CHIC-Ser and VLB primers (Table 3) for V_L domains amplification. The scFv fragments were then assembled with purified V_H and V_L domains by SOE-PCR (splicing by overhang extension-PCR) using VHF and VLB primers.

After SOE-PCR reaction, the full-size scFv genes were purified and digested with SfiI and Not I restriction enzymes (NEB), and cloned into the pHENHi phagemid vector [32] with the same restriction enzyme sites. The resulting recombinant phagemids were then transformed into E. coli XL1-Blue MRF’ (Stratagene) by electroporation. The transformed cells were plated onto Luria-Bertani agar medium supplemented with 1% (w/v) glucose and 100 μg/mL ampicillin. All clones were scraped from the plates in Luria-Bertani broth containing 100 μg/mL ampicillin and 25% (v/v) glycerol and stored at −70 °C.

An aliquot of 800 μL recombinant E. coli cells of the constructed library was inoculated into 50 mL of fresh 2 × TY broth (1.6% (w/v) tryptone, 1.0% (w/v) yeast extract, 0.5% (w/v) NaCl) supplemented with 1% (w/v) glucose and 100 μg/mL ampicillin and incubated at 37 °C with shaking at 200 r/min until the OD_600nm reached 0.5. Approximately 5 × 10¹⁰ M13-KO7 helper phages (Amersham Biosciences) were added into 5 mL culture and incubated at 37 °C for 30 min without shaking. After centrifugation, the infected cells were resuspended in 140 mL 2 × TY broth containing 100 μg/mL ampicillin and 25 μg/mL kanamycin and cultured at 30 °C for 16 h with shaking at 200 r/min. The recombinant phages were recovered by precipitation with PEG/NaCl as described [65].

Solid phase panning was carried out according to Andris-Widhopf et al. [42] with modifications. Briefly, 20 wells of microtiter plate were coated with CWPs (200 μg/mL in sterile PBS and 50 μg/mL descending in the succeeding rounds of panning) at 37 °C for 2 h. After blocking with 2% (w/v) skimmed milk in PBS, 100 μL of the freshly prepared phages suspension was added to each well and incubated at 37 °C for at least 2 h. The wells were washed five times (10 and 15 washing times in the second and third round of panning) with PBST (0.1% (v/v) Tween-20 in PBS) and PBS. The bound phages were eluted by applying 100 μL 100 mM triethylamine at room temperature for 10 min without shaking. Then, 50 μL Tris-HCl (1 mol/L, pH 7.4) was added immediately for quick neutralization. The log-phase bacteria infected with eluted phages were plated onto TYE-GA agar plates (1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.8% (w/v) NaCl, 1% (w/v) glucose, and 100 μg/mL ampicillin) and incubated at 37 °C overnight. The clones were collected for the next round of panning. In the panning procedure, one well was blocked with 2% (w/v) skimmed milk and 10 μL of prepared phages was added to serve as a control.

The 96-well microtiter plates were coated with 100 μL CWPs (20 μg/mL) or PBS (control) and blocked with 150 μL 2% (w/v) skimmed milk at 37 °C for 2 h. 50 μL of the blocking solution and 50 μL of phages prepared from the clones of the third round of panning were added into wells and incubated for 2 h at 37 °C. After three washings with 0.1% (v/v) PBST and PBS, the bound phages were detected with 1:5000 diluted HRP-conjugated mouse anti-M13 antibody (GE Healthcare) at 37 °C. The 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution was added to each well and the plates were incubated for 15 min for color development. The reaction was stopped with 2 mol/L H₂SO₄ and absorbance at 450 nm was measured.

The positive clones identified in phage ELISA were selected for DNA sequencing (Invitrogen). The forward primer pHENpel and the reverse primer pHENmyc (Table 3) were designed based on the pHENHi phagemid vector and used for sequencing scFv genes. Sequences were analyzed using BioEdit software (Ibis Biosciences).

The colony culture from a −70 °C glycerol stock was inoculated into 20 mL 2 × TY broth containing 1% (w/v) glucose and 100 μg/mL ampicillin and cultured at 37 °C overnight with shaking at 200 r/min. The next day, an aliquot of 8 mL of the bacteria culture was transferred into 160 mL 2 × TY broth with ampicillin and grown to an OD_600nm of 0.5 to 0.6. A final concentration of 0.1 mmol/L IPTG was then added into the culture and the cells were incubated at 25 °C and 200 r/min for 2 h for scFv expression. The periplasmic proteins were extracted by osmotic shock method [40,66] and dialyzed against PBS buffer. The soluble scFv antibodies were purified by immobilized metal affinity chromatography (IMAC, Qiagen).

For affinity analysis of scFv antibodies, 96-well microtiter plates were coated with 100 μL 20 μg/mL CWPs in PBS and blocked with 150 μL 2% (w/v) skimmed milk at 37 °C for 2 h. After three washings with PBS, 200 nmol/L of purified scFv antibody was added and incubated for 1.5 h at 37 °C. Subsequently, the plate wells were incubated with 100 μL 1:5000 diluted monoclonal anti-polyhistidine (His) antibody (Sigma) and AP-conjugated goat anti-mouse IgG antibody (Sigma) for 1.5 h at 37 °C. Colorimetric detection was performed with 0.2% (w/v) pNPP substrate solution and absorbance at 405 nm was measured. For analysis of specificity, the wells were coated with germinated conidiospores (10⁷/mL) of Fusarium and other genera fungi in YPG broth (0.3% (w/v) yeast extract, 1% (w/v) peptone, and 2% (w/v) glucose), and then ELISA detection was performed as described above. The YPG broth and PBS were taken as control separately and each sample was done in triplicate.

The CWPs from F. verticillioides, F. asiaticum, Aspergillus flavus, and Sclerotinia sclerotiorum or the periplasmic proteins from scFv expression system were separated by 12% (w/v) SDS-PAGE and transferred to nitrocellulose membranes. Immunoblot containing CWPs was developed using the purified scFv antibody (50 nmol/L), a 1:5000 diluted monoclonal anti-His antibody and AP-conjugated goat anti-mouse IgG antibody. The membrane with periplasmic proteins was detected with the antibodies described above, omitting the scFv antibody. The colorimetric reaction was carried out by using the BCIP/NBT Color Development Kit (Boster). The immunoblot membranes were scanned with a Bio-Rad GS-800 Densitometer.

The wells of a 12-well cell culture plate (Corning) were blocked with 2% (w/v) BSA in PBS at 37 °C for 2 h followed by three washings with PBS. The poly-l-lysin covered nummular cover slides were placed into the wells and 1 mL of germinated conidiospores was added. The supernatant was discarded after centrifugation at 2000 g for 15 min. 500 μL of the purified scFv (1 μg/mL), monoclonal anti-His antibody (1:3000 dilution in PBS) and Cy3-conjugated affinipure goat anti-mouse IgG (H + L) (1:100 dilution, ProteinTech Group) was added in turn. Unbound antibodies were washed away with 0.1% (v/v) PBST and PBS each three times. Then the prepared coverslips were transferred with the embedded antigen down on the slides by adding Antifade Mounting Medium (Beyotime). A Nikon Eclipse 90i fluorescence microscope with a TRITC model and the NIS-Elements AR 3.2 software (version 3.2; Nikon: Tokyo, Japan, 2011) was used for microscopic detection and analysis.

To obtain the detection limit to mycelium, different concentrations of F. verticillioides mycelium ground in PBS (10⁻², 10⁻¹, 1, 10, 10², 10³, 10⁴ μg/mL) were added into plate wells and ELISA detection was performed as described above. For determining the minimum level of mycelium biomass present in maize grains, 0.1 g of maize powder suspended in 10 mL PBS containing 0.05% (v/v) Tween-20 was mixed with different amounts of mycelium (10⁻³, 10⁻², 10⁻¹, 1, 10, 10², 10³ mg). Then, 100 μL aliquot of the homogenates was added into plate wells for ELISA detection.

To determine the feasibility of Fusarium contamination detection using the bacteria expressed scFv antibody, 10 naturally and artificially infected maize samples were collected from different areas of China and ground in liquid nitrogen. The powder was mixed vigorously in PBS and the supernatants were added into plate wells. The test assay was developed as described for ELISA assays. For thermostability analysis, the molded samples were treated by roasting or boiling at 100 °C for 10 min. Also, healthy samples were similarly treated to serve as negative controls and each sample was done in triplicate.