Detection of antibodies to U.S. isolates of avian pneumovirus by a recombinant nucleocapsid protein-based sandwich enzyme-linked immunosorbent assay

Abstract

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the approximately 47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detection of APV/US antibodies in turkey sera.

FIG. 1

Western immunoblot analysis of recombinant N protein and partially purified APV with hyperimmune antiserum to N protein raised in rabbits. Recombinant N protein (lanes 1 and 2) and partially purified APV proteins (lanes 3 and 4) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with a 1:20,000 dilution of N-protein-specific rabbit hyperimmune serum or normal rabbit serum, followed by incubation with a 1:5,000 dilution of anti-rabbit IgG horseradish peroxidase. Immunoreactive bands were visualized with the tetramethylbenzidine substrate system. While hyperimmune N-protein-specific antisera detected a single band with an M_r of ≈47,000 in lanes containing N and APV proteins (lanes 1 and 3, respectively), no reaction was observed with N or APV proteins by using preimmune rabbit serum (lanes 2 and 4, respectively).

FIG. 2

Sandwich N-ELISA showing reactivity with different subgroups of avian pneumovirus. Twofold serial dilutions of antiserum against APV subgroups A, B, and C were tested by the N-ELISA as described in the text. The cutoff value of the absorbance at 490 nm for a positive result was 0.15.

FIG. 3

Western immunoblot analysis of turkey sera suspected of being infected with APV by using recombinant N protein (A) and partially purified APV proteins (B). Nitrocellulose membrane strips were reacted with sera from two turkeys that were APV antibody positive by routine ELISA but negative with the capture N-ELISA system (lanes 1 and 2, respectively) and with known APV-positive and -negative sera (lanes 3 and 4, respectively). The results demonstrate that the two samples positive by the routine ELISA do not show any evidence of APV-specific antibodies and thus have false-positive results.