False-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid enzyme-linked immunosorbent assay due to HCoV-OC43 and HCoV-229E rectified by Western blotting with recombinant SARS-CoV spike polypeptide

Abstract

Using paired serum samples obtained from patients with illness associated with increases in anti-human coronavirus OC43 (HCoV-OC43) or anti-HCoV-229E antibodies, we examined the possibility of false-positive results detected in a recombinant severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Three of the 21 and 1 of the 7 convalescent-phase serum samples from persons with increases in antibodies against HCoV-OC43 and HCoV-229E, respectively, tested positive by the recombinant SARS-CoV nucleocapsid protein-based ELISA. None of these samples were found to contain a specific antibody in the recombinant SARS-CoV spike polypeptide-based Western blot assay.

FIG. 1.

Recombinant SARS-CoV nucleocapsid protein-based IgG antibody ELISA for serum samples positive for the anti-HCoV-OC43 or anti-HCoV-229E antibody.

FIG. 2.

Western blot analysis of purified recombinant SARS-CoV nucleocapsid protein (A) and spike polypeptide (B) using human sera that tested positive by the recombinant SARS-CoV nucleocapsid protein-based IgG antibody ELISA. Two of the three serum samples with the anti-HCoV-OC43 antibody (lanes 1 to 3) and the serum sample with the anti-HCoV-229E antibody (lanes 4) produced very faint bands in the recombinant SARS-CoV nucleocapsid protein-based Western blot assay, but not the recombinant SARS-CoV spike polypeptide-based Western blot assays. Also shown are a positive control using serum of a patient with SARS-CoV pneumonia (lanes 5) and a negative control using serum of a healthy blood donor (lanes 6).