Sandwich antibody arrays using recombinant antibody-binding protein L

Abstract

Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a "sandwich-type" antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months.

Scheme 1. Construction of a Sandwich Antibody Array

Figure 1

Specific capture-detection antibody binding on proL-CVIApf coated slides. ProL-CVIApf coated slide was incubated with mouse anti-goat IgG (0.01–100 μg/mL) followed by treatment with Alexa 680-labeled goat anti-rabbit IgG (1.0 μg/mL). Part a: fluorescence intensities measured at excitation/detection 633/670 nm (Alexa 680-labeled goat anti-rabbit IgG, Red). Part b: plots of relative fluorescence intensities versus the concentration of mouse anti-goat IgG.

Figure 2

Variation of [proL-CVIApf]. Slides were treated with 0.001–100 μM proL-CVIApf at rt for 2 h and subsequently incubated in sequence with mouse anti-GFP IgG (55 μL, 1 mg/mL), GFP (20 μM), and DyLight 680-labeled goat anti-GFP IgG (1.0 μg/mL). Part a: fluorescence intensities measured at excitation/detection 532/526 nm (GFP) or 633/670 nm (DyLight 680-labeled goat anti-GFP IgG). Part b: plot of relative fluorescence intensity versus [proL-CVIApf].

Figure 3

Variation of [mouse anti-GFP IgG]. Slides were treated with 100 μM proL-CVIApf at rt for 2 h and incubated in sequence with mouse anti-GFP IgG (10 μL, 0.001–100 μg/mL), GFP (20 μM), and DyLight 680-labeled goat anti-GFP IgG (1.0 μg/mL). Part a: fluorescence intensities measured at excitation/detection 532/526 nm (GFP) and 633/670 nm (DyLight 680-labeled goat anti-GFP IgG). Part b: plot of relative fluorescence intensity versus [mouse anti-GFP IgG].

Figure 4

Detection of GFP with a sandwich-type antibody array. A slide was surface-coated with 100 μM proL-CVIApf and incubated in turn with mouse anti-GFP IgG (55 μL, 1 mg/mL), GFP (10 μL, 0.001, 0,01, 0.1, 1.0, 10, and 100 μM), and DyLight 680-labeled goat anti-GFP IgG (1.0 μg/mL). Part a: fluorescence with excitation/detection at 633/670 nm (DyLight 680-labeled goat anti-GFP IgG). Part b: plot of fluorescence intensity versus [GFP].

Figure 5

Comparison of epoxy-modified (blue) and an azido-modified (red) slides. Part a: slides were treated with 0.1–100 μM proL-CVIApf at rt for 2 h and incubated with mouse anti-goat IgG (55 μL, 1 mg/mL) and Alexa 680-labeled goat anti-rabbit IgG (1.0 μg/mL) at rt for 1 h. The slides were visualized and treated with Alexa 680-labeled goat anti-rabbit IgG (excitation/emission at 633/670 nm). The slides were treated with 0.1 M glycine at pH 2.6 for 40 min, and the treatment/visualization steps were repeated. Part b: plot of relative fluorescence intensity versus [proL-CVIApf].

Figure 6

Regeneration of ProL-CVIApf-coated slides. Slides were treated with 10 μM μM proL-CVIApf at rt for 2 h and incubated with Texas Red-labeled monoclonal mouse anti-GFP IgG (1 μg/mL) at rt for 1 h. Fluorescence intensities were measured by excitation/emission at 532/580 nm.

Figure 7

Multiple reuse of proL-CVIApf in sandwich-type antibody array. The slides were immobilized with 100 μM proL-CVIApf at rt for 2 h and subsequently incubated with mouse anti-GFP IgG (55 μL, 1 mg/mL), GFP (0.01–10 μM), and DyLight 680-labeled mouse anti-GFP IgG (1.0 μg/mL) at rt for 1 h. Fluorescence intensities were measured by each excitation/detection at 532/526 nm (GFP) and 633/670 nm (DyLight 680-labeled goat anti-GFP IgG).