Development of an Immunoassay for Detection of Torque Teno Virus (TTV) Antibodies Using the N22 Expression Product from TTV Genotype 2

Abstract

Aims: This study describes an immunoassay to detect anti-torque teno virus (TTV) antibodies using a peptide obtained from expression of the N22 region of TTV genotype 2. Methods: The N22 region (∼500 bp) of TTV genotype 2 was cloned in a pET-28a(+) vector and expressed in ZYM-5052 autoinduction medium. Following metal affinity chromatography, a purified polypeptide was used as an antigen for the development of an immunoassay to detect anti-TTV antibodies in human sera. Results: Recombinant protein (∼25-kDa) was obtained after 24 h of incubation at 25°C in ZYM-5052 autoinduction medium. A blot assay developed using this polypeptide as an antigen and TTV-positive sera as the primary antibody produced a distinct spot on the nitrocellulose membrane. Serum samples from 36 of 42 patients with renal disease and 29 of 48 patients with liver diseases produced a positive signal using this immunoassay. Simultaneously, 18 of 48 healthy controls were also detected to be positive for anti-TTV antibodies. These results were found to be comparable with TTV detection using PCR, and the assay showed a high sensitivity and specificity (i.e., 97.44 and 91.67%, respectively). Moreover, this assay could detect TTV infection irrespectively of the genotype, including cases of mixed infection. Conclusion: The present immunoassay using the N22 expression product may be used as an alternative to PCR to detect TTV infection in large populations.