Serodia htlv 1

Plasma samples were evaluated for STLV-1-specific antibody titers (ABTs) with a particle agglutination (PA) assay using Serodia-HTLV-1 (Fujirebio Inc. Tokyo, Japan) as previously described [52 (link)]. The plasma cut-off titer was a 1:16 dilution. In case of the macaques whose blood samples were positive for the antibody while negative for the proviral DNA were retested for the PA assay with and without absorption procedure according to the manufacturer’s instructions. Only the individuals whose antibody positivity was confirmed by the absorption procedure were indicated as positive for the antibody while negative for the proviral DNA.

HTLV-1 antibodies in plasma were first detected by a particle agglutination (PA) technique (Serodia HTLV-1, Fujirebio, Tokyo, Japan) at the Institut Pasteur (IP) in New Caledonia and then transferred to the IP in Paris for serological confirmatory assays. An indirect immunofluorescence assay (IFA) using the HTLV-1 and HTLV-2 transformed cell lines MT2 and C19 respectively, was performed. Anti-HTLV-1 antibody titers were determined by successive 2-fold dilutions. All positive samples were further tested by Western blot (WB) assay (HTLV-I/II Blot 2.4, MP Diagnostic, Illkirch, France). A sample with reactivity to the two gag proteins (p19 and p24) and both env-coded glycoproteins (the HTLV-1 recombinant gp46-I peptide [MTA-1] and the HTLV-1/HTLV-2 recombinant [rGD21] protein) was considered to be positive for HTLV-1 antibodies.

Buffy coats and plasma were shipped to the National Serology Reference Laboratory (NRL), Melbourne, in batches at intervals during the study. HTLV-1 status was confirmed by Western blot analysis (WB) (HTLV-I/II Blot 2.4, MP Diagnostics) on samples reactive on screening either by enzyme immunoassay (Murex HTLV-I + II, DiaSorin, Italy) or particle agglutination assay (Serodia HTLV-1, Fujirebio, Tokyo, Japan). HTLV-1 PVL was determined by polymerase chain reaction (PCR) using DNA extracted from PBBC cells. Primers and probes that targeted a highly conserved region at the 5′ end of the gag gene in the p19 coding region of HTLV-1c (Mel5; accession number, L02534) were expressed as HTLV-1 copies per 105 peripheral blood leucocytes (PBL). Consistent with previous studies [25 (link)], HTLV-1 PVL was stratified as low (<1000 copies per 10⁵ PBL) and high (≥1000 copies per 10⁵ PBL). The lower limit of detection was 6.5 copies for HTLV-1 (95% confidence interval [CI], 5.4–8.4) [18 (link)]. HTLV-1 infection was defined as a positive WB test or a positive HTLV-1 PCR test.

Peripheral blood mononuclear cells, serum and CSF samples were prepared as described previously (Sato et al., 2013 (link)). Briefly, PBMCs were isolated with standard procedures using Pancoll^® density gradient centrifugation (density 1.077 g/mL; PAN-Biotech GmbH, Aidenbach, Germany). Serum was obtained from venous blood samples by centrifugation after clotting. CSF was obtained by lumbar puncture. A small amount of CSF was used for routine laboratory tests, which included total protein, glucose, and cell counts. The remaining CSF was aliquoted into cryotubes and stored at -80°C until undergoing further analysis. The serum concentration of soluble IL-2 receptor (sIL-2R) was determined using a chemiluminescent enzyme immunoassay (LSI Medience Corporation, Tokyo, Japan). HTLV-1 proviral load was measured using real-time PCR, following DNA extraction from PBMCs, as previously described (Yamano et al., 2002 (link)). The anti-HTLV-1 antibody titer in CSF was determined using the gelatin particle agglutination test (Serodia-HTLV-1; Fujirebio, Tokyo, Japan). CSF neopterin level was measured using high-performance liquid chromatography at a commercial laboratory (SRL Inc., Tokyo, Japan). CXCL10 in CSF was measured using a cytometric bead array (BD Biosciences, Franklin Lakes, NJ, United States).

The anti-HTLV-1 Ab titers in plasma and CSF were determined by the particle agglutination method using Serodia HTLV-1 (Fuji Rebio, Inc.). Antigen-specific antibodies anti-Gag Ab and anti-Env Ab in plasma samples at 1:100 dilutions were confirmed by the Inno-LIA HTLV I/II Score line blot assay (Fuji Rebio, Inc.).

Antibody screening for HTLV-1/2 was performed by particle agglutination technique (SERODIA-HTLV-I, Fujirebio, Tokyo, Japan) and/or by enzyme-linked immunosorbent assay (ELISA) (Murex HTLV-I+II, Abbott Laboratories Argentina, Buenos Aires, Argentina or HTLV I&II Ab v. ULTRA, Dia.Pro, Milan, Italy). Reactive samples were subjected to WB confirmation (HTLV blot 2.4, Genelabs Diagnostics, Science Park, Singapore). A WB was scored as HTLV-1 or HTLV-2 positive, untypeable, indeterminate, or negative according to the manufacturer’s criteria.
For molecular confirmation of indeterminate or HTLV-positive samples by WB, DNA was extracted from peripheral blood mononuclear cells (PBMCs) by column extraction (ADN PuriPrep-S kit, Highway^®, Inbio, Tandil, Argentina) and analyzed with ‘‘in-house’’ n-PCR for HTLV-1 and 2 pol and tax regions as previously described [19 (link),20 (link)]. PCR was considered positive when amplicons from at least one amplification reaction were clearly detectable following agarose gel analysis [11 (link)]. Samples with non-reactive results by PA or ELISA were also further confirmed by n-PCR in order to avoid misdiagnosis in patients on retroviral treatment (because of other infections) and immune-compromised patients.

Anti-HTLV-1 antibodies were examined by line immunoassay (INNO-LIA HTLV I/II; Fujirebio) and by particle agglutination assay (Serodia HTLV-I; Fujirebio). Plasma samples were heat inactivated at 56°C for 30 min. In INNO-LIA, 10 μL of heat-inactivated plasma samples were dispensed to 1 mL of kit-provided diluent and incubated with HTLV-1 antigen-coated strip overnight, followed by incubation with kit-provided conjugate solution and substrate solution. Then, the reaction was stopped, and the scores for relative levels of antibodies specific for individual antigens (Gag [p19 and p24] and Env [gp46 and gp21]) were determined based on the band (line) intensity according to the manufacturer's instructions. In Serodia, heat-inactivated plasma samples were serially diluted with kit-provided diluent and mixed with sensitized particles. Two hours later, the patterns of particles were evaluated. Endpoint titers were determined according to the manufacturer’s instructions. The lower limit of detection is a 1:16 dilution.