Figure 1.
Analysis of DENV2-rNS1 refolded protein.
(A) SDS-PAGE of refolded rNS1 protein, M: protein marker, lane 1: protein after refolding. (B) Correct refolding of rNS1 by fluorescence spectroscopy. The dashed line represents denatured protein with a peak at 355 nm whereas the solid line represents refolded protein with a peak at approximately 340 nm. The emission spectrum was recorded from 300 nm to 420 nm by fixing the excitation wavelength at 278 nm. (C) The CD of refolded rNS1. The experiment was performed at 20°C and samples were scanned at 30 nm/min.
Figure 2.
Antibody titer of monoclonal antibody (3B3) against rNS1 by indirect ELISA.
The positive control was another mouse monoclonal antibody against dengue type 2 rNS1 provided by Wondfo (Guangzhou, China). Values are the means of three replicates. Mean ± SE values with the same letter within a column are not significantly different (Student–Newman-Keuls test; P<0.05).
Figure 3.
Amino acid sequence analysis of four different strains of VHH antibodies (P2, P13, P10 and P9).
Complementarity determining regions (CDR), framework regions (FR) and dot gaps are defined and numbered according to the IMGT unique numbering system (GenBank accession number: KF193086–KF193089).
Figure 4.
Double digestion and purification of VHH antibodies.
(A) Agarose gel electrophoresis of VHH antibodies double digested with BamHI and HindIII. Upper bands of 4500 bp correspond to vector pCANTAB5E whereas lower bands of 400 bp correspond to VHH fragments of P2 (lane 1), P9 (lane 2), P10 (lane 3), P13 (lane 4), respectively, and M stands for marker. (B) 12% polyacrylamide SDS electrophoresis gel of VHH antibodies purified from E. coli BL21 using a Ni-NTA column. A single band of about 15 kDa is observed for each purified VHH: P2 (lane 1), P10 (lane 2), P9 (lane 3), P13 (lane 4); M stands for marker.
Figure 5.
SPR sensorgrams of VHH antibody P2 and monoclonal antibody 3B3.
(A) Sensorgram showing association and dissociation of ligand P2 to analytes rNS1 and recombinant HIV-p24. (B) Sensorgram showing association and dissociation of ligand 3B3 to analytes rNS1 and recombinant HIV-p24.
Table 1.
Kinetic constants for monoclonal antibody (3B3) and VHH antibody (P2) against the recombinant protein NS1.
Figure 6.
Sensitivity of antibody immobilized kits to rNS1 antigen.
Test strips were treated with different concentrations of rNS1 antigen. Upper strip (A) was VHH antibody immobilized while lower strip (B) was monoclonal antibody immobilized at each tested concentration. T and C represent test and control lines, respectively. Detection limits visually observed were 4.5 ng/ml and 9 ng/ml for VHH and monoclonal antibodies immobilized strips, respectively.
Table 2.
Sensitivity and specificity of monoclonal and VHH antibodies based on immunochromatographic tests.
Figure 7.
Sequence similarity between binding peptides and dengue type2-rNS1.
(A) Clones V1–V7 represent different binding peptides of VHH antibody (P2). All selected binding peptides were homologous to the 224HWPKPHTLW232 amino acid region of rNS1. (B) Clones M1–M5 represent different binding peptides of monoclonal antibody (3B3). All selected binding peptides were homologous to the 224HWPKPHTLW232 amino acid region of rNS1.