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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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Related in: MedlinePlus

Differential binding to SPRi surfaces.Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in [2], 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.
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pone-0012152-g004: Differential binding to SPRi surfaces.Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in [2], 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.

Mentions: 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 [2], and the other using the GLISS surfaces used in the current GAD65 study. As can be seen in Figure 4 there is a qualitative and quantitative difference between non-specific binding to the two surfaces with a much reduced binding to the GLISS surface. The almost negligible amount of material retained at the GLISS surface compared to the classical SAM surface is even more evident from the SPR curves derived from the images.


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)

Differential binding to SPRi surfaces.Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in [2], 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012152-g004: Differential binding to SPRi surfaces.Two independent surfaces were created either using classical SAM constructed from undecanoic acid as described in [2], 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.
Mentions: 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 [2], and the other using the GLISS surfaces used in the current GAD65 study. As can be seen in Figure 4 there is a qualitative and quantitative difference between non-specific binding to the two surfaces with a much reduced binding to the GLISS surface. The almost negligible amount of material retained at the GLISS surface compared to the classical SAM surface is even more evident from the SPR curves derived from the images.

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