Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery

Abstract

Background: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules.

Methodology/principal findings: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding.

Conclusions: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers.

Figure 1. Structural characteristics of GAD65.

Location of Cys residues (orange) on the GAD65 molecule. GAD65 molecules are coupled to the biosensor using Cys101 and coupling agent/linker. Domains are coloured separately. Putative epitope regions are indicated by red ellipses. Cysteine residues are indicated in orange, showing their accessible surface area (ASA), Cys101 being the only cysteine residue accessible for coupling to the biosensor surface. The PDEA coupling agent is indicated by black sticks.

Figure 2. Difference images obtained from SPRi of antibodies binding to GAD65 immobilised at a gold/GLISS surface.

Spots (400 µm diameter) containing GAD65 protein were immobilised by spotting at the surface using Hamilton Starlet equipped with a PinTool tip and modified software. GAD1 antibody (4.8 nM in 200 µl PBS) was flowed across the surface at 20 µl/min as described in Methods and differential images obtained using the GenOptics SPRi device. The red circle illustrates the area that is generally chosen to calculate the pixel density at any given time in order to generate curves of the type shown in Figure 3.

Figure 3. Changes in % reflectivity as a function of time as GAD1 antibody reacts with immobilised GAD65 protein.

Curves were generated from calculating the change in % reflectivity in time across the spots of the sort shown in Figure 2. A) GAD1 antibody (4.8 nM) injected over the surfaces. The wtGAD65 curve was fitted to a simple binding model (red line) as described in Data Analysis to give the following dissociation (k_off) and association (k_on) values: k_off = 1.29±0.02·10⁻³ s⁻¹; k_on = 9.41±0.29·10⁵ M⁻¹s⁻¹ from which an overall equilibrium dissociation constant, K _d of 1.37±0.06 nM, could be calculated. The background curve is taken from changes in surface pixel density at equivalent areas on the surfaces not containing immobilised GAD65; B) GAD1 antibody (48nM) injected over the surfaces. The association and dissociation phases gave a poor fit to a single binding site model and were clearly not monophasic.

Figure 4. Differential binding to SPRi surfaces.

Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in , or the GLISS protocol described here. Serum (stock concentration 60mg/ml diluted to a final concentration of 0.6 mg/ml) in PBS was passed across the surfaces at 25 µl/min. The three images for each surface refer to images prior to, during and after injection of the serum. A) Differential images of classical undecanoic based SAM surfaces B) Differential images of GLISS prepared surfaces C) Changes in % reflectivity as a function of time derived from images of the type shown in A and B.