一种用于检测金黄色葡萄球菌A型肠毒素的四聚化单链抗体

A tetravalent single-chain variable fragment antibody for the detection of staphylococcal enterotoxin A 一种用于检测金黄色葡萄球菌A型肠毒素的四聚化单链抗体 1 2 equal first-author https://orcid.org/0000-0001-9948-5826 2 equal 2 2 corresp Xiaohong WANG, wxh@mail.hzau.edu.cn Xiaohong WANG, wxh@mail.hzau.edu.cn wxh@mail.hzau.edu.cn School of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou450046, China School of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou450046, China Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan430070, China Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan430070, China Staphylococcal enterotoxin A (SEA) synthesized by Staphylococcus aureus is a foodborne and heat-stable toxin, which is a great threat to human health ( Pexaraet al., 2010 Pexara et al., 2010 ). Highly sensitive antibodies are a key factor in the immunological detection of SEA, which is one of the most effective ways to detect SEA because of its accuracy, agility, and efficiency ( Nouri et al., 2018 Nouri et al., 2018 ). In this study, we constructed a tetravalent anti-SEA antibody gene by linking the tetramerization domain of human p53 to the C-terminus of the anti-SEA single-chain variable fragment (scFv), which was then transformed into Escherichia coli BL21 (DE3) for the production of a SEA-specific tetravalent antibody. Successful expression of the tetravalent antibody was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. An indirect non-competitive enzyme-linked immunosorbent assay (ELISA) revealed that the tetravalent antibody exhibited SEA-specific binding activity. A sandwich ELISA demonstrated that compared to the scFv monomer, the tetravalent antibody was more sensitive in detecting SEA. Molecular docking analysis revealed that the SEA interacted with the scFv mainly on the opposite side of the residue linked to p53. Thus, this study indicated that genetically engineered tetramerization is a potential way to improve the sensitivity of SEA-specific scFv. 免疫学方法可以用于金黄色葡萄球菌A型肠毒素的准确而快速的检测，而快速生产高亲和力抗体是其中的关键步骤。在本研究中，我们通过p53的四聚化功能结构域，在金黄色葡萄球菌A型肠毒素特异性单链抗体的基础上，成功制备了四聚化的单链抗体。酶联免疫吸附试验表明该四聚化抗体对金黄色葡萄球菌A型肠毒素的亲和力显著高于其单体形式，表明基于基因工程的四聚化是提高单链抗体亲和力的一种潜在的可行方法。 金黄色葡萄球菌A型肠毒素 酶联免疫吸附试验 单链抗体 四聚体 Among staphylococcal enterotoxins (SEs), SEA is the most frequent type recovered from foodborne outbreaks. It causes several symptoms, including but not limited to nausea, vomiting, diarrhea, cramps, and abdominal pain ( Pinchuk et al., 2010 Pinchuk et al., 2010 ). The close connection between SEA and human infections was confirmed by genome analysis in our study. Genome sequences were downloaded from the United States National Center for Biotechnology Information (NCBI) for S. aureus strains whose complete genome sequences are available, and used to determine whether SEA was produced in these strains. A total of 525 strains were tested and 85 of them (16.2%) were SEA-positive ( Fig. 1 Fig. 1 a). Next, we determined the host information of these SEA-producing strains and found that 62 of them (72.9%) were involved in human infections ( Fig. 1 Fig. 1 b). Detailed host information on SEA-producing strains is shown in Fig. 1 Fig. 1 c. These results indicated that SEA-producing strains were highly risky for humans. Given that SEA is a kind of heat-stable toxin and still has ability to induce severe symptoms in the digestive tract after ingestion ( Ortega et al., 2010 Ortega et al., 2010 ), accurate detection is undoubtedly important. The first and most critical step for the immunological detection of toxins is obtaining stable and highly sensitive antibodies and producing these antibodies more quickly ( O'Kennedy, 2019 O'Kennedy, 2019 ). For this study, we chose the multimerization peptide of human tumor suppressor protein p53 to fuse with the scFv gene obtained in our previous research and construct the tetravalent antibody against SEA ( Chen et al., 2014 Chen et al., 2014 ). The anti-SEA monoclonal antibody (mAb) used for generating the scFv was also obtained from our previous study ( Liang et al., 2011 Liang et al., 2011 ) and preserved in our laboratory. The coding regions of the fusion plasmids are shown in Fig. 2 Fig. 2 a and the schematic diagram of the tetravalent scFv antibody assembly is represented in Fig. 2 Fig. 2 b. The amplified scFv and p53 gene fragments were digested separately with corresponding restriction enzymes and then were ligated with digested pET-22b plasmid. The successful construction of the pET-22b-scFv/p53 plasmid was confirmed by polymerase chain reaction (PCR) and sequencing ( Fig. 2 Fig. 2 c). The amino acid sequence of anti-SEA scFv revealed that it contains a variable heavy (VH) chain and a variable light (VL) chain, which are connected by a peptide linker ( Fig. 2 Fig. 2 d). Each chain contains three complementarity-determining regions (CDRs) ( Fig. 2 Fig. 2 d), which play a vital role in specific antibody binding ( Polonelli et al., 2008 Polonelli et al., 2008 ). The constructed expression vector pET-22b-scFv/p53 was then transformed into E. coli BL21 (DE3) for protein expression. The soluble target protein was at its highest concentration when isopropyl β‍-d-1-thiogalactopyranoside (IPTG) concentration was 1 mmol/L and the temperature was 16 °‍C (data not shown). SDS-PAGE analysis demonstrated that the constructed plasmid expressed an obvious protein band with a relative molecular weight of 30 kDa ( Fig. 3 Fig. 3 a). Western blot yielded two detectable protein bands around 30 and 60 kDa, corresponding to the monovalent products and bivalent form of the antibody, respectively ( Fig. 3 Fig. 3 b). The recombinant protein was purified by metal affinity chromatography using Ni-nitrilotriaceate (Ni-NTA), and the concentration of purified protein was quantified by Bradford assay. The typical yield of nickel resin-purified target protein was about 3.6 mg/L of expression media. The purified protein samples were loaded in the non-reducing buffer and treated at different temperatures (room temperature, 60 and 100 °‍C) for 10 min. Then they were analyzed by SDS-PAGE. The purified protein existed mainly in the form of tetramers and dimers (protein bands at around 120 and 60 kDa which are consistent with the theoretical values) rather than monomers ( Fig. 3 Fig. 3 c). Howeve, when the protein samples were treated with 1% (volume fraction) β‍-mercaptoethanol, the tetravalent antibodies were reduced from tetramer to dimer form regardless of treatment temperatures ( Fig. 3 Fig. 3 c). These results indicated that the tetramer form could be easily reduced down to dimer form by β‍-mercaptoethanol but was relatively stable at different temperatures. The tetravalent antibodies were then concentrated and verified by SDS-PAGE ( Fig. 3 Fig. 3 d). The reactivity of the genetically engineered antibody after purification against SEA was tested by indirect ELISA (iELISA), which revealed that the tetravalent scFv antibody could specifically bind to immobilized SEA in a concentration-dependent manner ( Fig. 4 Fig. 4 a). This result was in accordance with the SEA-binding activity observed in the scFv monomeric form (without binding to p53) ( Chen et al., 2014 Chen et al., 2014 ). It should be pointed out that in the previous study we tested the specificity of the scFv monomer and found that the monomer was highly specific to SEA and showed no affinity to other SEs such as SEB and SEC1 ( Chen et al., 2014 Chen et al., 2014 ); this provided a foundation for the SEA-specificity of the scFv tetramer. A sandwich ELISA has been developed for SEA detection, which is more effective than other types of ELISA for the detection of large molecules ( Dasgupta, 2019 Dasgupta, 2019 ). The sandwich ELISA of our samples indicated that the tetravalent scFv antibody was more sensitive than the scFv monomer at SEA concentrations of 1 and 2 μg/mL ( Fig. 4 Fig. 4 b). The effect of polyclonal antibody (pAb) concentration on the performance of the tetravalent scFv antibody was then tested. The signal intensity of both scFv tetramers and monomers to SEA was increased following an increase in pAb concentration; and when pAb concentration was higher than 1 μg/mL, the tetravalent scFv antibody consistently generated stronger signals than the scFv monomers ( Fig. 4 Fig. 4 c). These results revealed that the tetramerization of the scFv antibody successfully improved its sensitivity to SEA. However, sensitivity of the tetravalent scFv antibody was still significantly lower than that of its mAb ( Liang et al., 2011 Liang et al., 2011 ), which indicated that further attempts, such as point-mutation ( Thomas et al., 2002 Thomas et al., 2002 ), should be made to improve the sensitivity of the SEA-specific tetravalent scFv. To understand the effect of tetramerization on scFv sensitivity in a molecular insight, we performed scFv-SEA docking analysis. We used chain A of the scFv antibody 4B08 (Protein Data Bank (PDB) entry 5YD5) as the template to predict anti-SEA scFv structure ( Miyanabe et al., 2018 Miyanabe et al., 2018 ). The prediction revealed that 243 residues of anti-SEA scFv (82%) were modeled with 100% confidence by the template ( Fig. 5 Fig. 5 a). Molecular docking analysis is visualized in Fig. 5 Fig. 5 b. The CDRs play a vital role in the binding of antibodies to specific toxins ( Liu et al., 2007 Liu et al., 2007 ; Murase et al., 2014 Murase et al., 2014 ). The specific binding of the anti-SEA scFv monomer to SEA was experimentally confirmed in our previous study ( Chen et al., 2014 Chen et al., 2014 ). The current docking analysis revealed that SEA interacted with scFv mainly on the side with CDRs, whereas the residue linked to p53 (C-terminus) was located on the opposite side ( Fig. 5 Fig. 5 b); this indicated that the tetramerization did not negatively affect the scFv-SEA interaction in three dimensions. In summary, we used the tetramerization domain of human p53 for the multimerization of the anti-SEA scFv antibody. SDS-PAGE and western blot showed that the scFv was successfully expressed and the monomers could self-assemble into tetramers. The iELISA demonstrated that the tetrameric scFv specifically reacted with SEA. The sandwich ELISA for detection of SEA was established using the tetrameric scFv and results showed that the tetramer of scFv was more effective than the monomer in SEA detection. This study revealed that genetically engineered tetramerization is a potential way to improve the sensitivity of the scFv in detecting foodborne heat-stable toxins. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References Chen WF , Hu L , Liu AP , et al. , 2014 . Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli . Can J Microbiol , 60 ( 11 ): 737 - 743 . https://doi.org/10.1139/cjm-2014-0468 https://doi.org/10.1139/cjm-2014-0468 Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli Dasgupta A , 2019 . Biotin and Other Interferences in Immunoassays: A Concise Guide . Elsevier, St. Louis . https://doi.org/10.1016/C2018-0-00537-X https://doi.org/10.1016/C2018-0-00537-X Biotin and Other Interferences in Immunoassays: A Concise Guide Liang B , Zhang YX , Liu AP , et al. , 2011 . Production of a monoclonal antibody by simultaneous immunization of staphylococcal enterotoxin A and B . Appl Biochem Biotechnol , 164 ( 6 ): 831 - 840 . https://doi.org/10.1007/s12010-011-9177-3 https://doi.org/10.1007/s12010-011-9177-3 Production of a monoclonal antibody by simultaneous immunization of staphylococcal enterotoxin A and B Liu JL , Anderson GP , Goldman ER , 2007 . Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library . BMC Biotechnol , 7 : 78 . https://doi.org/10.1186/1472-6750-7-78 https://doi.org/10.1186/1472-6750-7-78 Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library Miyanabe K , Akiba H , Kuroda D , et al. , 2018 . Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody . J Biochem , 164 ( 1 ): 65 - 76 . https://doi.org/10.1093/jb/mvy032 https://doi.org/10.1093/jb/mvy032 Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody Murase T , Eugenio L , Schorr M , et al. , 2014 . Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile . J Biol Chem , 289 ( 4 ): 2331 - 2343 . https://doi.org/10.1074/jbc.M113.505917 https://doi.org/10.1074/jbc.M113.505917 Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile Nouri A , Ahari H , Shahbazzadeh D , 2018 . Designing a direct ELISA kit for the detection of Staphylococcus aureus enterotoxin A in raw milk samples . Int J Biol Macromol , 107 : 1732 - 1737 . https://doi.org/10.1016/j.ijbiomac.2017.10.052 https://doi.org/10.1016/j.ijbiomac.2017.10.052 Designing a direct ELISA kit for the detection of Staphylococcus aureus enterotoxin A in raw milk samples O'Kennedy R , 2019 . Rapid Antibody-based Technologies in Food Analysis . The United Kingdom by CPI Group (UK) Ltd., Croydon, UK . https://doi.org/10.1039/9781788016322 https://doi.org/10.1039/9781788016322 Rapid Antibody-based Technologies in Food Analysis Ortega E , Abriouel H , Lucas R , et al. , 2010 . Multiple roles of Staphylococcus aureus enterotoxins: pathogenicity, superantigenic activity, and correlation to antibiotic resistance . Toxins (Basel) , 2 ( 8 ): 2117 - 2131 . https://doi.org/10.3390/toxins2082117 https://doi.org/10.3390/toxins2082117 Multiple roles of Staphylococcus aureus enterotoxins: pathogenicity, superantigenic activity, and correlation to antibiotic resistance Pexara A , Bourriel A , Govaris A , 2010 . Staphylococcus aureus and staphylococcal enterotoxins in foodborne diseases . J Hellenic Vet Med Soc , 61 ( 4 ): 316 - 322 . https://doi.org/10.12681/jhvms.14904 https://doi.org/10.12681/jhvms.14904 Staphylococcus aureus and staphylococcal enterotoxins in foodborne diseases Pinchuk IV , Beswick EJ , Reyes VE , 2010 . Staphylococcal enterotoxins . Toxins (Basel) , 2 ( 8 ): 2177 - 2197 . https://doi.org/10.3390/toxins2082177 https://doi.org/10.3390/toxins2082177 Staphylococcal enterotoxins Polonelli L , Ponton J , Elguezabal N , et al. , 2008 . Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities . PLoS ONE , 3 ( 6 ): e2371 . https://doi.org/10.1371/journal.pone.0002371 https://doi.org/10.1371/journal.pone.0002371 Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities Thomas R , Patenaude SI , MacKenzie CR , et al. , 2002 . Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity . J Biol Chem , 277 ( 3 ): 2059 - 2064 . https://doi.org/10.1074/jbc.M104364200 https://doi.org/10.1074/jbc.M104364200 Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity Weifeng CHEN: formal analysis, investigation, data curation, writing original draft, writing review, editing, and funding acquisition. Zhiwei LI: formal analysis, investigation, data curation, writing original draft, writing review, and editing. Xingxing DONG: formal analysis and investigation. Xiaohong WANG: conceptualization, methodology, resources, writing review, editing, supervision, project administration, and funding acquisition. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Key R&D Program of China (No. 2017YFC1600100), the National Natural Science Foundation of China (No. 31772054), the project of the Department of Education of Henan Province (No. 18A550010), the Science and Technology Department of Henan Province (No. 182102110303), and the Doctor Start-up Fund (No. 53000153) of Henan University of Animal Husbandry and Economy. We are grateful to Dr. YQ JIANG from the Academy of Military Medical Sciences (Beijing, China) for providing the SEA. 10.1631/jzus.B2000661 1673-1581（2021）04-0305-05 2020-10-23 2020-11-19 2021-02-23 2021-04-15 2022-05-09 © Zhejiang University Press 2021 Weifeng CHEN, Zhiwei LI, Xingxing DONG, and Xiaohong WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 4 22