Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

Abstract

The present study describes the cloning and expression of ORF-2 region of TTV genome and the use of expressed peptide in developing immunoassay for detection of anti-TTV antibodies in serum. Presence of TTV-DNA in serum was detected by PCR amplifying N-22 region of ORF-1 of TTV genome. This was followed by genotyping of TTV by RFLP using N-22 amplicon. Using genotype-1 positive serum as the source of TTV, the ORF-2 region was amplified by PCR and subsequently cloned and expressed in pET-19b vector. The expressed protein, identified as 20 kDa protein on SDS-PAGE gel, was purified by affinity chromatography and then used as antigen to develop western blot assay for detection of anti-TTV antibodies in serum. Analysis of sera for anti-TTV antibodies and their comparison with presence of TTV-DNA, produced encouraging results. There was a good relation between presence of anti-TTV and TTV-DNA in these sera samples. Anti-TTV antibodies could be detected in all TTV-DNA positive sera irrespective of the presence of TTV-genotype. This investigation demonstrates that ORF-2 peptide may be used in developing immunoassay for identification of TTV infection.

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

“…Serum from a known TTV-positive sample was tested and this produced a clear spot on the strip after development with the DAB substrate solution. No spot was observed on the strip when a known TTV-negative sample [32,30] was used in the assay as the primary antibody. The present assay was used to detect anti-TTV antibodies in a panel of sera from patients with liver and renal diseases as well as healthy individuals.…”

“…Although similar attempts were made in the past using various subgenomic regions of TTV-DNA, including ORF-1 [40,43,44] and ORF-2 [15,28,32]; however, we envisaged that N22, being a conserved region, could be a better subgenomic region for use in the development of immunoassay. Furthermore, all of the studies reported so far are based on genotype 1 since it is highly prevalent globally.…”

“…The reaction contained digested vector and digested insert DNA in a 1: 3 ratio along with 3 Weiss units of T4 DNA Ligase (Promega Corporation, USA) and was incubated overnight at 16 ° C. The resulting recombinant plasmids were transformed into E. coli DH5α-competent cells (Invitrogen, USA) via the heat shock method and screened on LB-agar medium plates containing 50 μg/mL kanamycin (Sigma-Aldrich, USA). Insertion was confirmed by colony PCR and nucleotide sequencing, as described in our previous publications [30][31][32]. Database searches were performed using the BLAST program (www.ncbi.nlm.nih.gov/BLAST) of the National Center for Biotechnology Information (NCBI).…”

“…Each strip was coated with 4 µg purified protein as antigen. After blocking the strips overnight at 4 ° C in 3% BSA, dilutions of human sera in PBS (1: 500; 1: 1,000; 1: 2,000; 1: 3,000; and 1: 5,000) were used as the primary antibody and incubated at room temperature for 2 h. A known TTV DNAnegative serum sample [30,32] was used in the negative control strip. After washing the strips in PBS, they were incubated for 2 h at room temperature in goat-raised anti-human IgG-HRP-conjugated secondary antibody (1: 4,000; Santa Cruz Biotechnology).…”

“…The results of the dot blot assay were compared to detection of TTV-DNA by heminested PCR in the same panel of sera using the NG059, NG061, and NG063 primers, as described in our previous reports [32,36]. For this, viral nucleic acids were extracted from 200 µL sera from the patient group as well as healthy controls using a High Pure Viral Nucleic Acid kit (Roche Applied Science, Germany) following the manufacturer's instructions.…”