(A) Scheme depicting strategy to synthesize EuK-labeled 2D7 mAb (9). EuK (1) is activated by addition of a sulfhydryl by reaction with SPDP (2) and reduction by TCEP (top). Separately, 2D7 mAb (6) is immobilized on Ni-NTA resin and reacted with sulfo-SMCC (7) to generate a maleimide derivative (8). A crystal structure of IgG (PDB ID: 1IGY) is shown as a model. The two activated reagents are combined and labeled 2D7 is eluted from the resin with imidazole. (B) Left: Size-exclusion chromatography with monitoring of absorbance at 280 nm (blue; protein) and 305 nm (red; EuK) to determine yield and labeling ratio. Right: Coomassie blue-stained non-reducing SDS polyacrylamide gel electrophoresis showing a single band of the intact 2D7-EuK after labeling. Note the impurities of the initial 2D7 sample have been largely removed by the procedure.

(A) Chromatogram showing co-elution of protein (blue; 280 nm) and fluorescent lipids (red; 570 nm). The 1D4 immunoblot below shows that CCR5-NABBs elute in the peak centered at 15.6 mL, exhibiting larger hydrodynamic radius than the majority of the NABBs at 16.1 and 17.0 mL. The anti-His₆ blot detects His-tagged zap1 belt protein. Free zap1 elutes at 18.6 mL (inset). (B) CCR5-NABBs were incubated with Protein G beads and 2D7, and the supernatant fraction was probed with a 1D4 immunoblot. The majority of CCR5 in NABBs is immunoprecipitated by 2D7, indicating properly folded receptor. (C) HTRF signal from a serial dilution of CCR5-NABBs, demonstrating the ability to quantify properly folded CCR5 in NABBs. (D) The HTRF signal is efficiently competed with 1 μM 1D4, showing signal specificity.

(A) HTRF sandwich immunoassay schematic. A hypothetical model of CCR5 based on the crystal structure of rhodopsin (PDB ID: 1U19) is shown. 2D7-EuK recognizes a conformation-sensitive split epitope on the extracellular side of CCR5. Biotinylated 1D4 (1D4-biot) binds an engineered nine-residue C-terminal epitope (red) and is linked to streptavidin-conjugated XL665. FRET is observed between EuK and XL665 when 2D7 binds properly folded CCR5. The Förster radius for this donor-acceptor pair is approximately 95 Å. (B) Assay controls with fluorescence counts at 615 nm (blue) and 665 nm (red) after excitation at 320 nm. CCR5-specific signal is seen as a signal increase at 665 nm and decrease at 615 nm. (C) A serial dilution of CCR5 shows the dynamic range of the assay. The signal saturates at ∼200% enhancement over background. ΔF is defined as:
(D-F) Competition experiments with the 1D5 nonapeptide (D), 1D4 mAb (E), and 2D7 mAb (F) demonstrate signal specificity. The apparent IC₅₀ values are 130 nM, 2.7 nM, and 0.79 nM for the three competitors, respectively.

The HTRF assay was applied to make high-throughput thermal stability measurements with femtomole quantities of CCR5. A range of temperatures was applied to detergent-solubilized CCR5 or CCR5-NABBs before adding to labeled HTRF components in a 384-well plate. (A) Melting curves of unliganded CCR5 (black) and CCR5 preincubated with the small molecule antagonist maraviroc (red). Maraviroc shifts the T_M of detergent-solubilized CCR5 from 47.1°C to 66.0°C (rightward-pointing arrow) and appears to display a two-step binding profile. The first ligand-receptor state reduces the accessibility of the 2D7 epitope on the EC2 loop compared with unliganded receptor (downward-pointing arrow). The second ligand-receptor state results in a higher HTRF signal (upward-pointing arrow). (B) Thermal denaturation of CCR5-ligand complexes. CCR5 was preincubated with maraviroc (red), AD101 (green), vicriviroc (blue), and CMPD 167 (cyan). All of these antagonists appear to have two binding states. The calculated T_M for maraviroc, AD101, vicriviroc, and CMPD 167 is 66.0°C, 59.9°C, 59.5°C, and 62.7°C, respectively. The unliganded receptor, which melts at a lower temperature, is shown as a black dashed line for reference. (C) Molecular structures of the CCR5 antagonists tested. (D) Melting curve of CCR5-NABBs. The assembly denatures at 54.5°C. CCR5-NABBs melt over a much broader range than CCR5 in detergent solution, suggesting some sample heterogeneity or a distinct denaturation pathway.