Characterization of Gag CA-positive plasma samples. (A) Sensitivity of immunoblot assay with GST-fused recombinant Gag CA (GST-CA) protein. GST-CA protein (300 ng per lane) was analyzed by 5-20% SDS-PAGE and detected with serially diluted R187 anti-Gag mAb. The concentration of the detection limit was 0.78 ng/ml (1:5,120,000). (B) Immunoblot assay of plasma samples that tested positive (D7, D20, D51, D98, D183, D184, D359, D385 in blood donors; P24 and P28 in PC patients; C4 and C32 in CFS patients) or negative (D306 and D307 in blood donors) for the screening immunoblot assay with 3 μl of VP62 virus lysate (upper panel) or 300 ng of the GST-CA recombinant protein (lower panel). For positive control, 8.5 μg/mL (1:1,000) of anti-Env pAb and 0.8 μg/ml (1:5,000) of anti-Gag mAb, R187, were used. (C) Immunoblot assay using recombinant Env SU (rSU) protein of xenotropic MLV. The detection limit of 300 ng of rSU protein was 1.1 μg/ml (1:8,000) by anti-Env pAb (left). One hundred diluted plasma samples tested positive for the screening assay were negative for rSU protein (right). (D) Immunoblot assay in a native-PAGE using 5 μl of the concentrated VP62 lysate in native sample buffer. Plasma samples testing positive (D7 to C32) and negative (D306 and 307) for the screening assay were examined. α-Env, anti-Env pAb (1:200, 42.5 μg/ml); α-Gag, R187 mAb (1:80,000, 50 ng/ml). (E) MoMLV particles with (M) or without (M') amphotropic Env were produced and subjected to an immunoblot assay to examine their cross-reactivity with XMRV-positive plasma. PC, a mixture of anti-Gag mAb (R187, 0.4 μg/ml) and anti-Env pAb (8.5 μg/ml) as the positive control. Arrow head, GST-fused Gag Capsid protein; SU, Env surface subunit; rSU, recombinant Env surface subunit of xenotropic MLV; TM, Env TM subunit; CA, Gag capsid protein.