title: Detection of neutralizing antibodies against human T-cell leukemia virus type 1 using a cell-free infection system and polymerase chain reaction.
abstract: We have developed a cell-free infection system to titrate neutralizing antibodies against human T-cell leukemia virus type 1 (HTLV-1) using the polymerase chain reaction (PCR). S+L-CCC (8C) feline kidney or U-251 MG human glioma cells were infected with a cell-free culture supernatant derived from HTLV-1-infected c77 feline cells. DNA was extracted from 8C or U-251 MG cells after incubation for 24 hr and amplified by PCR. The c77 cell supernatant gave discrete bands, whereas those of HTLV-1-positive T cells did not. When the inocula were treated with HTLV-1 antibody-positive human sera or the monoclonal or polyclonal antibody against the peptide 190-199 of HTLV-1 envelope protein gp46, the subsequent formation of HTLV-1 proviral DNA was inhibited. We determined the titers of neutralizing antibodies by densitometrically scanning the intensity of the PCR bands. These titers correlated well with those determined by the plaque assay using a pseudotype of vesicular stomatitis virus bearing the envelope antigens of HTLV-1. At high serum concentrations, many seronegative samples markedly inhibited the plating of the HTLV-1 pseudotype whereas they barely affected results obtained by PCR. Thus, the c77-PCR system can detect neutralizing antibodies against HTLV-1 even at low titers.
authors.affiliation: Department of Hygiene and Virology, Gunma University School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Haraguchi
authors.forename: D W
authors.initials: DW
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Handa
authors.forename: N
authors.initials: N
authors.lastname: Shimizu
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.forename: H
authors.initials: H
authors.lastname: Hoshino
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002415:Cats; D002460:Cell Line; D002474:Cell-Free System; D004279:DNA, Viral / Q000302:isolation & purification; D015686:Gene Products, env / Q000276:immunology; D015481:HTLV-I Antibodies / Q000032:analysis*; D015490:HTLV-I Infections / Q000175:diagnosis* / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D015458:Leukemia, T-Cell / Q000175:diagnosis; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D015682:Retroviridae Proteins, Oncogenic / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D015686:Gene Products, env; D015481:HTLV-I Antibodies; D015682:Retroviridae Proteins, Oncogenic; C055266:gp46 protein, Human T-cell leukemia virus type I