XMRV Ab screening. Immunoblot assay of proteins of HIV-1 Env-defective mutant (HIV ∆env) and XMRV clone VP62 for screening anti-XMRV antibodies in plasma. (A) Three different amounts of viral preparations (3, 6, or 9 μl/lane indicated by black triangles) were separated by 5-20% SDS-PAGE and stained with SYPRO Ruby. Asterisks represent Gag capsid (CA) proteins: *p24 in HIV and **p30 in XMRV. (B) Sensitivity of immunoblot assay used for screening. Viral lysates (3 μl) were detected with serially diluted control antibodies. An anti-spleen focus-forming virus (SFFV) Gag mAb (clone R187, left) and anti-XMRV Env pAb (right) was used for detection of Gag or Env proteins. Concentrations of detecting limit of each antibody were 6.3 ng/ml (1:640,000) in R187 mAb and 1.1 μg/ml (1:8,000) in anti-Env pAb. H, HIV∆env; X, XMRV; CA, Gag capsid; SU, Env surface subunit; TM, Env transmembrane subunit. (C-E) Ab screening by immunoblot assay of blood donor samples (C), PC patients (D), and CFS patients (E) using 3 μl of each viral lysate. Pairs of strips were incubated with 1:100 diluted plasma from individuals. XMRV-specific reactivity of substantial intensity was defined as a positive reaction (red squares).