After blocking with 1% foetal calf serum, the samples were reacted with a 1∶10,000 dilution of group 19 typing sera (Statens Seruminstitut, Copenhagen, Denmark) for 4 h.
The samples were reacted with anti-P.
Additionally, lysine residues in a bovine serum albumin (BSA) sample were reacted with a succinimidyl-activated sydnone derivative in vitro.
After a 1-h incubation, the plates were washed, and each serum and control sample was reacted with sucrose-acetone extracted yellow fever antigen and control antigen at optimal dilution.
Additionally, human melioidosis serum samples were found to react with Hcp1 protein, consistent with our observation (data not shown).
The 15 μL serum samples were allowed to react with 120 μL Hisens auto RPR LTIA R1 (buffer) and 60 μL Hisens auto RPR LTIA R2 (latex reagent containing cardiolipin lecithin cholesterol, 1.0 mg/mL) in a CA-400 autoanalyzer (Furuno Electric Co, Nishinomiya, Japan).
Plasma or serum samples were diluted 1∶50 and reacted separately to 150 ug of infected and uninfected cell lysates after protein separation through 4 12% polyacrylamide gels and then transfered to nytran membranes, as previously described [21], [39], [40].
Patient serum samples were tested for IgG-class antibodies reacting to A. phagocytophilum and E. chaffeensis at the Mayo Clinic, as previously described (11 ).
When all the positive serum samples were rescreened by endpoint titration, 118 samples (22.0%) reacted with the GIV.1 antigen at rates of 14.5% (78/535) at dilution of 1 200 and 7.4% (40/535) at dilution of 1 400.
Human serum samples were assayed for total amidated gastrin concentrations by radioimmunoassay using antibody L2 (which reacts with G-17 and G-34 but not progastrin or Gly gastrins) and I-labeled G-17 as previously described (4).
