Dataset Information

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

ABSTRACT: Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

SUBMITTER: Shriver-Lake LC

PROVIDER: S-EPMC6308234 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Publications

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

Shriver-Lake Lisa C LC Liu Jinny L JL Zabetakis Dan D Sugiharto Victor A VA Lee Cheng-Rei CR Defang Gabriel N GN Wu Shuenn-Jue L SL Anderson George P GP Goldman Ellen R ER

Scientific reports 20181227 1

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four ...[more]

PMID: 30591706

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Project description:A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Dataset Information

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

Publications

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

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