Conceived and designed the experiments: AMB MB. Performed the experiments: CN HL MB. Analyzed the data: CN HL AMB MB. Contributed reagents/materials/analysis tools: CN HL CGL RHPL AMB MB. Wrote the paper: AMB MB.
The authors have declared that no competing interests exist.
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.
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 ΔG, ΔS and ΔH 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.
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.
Surface plasmon resonance imagery (SPRi)
A major stumbling block to the goal of achieving high throughput, rapid, quantitative analysis of peptide microarrays using SPRi is the difficulty of eliminating non-specific interactions with the surface containing the microarray target molecules. We have devised a novel surface chemistry (Nogues
GAD65, but not GAD67, is a major autoantigen and autoantibodies to GAD65 are detected at high frequency in patients with newly diagnosed type 1 diabetes (T1D) (∼80%)
A multiplexed SPRi-based optical biosensor assay potentially offers many advantages over RIA and ELISA and has the potential to realize the high-throughput screening described above. We therefore decided to investigate the interaction between GAD65 and antibody using SPRi.
Protein chips were prepared on a glass prism (high refractive index n 1.7) activated by Reactive Ion Etching (RIE) prior to the thermal evaporation of a 50 nm gold layer as described in Nogues
The GAD65 protein was dialysed against phosphate buffered saline (PBS) in order to remove mercaptoethanol in the storage buffer and aliquots (100nL) at a final concentration of 0.5 mg/ml GAD65 were then deposited onto the fresh pre-treated prism surface using the Hamilton starlet robot and a modified pin tool protocol that minimised contact of the pin tool with the gold coated surface. Following GAD65 immobilization on the pre-treated surface via the covalent coupling of the thiol groups of the protein to the carboxylated-terminal of the surface, un-reacted groups on the surface were blocked as described in
Details of the expression and purification of recombinant human GAD65 have been published previously
The mouse mAb GAD1, prepared from a BALB/c mouse immunized with partially purified chicken brain GAD
The SPRi machine was purchased from GenOptics. The biological interface consists of a prism surface coated with a thin layer (∼50nm) of gold. An evanescent field referred to as a plasmon wave is created at the interface of this gold-coated surface and the dielectric from a light beam when the light beam arrives at the interface at an angle of total internal reflection (TIR). At TIR there is a resonance effect, leading to a decrease in reflectance at a given angle. This is measured by imaging the entire reflected light from a monochromatic polarized electroluminescent diode using a camera linked via a dedicated optical system. Thus the whole surface of the imaged field containing many discrete spots with immobilised ligands may be analysed simultaneously. A microcuvette system allows material to be flowed across the surface and the SPR response at predetermined spots can be assessed in parallel by a time resolved CCD that captures changes in percentage reflectivity at selected spots on the surface. These changes, averaged across the surface of each spot as a function of time, are related to changes in concentration of mass at each spot, and thus provide access to the kinetics of interactions at the surface at each immobilised ligand. A characteristic of this technique compared to other SPR based devices is that non-specific interactions of the molecules directly with the surface around selected spots can be simultaneously quantified and compared with specific interactions occurring with the target material in the spots, assuming that the amount of non specific interaction inside and outside the spots are identical. A recent study has shown that kinetic constants extracted from the kinetic curves collected with a Biacore SPR apparatus or from the SPRi apparatus are comparable when using identical surfaces and conditions
GAD1 antibody (200 µl at various concentrations) was flowed across the immobilised GAD65 proteins in the SPRi apparatus at 20µl/ml at room temperature in PBS buffer.
Prism software was used to fit curves and obtain apparent rate constants. Curves were fitted to a simple Langmuir binding model to obtain kinetic constants. The apparent dissociation rate constant koff may be obtained by fitting the dissociation phase to a simple exponential expression where the relative change in resonance response (R) as a function of time (t) with respect to R at time t = 0, (R(0)), results from
The biochip surface that we have developed (Nogues
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.
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
Curves were generated from calculating the change in % reflectivity in time across the spots of the sort shown in
In order to illustrate the degree of antifouling conferred by the General Liquid Interface Specific Surfaces (GLISS) we in fact passed human serum across two types of surface, one consisting of a classical self assembling monolayer (SAM) constructed from undecanoic acid as described in
Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in
The available crystallographic structure of GAD65
We clearly advocate the use of engineering surface exposed cysteines for immobilisation but recognize that this may not always be possible. A number of alternatives are available, and generally in SPR, coupling using amines, for the most part through accessible lysines, is advocated. It must be noted however, that this generally results in a reduced degree of activity of the immobilised target that may render quantitative analysis difficult. The GLISS surfaces used here may easily be functionalised with carboxyl, thiol or amine groups thus permitting a wide range of immobilisation techniques. However we would like to stress that although the expedient of engineering solvent accessible thiols is somewhat limiting it does optimise accessibility and that whilst this may restrict general applicability we strongly suggest that immobilisation strategies that aim at increasing accessibility and orientation be elaborated rather than blind immobilisation through solvent accessible amines for example. Alternatively one can immobilise on the GLISS surfaces, specific antibodies or haptens either through accessible cysteines or via other coupling techniques, that then allow mild capture of the target molecules.
Although the purpose of the present study was not to explore the detection levels of the SPRi technique we could detect and quantify GAD65-anti-GAD65 interactions between approximately 1010 molecules of target GAD65 on the surface and antibody at 4nM concentration in solution. Our limit of detection as discerned from the signal to noise ratio suggests that we can detect a change of approximately 0.01% reflectivity that corresponds to levels of detection of around 30 to 40 ng/ml of protein in solution.
We have characterised the binding of wild type GAD65 to the GAD1 monoclonal antibody using SPRi. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The implications of this are far reaching; in short not only does this approach obviate the dubious process of background subtraction but gives access to more accurate kinetic and equilibrium values that tend towards more affine measurements since any multiphase behaviour can be separated from non-specific binding. On a broader level, 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 ΔG, ΔS and ΔH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Finally, and this is particularly evident here, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. The observation that monoclonal antibodies can use multiple binding modes is intriguing. We are currently applying the technology developed here to analyse monoclonal antibody binding to microarrays containing selected mutants of GAD65 (Buckle
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.
We thank Merrill Rowley for the kind gift of GAD1 antibody.