Serological determination of hepatitis C virus subtypes 1a, 1b, 2a, 2b, 3a, and 4a by a recombinant immunoblot assay

Abstract

Serological determination of hepatitis C virus (HCV) subtypes has been hampered by the lack of suitable assays. Therefore, a recombinant immunoblot assay has been established for serological differentiation of HCV subtypes 1a, 1b, 2a, 2b, 3a, and 4a. It consists of recombinant HCV proteins from the NS-4 region propagated in Escherichia coli. To confirm the serotyping assay results, the results were compared with those obtained by nucleotide sequencing of the NS-5 region. Sera from 157 patients with chronic HCV infection were examined by this assay, and specific antibodies could be detected in 86% (n = 135) of them. The HCV genotype was determined correctly in all but one sample, and the subtypes determined by the serotyping assay corresponded to the HCV subtypes detected by nucleotide sequencing for 95% (n = 128) of the samples. These data indicate that HCV subtypes can be distinguished serologically. The assay that is described provides an easier means of identification of infection with different HCV subtypes for wider clinical and epidemiological applications.

FIG. 1

Comparison of amino acid sequences of recombinant proteins used for NS-4 IBA. Bars indicate identical amino acids between subtypes 1a and 1b and between subtypes 2a and 2b. Asterisks indicate amino acids that are conserved among all isolates. Dots within the amino acid sequences indicate deletions.

FIG. 2

Pattern of reactivity by NS-4 IBA with serum samples from patients infected with different HCV subtypes, as described in the Materials and Methods section. HCV subtypes are determined by the antibody reactivities against the respective subtype-specific recombinant protein. Lane 1a/3a, a serum specimen derived from a patient who was sequentially infected with two strains with different HCV subtypes (subtypes 1a and 3a). Antibodies against both subtypes can clearly be detected. IgG, immunoglobulin G.