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


RESULTS
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.
Nogues
Claude
C
Dynamics of Macromolecular Complexes, Laboratoire de Biologie et Pharmacologie Appliquée, UMR 8113 du CNRS, Institut d'Alembert, Ecole Normale Supérieure de Cachan, Cachan, France.
Leh
Hervé
H
Langendorf
Christopher G
CG
Law
Ruby H P
RH
Buckle
Ashley M
AM
Buckle
Malcolm
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000276:immunology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D040081:Protein Array Analysis / Q000379:methods*; D011487:Protein Conformation; D020349:Surface Plasmon Resonance / Q000379:methods*
D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2