Development of novel immunoglobulin G (IgG), IgA, and IgM enzyme immunoassays based on recombinant Puumala and Dobrava hantavirus nucleocapsid proteins

Abstract

Human infections with Asian and European hantaviruses can result in hemorrhagic fever with renal syndromes of differing severities characterized by renal dysfunction and sometimes by pulmonary symptoms. For the serological detection of human infections by hantaviruses relevant for Europe, we developed monoclonal antibody capture immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays (ELISAs) based on yeast-expressed nucleocapsid proteins of Puumala and Dobrava hantaviruses. Moreover, for diagnosis of acute infections, mu-capture IgM ELISAs were established with nucleocapsid proteins expressed in Drosophila melanogaster Schneider S2 cells. The cutoff values of the ELISAs were determined by investigation of up to 500 human anti-hantavirus-negative serum samples. The specificities of the Puumala and Dobrava virus-specific IgM, IgA, and IgG ELISAs were found to be 100%. The sensitivities of these ELISAs were determined to be 100% with panels of characterized anti-Puumala or anti-Dobrava virus-positive human serum samples. In most cases, Puumala and Dobrava virus infections could be differentiated by ELISA reactivity alone, i.e., endpoint titration with homologous and heterologous antigens.

FIG. 1.

Detection of the expression of rN proteins in yeast and insect cells by immunoblotting with cross-reactive MAb 1C12 (A), PUUV-specific MAb A1C5 (B), and HTNV-specific MAb B5D9 (C). Amounts of 0.5 or 1 μg of purified, yeast-expressed rN proteins of HTNV (lane 2), DOBV (lane 3), and PUUV (lane 4) and crude lysates of Drosophila Schneider S2 cells expressing the rN proteins of HTNV (lane 5), DOBV (lane 6), and PUUV (lane 7) were subjected to 10% SDS-PAGE. As a negative control protein, E. coli-expressed, carboxy-terminally truncated hepatitis B virus core antigen was used in the immunoblots (lane 1). Prestained molecular mass markers (Bio-Rad): phosphorylase B (97.4 kDa), BSA (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa).

FIG. 2.

Determinations of cutoff values, specificities, and sensitivities for PUUV and DOBV IgG, IgA, and IgM ELISAs. (A and B) IgG ELISAs based on DOBV (A) or PUUV (B) rN antigens. For determinations of cutoff values and specificities, 503 (A) or 504 (B) sera from blood donors and patients with viral infections other than hantaviruses were used. The sensitivities were determined with 60 and 69 sera originating from DOBV- and PUUV-infected HFRS patients, respectively. (C and D) IgA ELISAs based on DOBV (C) or PUUV (D) rN antigens. For determinations of cutoff values and specificities, 112 sera each from blood donors and patients with viral infections other than hantaviruses were used. The sensitivities were determined with 29 and 23 sera originating from DOBV- and PUUV-infected HFRS patients, respectively. (E and F) IgM ELISAs based on DOBV (E) or PUUV (F) rN antigens. For determinations of cutoff values and specificities, 200 (E) and 80 (F) sera from blood donors and patients with viral infections other than hantaviruses were used. The sensitivities were determined with 35 and 88 sera originating from DOBV- and PUUV-infected HFRS patients, respectively.

FIG. 3.

Time courses of the reciprocal titers of hantavirus-specific IgM, IgA, and IgG antibodies in 17 DOBV-infected (A) and 17 PUUV-infected (B) German HFRS patients during the acute and convalescent phases, starting at the time of hospitalization.

FIG. 4.

Levels of cross-reactivity (estimated as OD) of 74 anti-DOBV-positive sera (open circles) and 35 anti-PUUV-positive sera (closed circles) from German HFRS patients in IgG ELISAs based on rN proteins of PUUV versus DOBV (A) and HTNV versus DOBV (B). For calculations of r² values as a measure of the goodness-of-fit of linear regression, SPSS 12 software (SPSS Inc., Chicago, IL) was used. P values were found always to be <0.001.

FIG. 5.

Cross-reactivity patterns of acute-phase/early convalescent-phase and convalescent-phase sera of 17 DOBV-infected (A) and 17 PUUV-infected (B) German HFRS patients in respective IgM, IgA, and IgG ELISAs.