Evaluation of envelope domain III-based single chimeric tetravalent antigen and monovalent antigen mixtures for the detection of anti-dengue antibodies in human sera

A panel of 164 sera obtained from both dengue-endemic and non-endemic regions was pre-screened for evidence of infection by DENV, TBEV and a variety of non-flavivirus pathogens including Chikungunya virus, Plasmodium, Leptospira, and Salmonella using commercially available kits. This panel was used in indirect ELISAs to evaluate the performance of a mixture of monovalent EDIIIs, EDIII-T and b-EDIII-T as diagnostic reagents in detecting anti-DENV antibodies.

Goat anti-human IgG (γ-chain specific)-horseradish peroxidase (HRP), and goat anti-human IgM (μ-chain specific)-HRP conjugates were purchased from Calbiochem (La Jolla, CA, USA). HRP substrate 3, 3', 5, 5'-Tetramethylbenzidine (TMB) was from Sigma-Aldrich (St. Louis, MO, USA). Maxisorp polystyrene ELISA plates and immobilizer streptavidin ELISA plates were from Nunc-Thermo fisher scientific (Roskilde, Denmark). Dengue IgG/IgM capture ELISA test was from PanBio Diagnostics (Brisbane, Australia) and TBEV IgG/IgM ELISA was from Virion/Serion GmbH (Würzburg, Germany). The Advantage Pan Malaria Card test for Plasmodium LDH antigen and the Lepto IgM micro-ELISA test for Leptospira interrogans antibodies were from J. Mitra & Company Ltd. (New Delhi, India). The Typhi-Dot test for Salmonella typhi antibodies was from MBDr Diagnostics Sdn Bhd (Selangor, Malaysia) and the Insight Chik V rapid test for chikungunya antibodies was from Tulip Diagnostics Ltd. (Goa, India).

The human sera samples (n = 164) used in this study were from hospitals in India and Finland. Sera were drawn in the early symptomatic phase after informed written consent of the patients. Ethical permissions for the collection of blood samples were obtained from the ethical committees of the respective hospitals. All sera were pre-screened for anti-DENV antibodies using the PanBio Dengue IgG/IgM capture ELISA test. Eighty samples scored positive and 84 scored negative for DENV IgG whereas 81 samples scored positive and 83 scored negative for DENV IgM antibodies. Of the DENV IgM^-/IgG^- sera samples (n = 72), there was evidence of infection by TBEV (n = 18), Plasmodium (n = 6), Leptospira interrogans (n = 5), Salmonella typhi (n = 6) and Chikungunya virus (n = 4).

The monovalent EDIIIs antigens used in this study, corresponding to the four DENV serotypes, each contain 121 amino acid (aa) residues spanning DENV envelope aa296-aa416. Their expression and purification have been reported earlier [7, 8]. The design of the tetravalent antigen EDIII-T containing the EDIIIs of the four DENV serotypes, linked in tandem, its expression and purification have been described previously [5]. To produce the biotinylated tetravalent antigen b-EDIII-T, we used an in vivo method wherein the biotinyl moiety is added to a specific site enzymatically [8]. The chemical method which tends to add the biotinyl moiety in a random fashion to available acceptor groups is not quite suitable for achieving uniform and oriented binding on streptavidin coated surfaces. In order to carry out site-specific enzymatic biotinylation of the EDIII-T antigen in vivo, a biotin acceptor peptide (BAP) was cloned in frame with the amino terminus of the EDIII-T antigen and co-expressed with biotin ligase in E. coli. The engineered BAP moiety of the recombinant tetravalent antigen serves as the substrate for biotin ligase resulting in the production of in vivo biotinylated tetravalent antigen, b-EDIII-T [8].

Polystyrene ELISA plate wells were coated either with 100 μl of EDIII-T (3 μg/ml), b-EDIII-T (3 μg/ml) or a mixture of four EDIII antigens (0.75 μg/ml of each EDIII) in 0.1 M carbonate buffer, pH 9.5, and incubated overnight at 4°C. The wells were blocked with 200 μl of blocking solution (4% skim milk in 1x PBS, pH 7.2) at 37°C for 2 hours and washed 3x with 1x PBS, pH 7.2/1% Tween 20/0.5 M KCl. For DENV specific IgG detection in sera, washed wells were incubated with 100 μl patient serum diluted 1:100 in IgG assay diluent (1x PBS, pH 8.0/2.5% skimmed milk/2% polyvinyl pyrrolidone/10% goat serum/0.1% BSA/1% Tween 20/0.5 M KCl) at 37°C, for 45 minutes. Plates were washed and incubated with 100 μl of anti-human IgG-HRP conjugate (diluted 1:10,000 in IgG assay diluent) at 37°C, for 30 minutes. Next, wells were incubated with 100 μl of TMB soluble substrate at 37°C, for 15 minutes. The reaction was stopped by adding 100 μl of 1 M H₂SO₄ and the absorbance read at 450 nm. The IgM indirect ELISA was essentially similar, except for the following differences. Sera were diluted 1:80 in IgM assay diluent (IgG assay diluents lacking goat serum and KCl) and pre-incubated with Serion Rf-Absorbent as recommended by the manufacturer (Virion/Serion GmbH, Würzburg, Germany) to remove IgG antibodies before using in the assay. Bound anti-DENV IgM antibodies were detected using anti-human IgM-HRP conjugate (diluted 1:2,500 in IgM assay diluent). In some experiments, the sera were tested on streptavidin ELISA plates coated with 100 μl of b-EDIII-T antigen (250 ng/ml, in 1x PBS, pH 7.0/0.5% Tween 20/1% skim milk) for 2 hours, at 30°C, with shaking, at 200 rpm. Wells were washed and used for IgG and IgM indirect ELISAs as above. For each of the ELISAs performed, the mean absorbance of the DENV IgM^-/IgG^- serum samples (n = 72) plus three times the standard deviation was designated as the cut-off value (see footnotes to Tables 1 and 2). We used this relatively stringent cut-off to minimize the possibility of picking up false positives. Results were expressed as signal to cut-off (S/Co) ratios. Sera with S/Co ratios ≥ 1.0 were designated as positive and those with ratios < 1 as negative.

To compare the performance of the single tetravalent EDIII-T antigen with that of a mixture of the monovalent EDIII antigens in detecting anti-DENV antibodies, the S/Co values were used to calculate a ratio by dividing the S/Co value obtained using EDIII-T antigen by the S/Co value obtained using the EDIII monovalent antigen mixture. Similarly, the performance of b-EDIII-T in streptavidin-coated ELISA plates versus polystyrene plates was assessed from a ratio of the corresponding S/Co values. The significance of the relative sensitivity was assessed by obtaining the 95% confidence interval (CI) using an alpha value of 0.05, the calculated standard deviation and the sample size. Statistical calculations were performed using MS Excel 2003 software.

The reactivity of an antigen mixture comprising the EDIIIs of the four DENV serotypes towards anti-DENV antibodies was compared to that of a single tetravalent antigen incorporating all these four EDIIIs in a single molecule [5]. For this purpose indirect IgM and IgG ELISAs were performed using 164 human sera collected from endemic and non-endemic countries which were pre-characterized for the presence or absence of anti-DENV IgM and IgG antibodies using a commercial kit from PanBio Diagnostics. As the cut-off absorbance values for different antigens were different for the same class antibodies (Tables 1 and 2), results are represented as S/Co ratios to interpret the data correctly (Figure 1). The data reveal that, both the sensitivity and specificity of a physical mixture of 4 monovalent EDIII antigens in detecting anti-DENV antibodies are comparable to those obtained using the single EDIII-T antigen. In fact, the EDIII-T antigen resulted in a modest, yet significant increase in sensitivity (Table 1). The S/Co ratios for DENV positive samples tended to be slightly higher in EDIII-T based ELISAs (compare Figure 1A with 1B for IgG and 1E with 1F for IgM).

It has been suggested that specific immobilization of antigen on the ELISA plate well can improve the performance of the immunoassay compared to passive coating of antigen [9‐11]. To test if this may be true for the detection of anti-DENV antibodies using the tetravalent antigen, we used a biotinylated version of this antigen to achieve specific and oriented immobilization on streptavidin treated ELISA plates. This demonstrated that, indeed, b-EDIII-T coated on streptavidin plates was superior in sensitivity in comparison to passively coated non-biotinylated EDIII-T antigen in detecting IgG as well as IgM antibodies (compare Figure 1B with 1D for IgG; and 1F with 1H for IgM; p < 0.05 in both cases). This enhancement in assay sensitivity was not evident if the b-EDIII-T antigen was passively coated on polystyrene plates (compare Figure 1B with 1C for IgG; and 1F with 1G for IgM). The data in Table 2 show that the use of b-EDIII-T coated on streptavidin can enhance the efficiency of detection of both IgG and IgM anti-DENV antibodies, as evidenced by 30% increase in the mean S/Co values.