ANTIGEN CAPTURE ARRAYS
Antigen Capture Microarrays harness the power of ELISA Assays with the added dimensions of multiplex capability, small volume fluidic sampling, and very high sensitivity and dynamic range. The overriding performance driver for antigen capture microarrays is the functional integrity of the capture ligand on the surface of the assay device, in terms of both the structural functionality and the density of bound material on the microarray feature. Both of these functional requirements are exemplified by AVID, NOVA, and SuperNOVA porous Nitrocellulose microarray slides.
Porous Nitrocellulose membrane slides bind up to 500x more protein per unit area than two-dimensional microarray surfaces such as epoxysilane or aminosilane. Higher protein binding provides higher local concentration of binding sites and ultimately higher signal to noise. The binding interaction is non-covalent, reducing the chance that covalent binding will cause antibody inactivation. The 3-dimensional structure of porous nitrocellulose provides a multidentate ionic binding scaffold, resulting in cooperative binding interactions which are stable to conditions of antigen capture assays.
The 3-dimensional matrix of Porous Nitrocellulose film creates a binding scaffold in which the tertiary structure of proteins is not stressed, allowing maintenance of native conformation and structure-function activity- an important consideration for antigen capture arrays in which specific binding of antibodies is dependent on proper native structure. In contrast, 2-dimensional microarray surfaces tend to cause disruption of native structure resulting in a lower fraction of active binding sites, decreasing the binding activity of capture antibodies and lowering overall signal and signal-to-noise.
Antigen microarrays allow comprehensive screening of patient or other blood-derived samples for antibody responses to panels of known or potential antigenic proteins. With the advent of complete genomic sequencing of bacterial and viral agents, it is now possible to express virtually every protein encoded within an organism’s genome, which in turn allows simultaneous screening for antigenic reactivity from samples of known or unknown disposition. This opens the potential to unlock disease pathway mechanisms, reveal details of immune responses and resistance, identify antigens suitable for vaccine development, and to create new types of diagnostic technologies for many infectious conditions currently without effective means of diagnosis.
Advantages of Nitrocellulose Films
Porous Nitrocellulose Microarray Slides bind up to 500x more protein in a given spot than planar microarray substrates, resulting in a much higher capacity to capture analyte from the sample solution. Further, the non-covalent nature of protein binding to nitrocellulose and the 3-dimensional support matrix results in a protein friendly environment, allowing maintenance of native protein structure and retention of structure dependent binding activity. Nitrocellulose microarray slides are compatible with existing array printing systems and processes and can be used in multi-chamber isolation systems to perform multiple assays simultaneously for the highest value and throughput processing.
Fluorescence detection of recombinant cytokines by microarray sandwich assay with Qdot 800. Microarrays were printed on ONCYTE NOVA Porous Nitrocellulose Films, blocked with Super G Blocking Buffer, and assayed for recombinant cytokine spiked in assay buffer. Results are n=8 replicate spots per dilution for 3 microarrays. Identical results were obtained with normal mouse serum.
Antibody pairs are components of R&D Systems Quantikine® Sandwich ELISA Kit (MAB400/BAF400 and MAB406/BAF406).
