Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides

Abstract

The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

Figure 1. Binding of phage-expressed scFvs affinity selected with GABA subunit peptides.

Equal amounts of biotinylated target peptides or non-target peptide (negative control) were captured on NeutrAvidin™ (NA) coated microtiter plate wells, and after washing the binding of equivalent amounts of phage particles, displaying different scFvs, was monitored by ELISA. A biotinylated anti-Flag antibody was used to normalize the amounts of scFv-displaying phage particles added to each well. Error bars correspond to the standard deviation of triplicate measurements of the optical density of the wells at 405 nm wavelength. A7, G8 and G11 are anti-β2 binders while A10 is the anti-α1 binder.

Figure 2. Binding affinities of anti-α1 and β2 scFvs measured using fluorescence polarization.

Purified scFv proteins were incubated with 6 or 4 µM (α1 or β2, respectively), of FITC-conjugated target peptide. Changes in polarization were measured in millipolarization (mP) units and plotted against concentration of the scFv. The K_d value of one representative trial is shown. Error bars correspond to standard deviations for duplicate measurements.

Figure 3. Immunofluorescence staining of GABA_A receptors expressed in Xenopus laevis oocytes.

X. laevis oocytes were injected with 50 nL of GABA_A receptor mRNA (A) or buffer (B), and later incubated with 70 nM anti-α1 (A10) and 100 nM β2 (A7, G8, G11) scFvs, and binding of the scFvs to the cell surfaces was detected with a mouse anti-Flag antibody conjugated to FITC. Representative images are shown for each condition; four independent replicates were performed for each condition. Scale bar depicts 150 µm.

Figure 4. Binding of scFvs to native GABA receptors in mouse retinal sections.

Immunohistochemistry of cryosections of mouse retina incubated with 600-α1 A10 scFv, (A and B), 300 nM biotinylated ZF130H1, negative control scFv (D) or 47 nM of anti-GABA_A positive control antibody (C). Streptavidin (SA)-Cy5 or anti-rabbit IgG-Cy5 was used as the secondary detection reagent. Panels A, C and D are images obtained under fluorescent light (Cy5 filter), while panel B shows the image acquired under bright light. Representative images from three independent trials are shown. Scale bar depicts 80 µm. Legend: GCL; Ganglion cell layer, INL; Inner nuclear layer, IPL; inner plexiform layer, ONL; outer nuclear layer, OPL; outer plexiform layer.

Figure 5. Comparison of detection limits of scFv and scFv-Fc.

A. Binding specificity of equimolar concentrations of scFv-Fc and scFv A10, to different biotinylated peptides was detected in an ELISA using anti-Flag antibody conjugated to HRP. Signal intensities were measured at 405 nm and error bars indicate standard deviation of duplicate trials. NA, NeutrAvidin™; negative control pep, retinal protein Pep 1 peptide; beta 2, GABA_A receptor subunit β2 peptide; rho 1, GABA_c receptor subunit ρ pep tide and alpha 1, GABA_A receptor subunit α1 peptide. B. Dot blots of varying concentrations of MBP-α1 proteins treated with 10 nM A10 scFv-Fc (top row) and scFv (bottom row). Circles around signal spots indicate pencil markings of protein addition sites. C. Signal intensities normalized to highest signal obtained for each trail are plotted against log of concentration for scFv-Fc and scFv. Error bars depict standard deviations. n = 3.