Design of r-Δgp160 and r-Trx-BAP-Δgp160 antigens and TRF assay. The left part of the figure shows a schematic representation of the native HIV-1 gp160 molecule. The black and grey boxes denote the signal sequence and the two internal hydrophobic regions in the C-terminal half of native gp160, respectively. Depicted in the middle part of the figure are the two recombinant antigens created for this study, r-Δgp160 and r-Trx-BAP-Δgp160. Both lack the signal sequence (black box) and the two internal hydrophobic regions (grey boxes) of native gp160. An additional sequence consisting of thioredoxin (Trx) and biotin acceptor peptide (BAP) was fused to the N-terminus in r-Trx-BAP-Δgp160. Both proteins were provided with 6x-His tags whose positions are indicated by the asterisks. The numbers indicate the starting and ending aa residues of the native gp160 sequences retained, and fused in-frame, in the recombinant antigens. Shown to the right is design of the double antigen sandwich assay: the r-Δgp160 and r-Trx-BAP-Δgp160 antigens were labeled with Eu3+-chelate and biotin isothiocyanate, respectively. These labeling reactions (denoted by 1 and 2) produced r-Δgp160-Eu3+ (3) and r-Δgp160-Bio (4). The biotinylated capture antigen r-Δgp160-Bio was bound to SA (6) coating the microtiter well surface (5). Serum anti-HIV-1 IgG (7) and IgM (8) that are captured in the wells are revealed using the tracer, r-Δgp160-Eu3+ (3), and visualized by excitation at 340 nm followed by time-resolved measurement of fluorescence at 615 nm.