Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

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.

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

“…Nanobodies are gaining a lot of interest in diagnostics, therapeutics, and basic research. They have been used for virus detection of HIV (30,31), vaccinia virus (32), Marburg virus (33), and Dengue virus (34). Nanobodies were shown to block virus attachment to cellular receptors in HIV and influenza virus (35)(36)(37).…”

Development and multi-use applications of dengue NS1 monoclonal antibody for early diagnosis

“…Several studies have been reported using the NS1 antigen detection method for the early diagnosis of dengue virus infection, however still the sensitivity and competency of NS1 antigen based diagnosis need more evaluation. 34,35 The sensitivity of our mAbs (D5, E4 & E7) were up to 0.0625 mg mL À1 on NS1 antigen detection.…”

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

“…After four rounds of panning on recombinant DENV 2 NS1 protein, 20 positive clones were selected. Affinity measurements with NS1 revealed a K D value of 2,79 × 10 −8 M for the best VHH antibody P2 [33].…”

“…Libraries originating from different species have been successfully employed to isolate virus specific antibodies in the past. Among others, libraries were constructed from macaque [105], mouse [68], chimpanzee [39], llama [33] and human origin [120].…”

“…Most commonly virus specific antibodies have been isolated from libraries in scFv format [41,131] but Fab libraries [132,134] and VHH libraries [33] were also successfully used.…”