Antibody responses of pigs to defined Erns fragments after infection with classical swine fever virus.
Antibody responses of pigs to defined Erns fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA). Selection of various E(rns) fragments was based on an immunodominant Erns region encompassing three overlapping antigenic regions, amino acids 65 to 145 (Erns(aa)65-145) (AR1), 84 to 160 (Erns(aa)84-160) (AR2), and 109 to 220 (Erns(aa)109-220) (AR3), identified earlier by our group (M. Lin, E. Trottier, J. Pasick, and M. Sabara, J. Biochem., in press). Defined Erns fragments, including AR1, AR2, AR3, Erns(aa)65-160 (AR12), Erns(aa)84-220 (AR23), Erns(aa)65-220 (AR123), Erns(aa)109-145 (the consensus region defined by the three overlapping regions), and Erns(aa)109-160 (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV. Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the Erns fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100%. Detection of antibody responses to refolded Erns(aa)109-145 and Erns(aa)109-160 by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational. When cutoff values were raised to give a specificity of 100%, four Erns fragments (AR2, AR23, Erns(aa)109-145, and Erns(aa)109-160) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%). Erns(aa)109-145 and Erns(aa)109-160 were capable of detecting antibody responses in infected pigs as early as 7 days postinfection. Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection. An advantage of such a test would be its utilization for serological survey in a classical swine fever-free country (e.g., Canada) in biocontainment level 2 laboratories.
Lin
Min
M
Animal Diseases Research Institute, 3851 Fallowfield Rd., Ottawa, Ontario, Canada K2H 8P9. linm@inspection.gc.ca
Trottier
Erin
E
Pasick
John
J
D000818:Animals; D000906:Antibodies / Q000097:blood*; D000956:Antigens, Viral / Q000276:immunology; D006691:Classical Swine Fever / Q000097:blood / Q000175:diagnosis*; D006692:Classical Swine Fever Virus / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D012372:ROC Curve; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods; D013552:Swine; D014759:Viral Envelope Proteins / Q000276:immunology*
D000906:Antibodies; D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins