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

Nogues C, Leh H, Langendorf CG, Law RH, Buckle AM, Buckle M - PLoS ONE (2010)

Bottom Line: 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.Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules.Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods.

View Article: PubMed Central - PubMed

Affiliation: Dynamics of Macromolecular Complexes, Laboratoire de Biologie et Pharmacologie Appliquée, UMR 8113 du CNRS, Institut d'Alembert, Ecole Normale Supérieure de Cachan, Cachan, France.

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.

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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.
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pone-0012152-g002: 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.

Mentions: The biochip surface that we have developed (Nogues et al., submitted) consists of a 50 nm thick gold surface functionalised with hydroxyl-terminated tetra (ethylene glycol) (EG4-OH) to which the proteins had been linked via cysteine residue 101 on GAD65 (Figure 1). Typical images of the interaction of the antibody (4.8 nM) as it flows across the spots containing the immobilised GAD65 protein are shown in Figure 2. The images are differential images and are taken every two seconds during the experiment. The background is low and this is also evident from curves in Figure 3A showing the % reflectivity changes for the wild type protein GAD65 containing spot and background spot as antibody passes over the surface. At relatively low concentrations of the antibody (4.8 nM) a simple binding curve was observed (Figure 3A) from which apparent rate constants (shown in the legend to Figure 3) could be calculated. At these concentrations of antibody the protein GAD65-antibody interaction gave an overall apparent equilibrium dissociation constant () of 1.37 nM. We note that these values are somewhat different from values reported previously in the literature for this type of interaction [18]; we consistently found lower equilibrium dissociation constants essentially due to more rapid association rates that we measure here. There may be many explanations for this related to the difference in the immobilisation procedure, the architecture of the SPR machine, and so forth. However we were surprised to note that at higher concentrations of antibody (48nM) the binding phase became multiphasic or at least biphasic (Figure 3B). This observation was clearly not due to non-specific binding to the surface (Figure 3B shows a difference curve after subtraction of background), and indeed the observation of multiphasic binding is due precisely to the fact that we have very low non-specific binding to non target areas on the surface. We note that the concentrations of anti-GAD antibody used in the Biacore study of [18] were in excess of 500nM, which is 10 to a 100 fold in excess of the values that we use in the current manuscript and that this strongly suggests that the values obtained in [18] were somehow influenced by a degree of non specific binding, if not necessarily to the surface then perhaps to an abnormal form of the immobilised GAD.


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

Nogues C, Leh H, Langendorf CG, Law RH, Buckle AM, Buckle M - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921342&req=5

pone-0012152-g002: 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.
Mentions: The biochip surface that we have developed (Nogues et al., submitted) consists of a 50 nm thick gold surface functionalised with hydroxyl-terminated tetra (ethylene glycol) (EG4-OH) to which the proteins had been linked via cysteine residue 101 on GAD65 (Figure 1). Typical images of the interaction of the antibody (4.8 nM) as it flows across the spots containing the immobilised GAD65 protein are shown in Figure 2. The images are differential images and are taken every two seconds during the experiment. The background is low and this is also evident from curves in Figure 3A showing the % reflectivity changes for the wild type protein GAD65 containing spot and background spot as antibody passes over the surface. At relatively low concentrations of the antibody (4.8 nM) a simple binding curve was observed (Figure 3A) from which apparent rate constants (shown in the legend to Figure 3) could be calculated. At these concentrations of antibody the protein GAD65-antibody interaction gave an overall apparent equilibrium dissociation constant () of 1.37 nM. We note that these values are somewhat different from values reported previously in the literature for this type of interaction [18]; we consistently found lower equilibrium dissociation constants essentially due to more rapid association rates that we measure here. There may be many explanations for this related to the difference in the immobilisation procedure, the architecture of the SPR machine, and so forth. However we were surprised to note that at higher concentrations of antibody (48nM) the binding phase became multiphasic or at least biphasic (Figure 3B). This observation was clearly not due to non-specific binding to the surface (Figure 3B shows a difference curve after subtraction of background), and indeed the observation of multiphasic binding is due precisely to the fact that we have very low non-specific binding to non target areas on the surface. We note that the concentrations of anti-GAD antibody used in the Biacore study of [18] were in excess of 500nM, which is 10 to a 100 fold in excess of the values that we use in the current manuscript and that this strongly suggests that the values obtained in [18] were somehow influenced by a degree of non specific binding, if not necessarily to the surface then perhaps to an abnormal form of the immobilised GAD.

Bottom Line: 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.Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules.Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods.

View Article: PubMed Central - PubMed

Affiliation: Dynamics of Macromolecular Complexes, Laboratoire de Biologie et Pharmacologie Appliquée, UMR 8113 du CNRS, Institut d'Alembert, Ecole Normale Supérieure de Cachan, Cachan, France.

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.

Show MeSH
Related in: MedlinePlus