Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

Wei Ye,Xiaoning Wang,Ying Wang,Haiying Wang,Xia Jin,Jufang Wang,Keren Kang,Aneela Fatima,Liliang Xia

doi:10.1371/journal.pone.0095263

Wei Ye, Xiaoning Wang + Show 7 more

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https://doi.org/10.1371/journal.pone.0095263

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Abstract

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Highlights

Dengue infection, commonly called bone breaker illness, is mosquito (Aedes aegypti/Aedes albopictus) borne
The objectives of the present study are to screen and purify nanobodies against recombinant NS1 protein of dengue virus (DENV) type 2 from a non-immune llama (Lama glama) library; determine the binding epitopes of VHH antibodies using the phage displaying peptides library (Ph.D12 kit); and investigate the possibility of developing VHH antibodies as a diagnostic tool as an alternative to conventional monoclonal antibodies
Expression, Purification and Refolding of Recombinant NS1 Protein (rNS1) The DENV2 NS1 gene was cloned into the expression vector pET-30a (+) in the correct open reading frame

Summary

Introduction

Commonly called bone breaker illness, is mosquito (Aedes aegypti/Aedes albopictus) borne. This disease ranges across tropical and sub-tropical regions around the world, especially in Asian and Latin American countries. It was recently estimated that 50–100 million dengue infections occur each year and almost half of the world’s population lives in countries where dengue is endemic [1,2]. Infection with any of these serotypes causes clinical symptoms including dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the absence of a suitable vaccine, effective, early and rapid diagnosis against all serotypes of DENV is of significance to reduce the morbidity and mortality of DHF and DSS, especially in developing countries [3,4]. DENV contains a single open reading frame of approximately 11 kb with three structural (C, prM and E)

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