Comparison of immunofluorescence and enzyme-linked immunosorbent assays for the serology of Hantaan virus infections

Abstract

Three enzyme-linked immunosorbent assay (ELISA) systems based upon different principles were developed for the serology of Hantaan virus infections and compared with an indirect immunofluorescence assay (IFA). The indirect IFA was carried out with γ-irradiated Hantaan virus-infected and uninfected Vero E6 cells fixed with ethanol (−70°C) or acetone (20°C) on drop slides and a FITC-coupled sheep anti-human Ig preparation. Atypical staining in the IFA was avoided by using ethanol (−70°C) instead of acetone (20°C) fixation. In the first ELISA (‘cell-assay’), Hantaan virus-infected or uninfected Vero E6 cells were used as antigens, which after γ-irradiation were seeded into microtiter ELISA strips. Serial dilutions of human sera were incubated and specific antibodies were demonstrated with a horseradish peroxidase (HRPO)-conjugated sheep anti-human Ig preparation. In the second ELISA (‘competition-assay’) an affinity-purified human Ig preparation was used as a capture antibody for Hantaan virus antigen. After incubation of serial dilutions of human sera with this coat, the reactivity of the affinity purified anti-Hantaan virus Ig coupled to HRPO was determined. In the third ELISA (‘complex trapping blocking [CTB]-assay’) the same capture antibody was used to react with a mixture of the antigen and serial dilutions of human sera. The reactivity with the same HRPO conjugate was then determined. The results obtained in the respective assay systems with sera from people at risk or suspected of Hantaan virus infection coincided well. The CTB-ELISA proved to be faster and more sensitive than both the other ELISA systems, without giving more non-specific reactions: it detected almost all the IFA positive samples. This was also confirmed by Western blotting analysis of sera showing discordant results in the respective assays. The sensitivity, specificity and rapidity of the CTB-ELISA makes it suitable for routine use for the serology of Hantaan virus infections of man and animals.