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Generation and Characterization of a nanobody Against SARS-CoV

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Virologica Sinica 2021年第6期36卷 1484-1491页

作者： Jiang-Fan Li Lei He Yong-Qiang Deng Shu-Hui Qi Yue-Hong Chen Xiao-Lu Zhang Shi-Xiong Hu Rui-Wen Fan Guang-Yu Zhao Cheng-Feng Qin State Key Laboratory of Pathogen and Biosecurity Beijing Institute of Microbiology and EpidemiologyAcademy of Military Medical Sciences(AMMS)Beijing 100071China College of Veterinary Medicine Shanxi Agriculture UniversityJinzhong 030801China Research Unit of Discovery and Tracing of Natural Focus Diseases Chinese Academy of Medical SciencesBeijing 100071China

The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

关键词： Severe acute respiratory syndrome coronavirus(SARS-CoV) Receptor-binding domain(RBD) nanobody Neutralizing antibody

Quantification of soluble epoxide hydrolase inhibitors in experimental and clinical samples using the nanobody-based ELISA

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Journal of Pharmaceutical Analysis 2023年第9期13卷 1013-1023页

作者： Huiyi Yang Meng Qi Qiyi He Sung Hee Hwang Jun Yang Mark McCoy Christophe Morisseau Suqing Zhao Bruce D.Hammock Department of Pharmaceutical Engineering School of Biomedical and Pharmaceutical SciencesGuangdong University of TechnologyGuangzhouChina Department of Entomology and Nematology and UCD Comprehensive Cancer Center University of CaliforniaDavisCA 95616USA Langfang Normal University LangfangHebei065000China

To ensure proper dosage of a drug,analytical quantification of it in biofluid is *** chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and ***,it requires expensive instrumentation and is not appropriate for bedside *** soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human *** optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and *** assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate *** addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory *** results indicate that nanobody-based ELISA methods can efficiently analyze drug like *** methods could be easily implemented by the bedside,in the field in remote areas or in veterinary *** work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and *** of nanobody based pharmaceutical assays are discussed.

关键词： nanobody Immunoassay Soluble epoxide hydrolase inhibitors Metabolites Small molecules

nanobody-based immunosensing methods for safeguarding public health

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The Journal of Biomedical Research 2021年第4期35卷 318-326页

作者： Jiarong Zhang Hui Sun Wei Pei Huijun Jiang Jin Chen Center for Global Health The Key Laboratory of Modern ToxicologyMinistry of EducationSchool of Public HealthNanjing Medical UniversityNanjingJiangsu 211166China School of Chemistry and Chemical Engineering Southeast UniversityNanjingJiangsu 211189China School of Pharmacy Nanjing Medical UniversityNanjingJiangsu 211166China Jiangsu Province Engineering Research Center of Antibody Drug Key Laboratory of Antibody Technique of National Health CommissionNanjing Medical UniversityNanjingJiangsu 211166China.

Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public *** advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical *** with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing *** minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.

关键词： immunosensing nanobody biomarker environmental pollutant food contamination

A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E

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Journal of Integrative Agriculture 2024年第4期23卷 1354-1368页

作者： Huanhuan Lü Pinpin Ji Siyu Liu Ziwei Zhang Lei Wang Yani Sun Baoyuan Liu Lizhen Wang Qin Zhao Department of Preventive Veterinary Medicine College of Veterinary MedicineNorthwest A&F University/Shaanxi Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic TechnologyMinistry of Agriculture and Rural AffairsYangling 712100China

Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry *** glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some *** has the advantages of small size and easy genetic engineering and has been a promising diagnostic ***,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE *** the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian ***,two nanobodies against PRV-gE were screened from the immunized camel by phage display ***,two nanobody-HRP fusion proteins were expressed with HEK293T *** PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE *** optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:*** cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,*** detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV ***,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were *** bELISA could be used for monitoring and eradicating PR.

关键词： nanobody nanobody-HRP blocking ELISA PRV-gE antibody

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

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Journal of Pharmaceutical Analysis 2022年第2期12卷 308-316页

作者： Xi Qin Maoqin Duan Dening Pei Jian Lin Lan Wang Peng Zhou Wenrong Yao Ying Guo Xiang Li Lei Tao Youxue Ding Lan Liu Yong Zhou Chuncui Jia Chunming Rao Junzhi Wang National Institutes for Food and Drug Control Beijing100050China Synthetic and Functional Biomolecules Center Peking UniversityBeijing100871China WHO Collaboration Centre for Biologicals Standardization and Evaluation Beijing100050China

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis *** current identification test for rhIFNa2b is *** this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of ***2b was used to immunize an alpaca,which established a phage nanobody *** five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector *** subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel *** develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test *** developed rhIFNa2b detection assay had a limit of detection of 1 mg/*** isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein *** principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

关键词： Recombinant human interferon a2b nanobody Phage display Screening Rapid test

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

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Virologica Sinica 2022年第6期37卷 922-933页

作者： Jiakai Zhao Jiahong Zhu Ying Wang Mengting Yang Qiang Zhang Chong Zhang Yuchen Nan En-Min Zhou Yani Sun Qin Zhao Department of Preventive Veterinary Medicine College of Veterinary MedicineNorthwest A&F UniversityYangling Observing and Experimental Station of National Data Center of Animal HealthMinistry of AgricultureYangling712100China Kunming Customs Technology Center Kunming650228China

African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig ***, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

关键词： African swine fever virus(ASFV) nanobody ASFV-p30 nanobody-HRP fusion Protein Competitive ELISA

An efficient method for the site-specific 99mTc labeling of nanobody

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Biophysics Reports 2021年第4期7卷 295-303页

作者： Qi Luo Hannan Gao Jiyun Shi Fan Wang Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory) Medical Isotopes Research Center and Department of Radiation Medicine State Key Laboratory of Natural and Biomimetic DrugsSchool of Basic Medical SciencesPeking University Key Laboratory of Protein and Peptide Pharmaceuticals CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of Sciences Beijing Translational Center for Biopharmaceuticals

Recently, there has been a lot of interest by using nanobodies（heavy chain-only antibodies produced naturally from the Camelidae） as targeting molecules for molecular imaging, especially for the nuclear medicine imaging. A radiolabeled method that generates a homogeneous product is of utmost importance in radiotracer development for the nuclear medicine imaging. The conventional method for the radiolabeling of nanobodies is non-specifically, which conjugates the radioisotope chelating group to the side chain ε-amine group of lysine or sulfhydryl of cysteine of nanobodies, with a shortcoming of produce of the heterogeneous radiotracer. Here we describe a method for the site-specific radioisotope;99m;Tc labeling of nanobodies by transpeptidase Sortase A. The radiolabeling process includes two steps: first step, NH;2;-GGGGK（HYNIC）-COOH peptide（GGGGK = NH;2;-Gly-Gly-Gly-Gly-Lys-COOH, HYNIC =6-hydrazinonicotinyl） was labeled with;99m;Tc to obtain GGGGK-HYNIC-;99m;Tc; second step, Sortase A catalyzes the formation of a new peptide bond between the peptide motif LPETG（NH;2;-Leu-Pro-GluThr-Gly-COOH） expressed C-terminally on the nanobody and the N-terminal of GGGGK-HYNIC-;99m;*** a simple purification process, homogeneous single-conjugated and stable;99m;Tc-labeled nanobodies were obtained in >50% yield. This approach demonstrates that the Sortase A-mediated conjugation is a valuable strategy for the development of site-specifically;99m;Tc-labeled nanobodies.

关键词： nanobody Sortase A Site-specific radiolabeling 99mTc

Two nanobody‑based immunoassays to differentiate antibodies against genotype 1 and 2 porcine reproductive and respiratory syndrome virus

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Animal Diseases 2024年第2期4卷 98-114页

作者： Xu Chen Yueting Chang Lu Zhang Xinyu Zhao Zhihan Li Zhijie Zhang Pinpin Ji Qingyuan Liu Jiakai Zhao Jiahong Zhu Baoyuan Liu Xinjie Wang Yani Sun Qin Zhao Department of Preventive Veterinary Medicine College of Veterinary MedicineNorthwest A&F UniversityYanglingShaanxi 712100China Shenzhen Branch Guangdong Laboratory of Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518100China

Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes significant economic loss to the global pig *** 1 and 2 PRRSV(PRRSV-1 and-2)infections have been reported in China,Europe and *** accurate prevention,nanobodies were first used as diagnostic reagents for PRRSV *** this study three nanobodies targeting both PRRSV-1 and-2,two targeting PRRSV-1 and three targeting PRRSV-2,were screened and *** develop two competitive ELISAs(cELISAs),the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA,to detect common antibodies against PRRSV-1 and-2,and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA,to detect anti-PRRSV-1 *** two cELISAs were developed using PRRSV-1-N protein as coating antigen,and the amounts for both were 100 ng/*** optimized dilution of testing pig sera was 1:20,the optimized reaction times were 30 min,and the colorimetric reaction times were 15 ***,the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6%and 35.6%,*** of them have high sensitivity,strong specificity,good repeatability,and *** addition,for the 1534 clinical pig sera,an agreement rate of 99.02%(Kappa values=0.97)was determined between the g1-2-cELISA and the commercial IDEXX ELISA *** the g1-cELSIA,it can specifically detect anti-PRRSV-1 antibodies in the clinical pig ***,combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and-2.

关键词： PRRSV Competitive ELISA nanobody Antigen epitope

Fast screening of nanobody and AIV detection

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生物物理学报 2009年第S1期25卷 502-503页

作者： Bin Mu ,Jianbing Zhang ,Xiyun Yan CAS-UT Joint Lab of Structural Virology and Immunology,Institute of Biophysics,Chinese Academy of Sciences Institute for Biological Sciences,National Research Council of Canada

nanobody is the variable region of heavy chain antibody, with obvious characteristics such as small size, good stability and solubility. Many useful nanobodies have been isolated by

关键词： nanobody fast screening AIV biosensor