Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken | Journal of Nanobiotechnology

Materials and reagents

The double blood bags used blood collection were obtained from Suzhou Laishi Transfusion Equipment Co., Ltd (Suzhou, China). The complete Freund’s adjuvant, incomplete Freund’s adjuvant and Amicon UltraCentrifugal Filter Units were procured from Sigma Aldrich (St. Louis, MO, USA). All the restriction enzymes utilized in the study were procured from New England Biolabs (Beijing) LTD (Beijing, China). The 96-well microplates were purchased from Corning (New York, NY, USA). The PCR Purification Kit, Gel Extraction Kit and TIANprep Mini Plasmid Kit used for plasmid preparation were obtained from TIANGEN (Beijing, China). All the reagents utilized in this study were analytical grade, unless otherwise indicated.

Cells, strains and vectors

The HEK293T cell lines were cultured in the Dulbecco’s modified Eagle’s medium (Life Technologies Corp, USA) supplemented with 10% fetal bovine serum (FBS, Gibco USA) and 100 IU/mL penicillin‒streptomycin solution (Gibco USA). All the strains were from our laboratory preserved strains (Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Chengdu, China) including S. Enteriditidis FY-04 (GenBank accession JAKIRO000000000), S. Enteriditidis ATCC13076, S. Typhimurium ATCC23566, S. Pullorum ATCC13036, S. gallinarum CICC21510, Staphylococcus aureus ATCC25923, Klebsiella pneumoniae (isolated by Laboratory), Campylobacter jejuni (isolated by Laboratory), Listeria monocytogenes ATCC19115, Escherichia coli ATCC 25922. The pET-25b vector (Novagen, USA) was utilized to express the recombinant nanobody fusions against S. Enteriditidis in the E. coli system. The pMECS vector and M13K07 helper phage were kindly gifted by Ph.D. Qizhong Lu from the Stata Key Laboratory of biotherapy, Sichuan University, and were used to construct the VHH library. The pEGFP-N1 vector (Clontech, Japan) stored at in our laboratory was used as a backbone to construct the pCMV-N1-HRP vector.

Inactivation of the S. Enteriditidis strain

To ensure the safety of the immunized Bactrian camel, the pathogenic S. Enteritidis FY-04 was inactivated with formaldehyde as previously described [24]. Briefly, the S. Enteriditidis was cultured overnight in LB media and harvested by centrifugation at 8000 rpm for 10 min and resuspended in PBS supplemented with 0.2% (v/v) formaldehyde, and placed at 37℃ for 48 h. The inactivated S. Enteriditidis was adjusted to 1 × 10⁹ CFU/mL of the final concentration. Finally, the inactivation efficacy was evaluated using the plating count. There were no colonies on the plate, suggesting that the strains were completely inactivated and could be used to inject the Bactrian camel for the experiment.

Bactrian camel immunization and VHH library construction

To obtain a specific VHH library against S. Enteriditidis FY-04, a previously described protocol was used [16, 25]. Briefly, a healthy 4–year-old Bactrian camel was subcutaneously immunized five times with the inactivated S. Enteritidis FY-04 mixed with equal volumes of adjuvant. Freund’s complete adjuvant was used for the first immunization, four times followed with Freund’s incomplete adjuvant at an interval of 2 weeks. To evaluate the titration of serum from the last immunized camel, an indirect ELISA with HRP-conjugated rabbit anti-camel IgG (SE283, Solarbio, Beijing, China) was performed where the inactivated S. Enteritidis as a coated antigen having a concentration of 1 × 10⁹ CFU/mL was used.

Three-four days after the last immunization, the peripheral blood mononuclear cell was collected from a 200 mL blood sample. The cDNA prepared from the total RNA was used for constructing the nanobodies (VHH) library using the procedures presented in Scheme 2. Briefly, the VHH fragments were amplified by a two-steps PCR, with primer pairs listed in Additional file 1: Table S1. The CALL001 and CALL002 were the first-round PCR amplification pair of primers, while VHH-FOR and VHH-REV constituted the second pair of primers [26]. The fragments of the second PCR were ligated into the phagemid vector pMECS (digested by the restriction enzyme PstI and NotI). Then, the recombinant phagemids (pMECS-VHHs) were electroporated into the pre-prepared E. coli strain TG1 electroporation-competent cells. The capacity of the VHH library constructed here was analyzed through the plate count method, and the LB plates were supplemented with 2% (w/v) final concentration of glucose and 100 µg/mL ampicillin. The randomly-selected 48 colonies were determined using the PCR amplification with the primer pair MP57 and GIII (Additional file 1: Table S1). Subsequently, the positive colonies were sequenced for identifying diversity. Finally, the VHH library was stored at −80 ℃ in the LB medium supplemented with 20% glycerol and 100 µg/mL ampicillin until further use.

Screening and identification of the specific nanobodies against S. Enteriditidis

The specific nanobodies were selected against S. Enteriditidis across four rounds of bio-panning using indirect ELISA (iELISA) as described previously [24, 27]. Briefly, a representative aliquot of the VHH library was cultured and infected with the M13K07 helper phages to obtain the rescue phage in every round of selection. The microtiter plates were coated with the inactivated-S. Enteriditidis of 1 × 10⁸ CFU/well in NaHCO₃ buffer (100 mM, pH = 8.2) at 4℃ overnight. The coated wells were washed three times with PBS containing 2% tween-20 (PBST, v/v), and then blocked with 3% skimmed milk (w/v). Then, 5 × 10¹¹ PFU of the rescue phage was added and incubated at 37℃ for 1 h. Then, each well was washed ten times with PBST, 100 μL TEA solution (100 mM triethylamine, pH = 11.0) was added and incubated at RT for 10 min to elute specific phage particles, which was immediately neutralized with 100 μL of 1.0 M Tris–HCl (pH = 7.4). Subsequently, for the next round of selection, the eluted phage particles were transferred to infect the TG1 cells for titration evaluation and amplification. The population of TG1 cells infected was counted to quantify the input and output phages and the enriched phage particles were detected using iELISA with an anti-M13 antibody (Hangzhou HuaAn Biotechnology Co., Ltd, Hangzhou, China). After four rounds of screening, 96 individual clones were randomly selected from the third, fourth round of eluted phages. They were then separately induced with 1 mmol/L IPTG for expressing the soluble nanobodies in the E. coli periplasm in the 96-well plates. Several freeze–thaw cycles yielded the periplasmic extract (PE) which consisted of Nbs with Hemagglutinin (HA) and His tags. Furthermore, the presence of specific nanobodies against S. Enteriditidis were determined using iELISA with mouse anti-HA monoclonal antibody (Sino Biological, Inc, Beijing, China). Finally, sequencing based on their complementary determining regions (CDRs) amino acid sequence were able to identify and classify positive colonies (P/N > 3.0).

Expression, purification and characterization of the nanobodies against S. Enteriditidis

To obtain nanobodies against S. Enteriditidis, the pET-25b⁺ vector with a signal sequence for expressing C-terminally HSV-tagged and His-tagged proteins in the periplasm was utilized for expressing high-yield nanobodies [23, 28]. The VHH genes were amplified by PCR using SE-Nb-F and SE-Nb-R primer pairs (as listed in Additional file 1: Table S1). To develop recombinant plasmids, named the pET-25b⁺-VHHs vector, the PCR products were digested and ligated into the pET-25b⁺ vector at the same restriction site. The E. coli BL21 (DE3) transformed positive vectors were induced with 0.2 mM IPTG for 16 h at 16 °C to obtain the nanobodies against S. Enteriditidis. Moreover, to evaluate the binding capacity of periplasm extract, an iELISA with anti-HSV tag monoclonal antibody (Bioss Inc, Beijing, China) was used. The Nbs were purified from periplasm extract using Ni-IDA 6FF Sefinose (TM) Resin Kit and imidazole were removed using the Sephadex DeSalting Gravity Column (Sangon Biotech, Shanghai, China). The expression and purification of the nanobodies was analyzed using SDS-PAGE and western blot. The binding capacity, specificity and cross-reactivity of the purified nanobodies, for the strains which include one S. Enteriditidis, and the other three Salmonellas, and five non-Salmonellas were confirmed by iELISA using the anti-HSV tag monoclonal antibody as a detection antibody.

Construction of the pCMV-N1-vHRP vector and producing nanobody‑HRP fusions against S. Enteriditidis

Construction and characterization of the pCMV-N1-vHRP vector

The pCMV-N1-vHRP vector was developed using the pEGFP-N1 vector as a backbone. This vector is designed to express nanobody fusions including the human Ig kappa chain signal peptide, HA tag, nanobodies coupled with the codon-optimized HRP, and the His tag in the HEK293T cells [20, 21, 23]. Briefly, the components like the secreting signal sequence (the human IgG kappa chain), HA tag, multiple cloning site (MCS), a short linker, vHRP coding sequence and 7 × His tag were synthesized by Sangon Biotech. Meanwhile, the validated sequence was completely digested using the enzymes NheI and XbaI before it was ligated into the commercial vector pEGFP-N1 (cut with the same enzymes) to create the pCMV-N1-vHRP vector. To determine whether inserting an exogenous gene could successfully express upon recombination pCMV-N1-vHRP vector in HEK293T cells, the EGFP coding sequence was amplified as a positive control using the primer pairs EGFP-F and EGFP-R (Additional file 1: Table S1) and subsequently inserted into the pCMV-N1-vHRP vector using the restriction enzymes PstI and HindIII. The recombination vector named pCMV-EGFP-vHRP was transfected into the HEK293T cell using Lipo8000™ Transfection Reagent (Beyotime Biotechnology, Shanghai, China). After 48 h of transfection, the EGFP-vHRP fusion was directly observed using fluorescence microscopy (Leica DMi8, Germany).

Expression and characterization of the nanobody‑vHRP fusion against S. Enteriditidis

The nanobody-HRP fusions were expressed in the HEK293T cells as described above. Briefly, the VHH coding genes were amplified by PCR using the Nb‑vHRP-F and Nb‑vHRP-R primers (Additional file 1: Table S1) and then ligated into the pCMV-N1-vHRP vector, to be ultimately named as pCMV-Nbs-vHRP. The positive plasmids were transfected to the HEK293T cells, and after 72 h of transfection, the supernatant of the culture was collected by centrifuging at 1000×g for 5 min to remove the cell debris. The secreted nanobody-HRP fusions supplemented with 0.02% NaN₃ (w/v) were then stored at 4℃ for direct use. Then, the expressions of the nanobody-HRP fusions in the HEK293T cells and culture supernatant were separately determined using indirect immunofluorescence assay (IFA) and Western blot assay. In addition, the anti-HA monoclonal antibody was not only treated as the first antibody with the FITC- and HRP-labeled goat anti-mouse antibodies (PROTEINTECH GROUP, Wuhan, China) but also as the second antibody in the two assays above. To evaluate the specificity, titers and cross-reactivity of the nanobody-HRP fusions in the cultured supernatant, the iELISA was performed using the anti-HA monoclonal antibody as the second antibody.

Indirect ELISA

The iELISA being a common immunoenzyme technique was frequently used in this studying for detecting the titer in the camel blood samples, screening of the specific nanobodies, and the specific binding and cross- reactivity of the nanobodies and nanobody-HRP fusions. Briefly, the inactivated S. Enteritidis or other strains (1 × 10⁸ CFU/well) were coated as antigens in the 96-well plate overnight at 4 ℃, where the NaHCO₃ buffer was used as a negative control. After blocking with 3% skimmed-milk, the primary antibodies (sera of different dilutions, the nanobodies of prokaryotic expression, periplasmic extracts, and the nanobody-HRP fusions) were added to the plates and incubated for 1 h at 37 ℃. For directly detecting the titer of the serum samples, the HRP-conjugated rabbit anti-camel antibody was employed as a detection antibody for direct use. The anti-HA and anti-HSV monoclonal antibodies were separately treated as the second antibody in the specific nanobodies screening and characterization, which were both followed by treatment with the HRP-conjugated goat anti-mouse IgG at 37℃ for 1 h. However, in the case of the nanobody-HRP fusions, no secondary and detection antibodies were used. After washing five times with PBST, 100 μL/well of TMB (Solarbio, Beijing, China) was added and incubated in the dark at 37 °C for 15 min for a colorimetric reaction, followed by stopping with 2 mol/L H₂SO₄ (50 μL/well). Finally, the optical density was measured at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA).

Immunofluorescence assay (IFA)

The procedure for IFA was modified based on a previously reported assay [23]. After 48 h of transfection, the transfected cells were fixed with 4% paraformaldehyde at RT for 30 min. Then, the cells were blocked with 2% bovine serum albumin and washed three times with PBS. The anti-HA monoclonal and FITC-conjugated goat anti-mouse antibodies were then used as the first and the second antibodies for incubating at 37 °C for 1 h. After DAPI staining, the cells were directly observed under a fluorescence microscope.

Establishment of the double nanobody‑based sandwich ELISA

The double nanobody‑based sandwich ELISA was established for detecting S. Enteriditidis, where the His-tagged Nbs and nanobody-HRP fusions were used as for capturing and detecting antibodies, respectively. The orthogonal assay was designed to select the best pair, procedure based on a previously reported procedure [24, 29]. Briefly, the plates were coated with the His-tagged Nbs (800 ng/well) overnight at 4 °C. After blocking with 3% (w/v) skim-milk for 1 h, the S. Enteriditidis (1 × 10⁸ CFU/well) was added and incubated in the plates for 1 h at 37 °C, an equal volume of NaHCO₃ buffer as the negative control. To this, 100 μL of different nanobody-HRP fusions (1:10 dilution ratio of original supernatant) were added to the plates and incubated for 1 h at 37 °C. The TMB (100 uL/well) which was used as a substrate of HRP was added to the plates and incubated in dark for 15 min at 37 °C. The color reaction was then stopped by adding 2 mol/L H₂SO₄ (50 μL/well) and the OD₄₅₀ nm value of each well was measured with a microplate reader (Thermo Fisher Scientific, USA). Henceforth, the best pair of nanobodies with the highest P/N value was selected. The optimal concentration for the capturing and detecting Nbs was determined for the different concentrations of the coated antigen by searching for the conditions with the highest P/N value as previously described [22]. Briefly, the sandwich ELISA was performed employing different amounts of the His-tagged Nbs (200, 400, 600, 800, 1000, 1500, 2000, 2500 ng/well) and the different dilution ratio of the nanobody-HRP fusions (1:1, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200 and 1:1000). S. Enteriditidis was employed as the positive control while NaHCO₃ buffer served as the negative control. Then, when the P/N ratio was the highest, the optimal amount of the capture nanobody and detection nanobody-HRP fusions were determined. To characterize the specificity and cross-reactivity of the double nanobody‑based sandwich ELISA, one S. Enteriditidis, the other three Salmonella and five non-Salmonella as mentioned above were used to evaluate. The standard curves were determined to quantify and determine the limit of detection (LOD) of the developed method. It used 3 aliquots of gradient dilution of S. Enteriditidis as detection antigen, with a primary concentration of 3.0 × 10⁹ CFU/mL.

Detection of S. Enteriditidis spiked in the milk

To validate the effectiveness of the double nanobody‑based sandwich ELISA, the skimmed milk from local supermarket ensured to be free of S. Enteriditidis using the plate counting method. The spiked milk sample was prepared by adding different concentrations of the S. Enteritidis FY-04 to the milk to attain final concentrations of 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ CFU/mL, respectively. The prepared samples were collected by centrifugation at 5000 rpm for 10 min. And after washing the pellet with PBS, the established sandwich ELISA was used to analyze the recovery rate. For detecting the enriched bacteria, 1 mL spiked milk with S. Enteritidis around 10 CFU were mixed with 9 mL LB liquid medium and enriched at 37 °C for 6 h, 8 h, 10 h, 12 h, 14 h at 37 °C, respectively. The samples were analyzed using the same method as described above, without adding S. Enteritidis to the milk sample as a negative control.

Analyzing of S. Enteritidis in vivo colonization in Chicken

The neonatal chickens were randomly divided into two groups under feeding conditions where Group A was orally challenged with 1.0 × 10⁸ CFU of S. Enteritidis FY-04, while group B was treated with the same volume of PBS as the blank control. On the fourth day after the oral challenge, all the chickens from each group were dissected to collect the tissues like the heart, liver, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, pancreas and kidney. For bacterial enrichment of these selected tissues, 1 g of every sample was cut and mixed with 25 mL pre-prepared buffered peptone water (BPW) and enriched at 37 °C for 12 h. Subsequently, the samples were centrifuged at 5000 rpm for 10 min and then were resuspended in PBS. The samples containing S. Enteritidis were determined using the established sandwich ELISA, qPCR detection and plate culture.

Real-time PCR

The bacteria genomic DNA was extracted using TIANamp Bacteria DNA Kit (Beijing, China) following the manufacturer’s instructions. The real-time PCR reactions were executed according to previous descriptions [30]. Precisely, the reaction mixture comprised 10 μL of SsoFast EvaGreen Supermix, 1 μL (10 μM) of each primer (qPCR-F and qPCR-R as listed in Additional file 1: Table S1), 2 μL of the DNA template, were added along with sterilized water to reach the final reaction volume of 20 μL. Subsequently, the assay was performed at a temperature of 95 °C for 2 min, with 40 cycles of 95 °C for 5 s, followed by 60 °C for 10 s, 72 °C for 20 s, and the fluorescence was assessed at 72 °C at the end of each cycle, calculating the Ct values (the cycle at which the fluorescence exceeds the background level). In this assay, the genomic DNA of S. Enteritidis ATCC 13,076 was used as a positive template and sterilized water as a negative control.

Statistical analysis

All the assays were independently repeated at least three times. The data presentation was represented using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). The Kappa values were calculated to estimate the coincidence between the developed sandwich ELISA assays, and the real-time PCR using the SPSS software (Version 20, http://www.spss.com.cn).