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Protein Microarray Detection Methods and Analysis

Protein and Antibody Microarrays

Detection Methods and Analysis for Protein and Antibody Chips


Detection Methods and Non-specific Binding
            Non-specific binding to the array needs to be minimized and this is typically done by immersing the arrays in a bovine serum albumin based buffer (BSA) (31).
            Analyte-binding and retention on protein arrays proceeds via thermodynamically driven binding mechanism similar to the hybridization of nucleic acid targets to probes.  However, the detection of bound targets to proteins is considerably more complex than that of DNA microarray detection (9).  Currently a variety of detection methods are being examined.  For example, ELISA was first used to detect proteins for both filter arrays (66,67) and glass arrays (68).  ELISA based detection methods have the disadvantage of non-specificity of protein-antibody interactions, leading to many false positives.   Radioisotope labeling was used by Ge et al. (22), radioisotope labeling to study protein–protein, protein–DNA, protein–drug interactions on filter arrays.  Zhu et al. used radioisotope labeling to conduct kinase assays of different substrates by using purified yeast kinase proteins on a array (36).  The preferred method of detection is fluorescence detection because these methods are generally safe, extremely sensitive, simple and can have very high resolution.  These detection methods are also compatible with standard microarray scanners. Generally, a chip is either directly probed with a fluorescent molecule (e.g. a fluorescently labeled protein or small molecule, by using a tagged probe (e.g. biotin), which can then be detected in a second step using a fluorescently labeled affinity reagent (e.g. streptavidin) (16,56). Another fluorescent labeling method is rolling circle amplification (RCA), which is also extremely sensitive (32).  
            Although the proteomes under comparison can be labeled in a comparable fashion with fluorophores, the reproducibility of these chemical reactions is poor and interference with the protein-antibody interactions presents an additional complexity (9). Also, non-uniform labeling of proteins can be addressed by performing a dual-colour ratiometric assay, where an internal standard is present for each target protein which is measured (9).          A disadvantage of labeling proteins with fluorophores is a reduction of the quantitative accuracy of the assay, as incorporation of the label may alter the binding properties of the proteins (9).

Although direct protein labeling detection methods are still widely used, the intrinsic problems mentioned has resulted in the increasing use of label free detection methods for protein microarrays.  These methods are mass spectrometry (MS), atomic force microscopy (AFM) (70), and surface plasmon resonance (SPR) (71). 
Non-labeling methods have advantages as a direct detection approach for antibody microarrays since labeling molecules affects protein activity. SELDI (surface-enhanced laser desorption/ionization) mass spectrometry has been used to detect low-density arrays of captured proteins (69). Proteins are captured on a metal surface array (SELDI protein array) and are vapourized using a laser beam.  Analysis using mass spectrometry data is then performed in order to reveal the identities of these proteins.

            Atomic force microscopy (AFM) method uses surface topological changes to identify the captured proteins on an antibody array (70).  When rabbit IgG is immobilized on a gold surface and binds to its complimentary antibodies, goat ant-rabbit IgG, AFM detects the increase in height, and thus is able to measure binding interactions.  However in order to study the kinetics of antigen–antibody interactions, real-time detection methods will be useful.  Surface plasmon resonance (SPR) has matured into a versatile detection tool to study the kinetics of receptor–ligand interactions with a wide range of molecular weights, affinities and binding rates (72-74). Commercial SPR chips are available however their detection resolution is limited.  A sensor surface with 64 individual immobilization sites in a single flow cell was developed (75).  An antibody array biosensor was also developed to study the kinetics of antigen binding using a planar waveguide as the detection method. Using this method, the group demonstrated that significant signal intensity could be achieved from spots as small as 200 mm in diameter. It is therefore expected that this approach will be suitable for high-throughput and parallel kinetics studies. (76).

Range of Detection
            Another difference between protein and DNA microarrays is that protein concentrations in a single biological sample or cells are several orders of magnitute greater than that for mRNAs.  Thus protein chip detector systems must have a very large range of detection operation – up to a factor of 1014, compared to 104 for mRNA.  Thus an antibody with nanomolar affinity to a particular target will be saturated by the presence of this target at micromolar concentrations and will fail to detect pico- or femtomolar target levels.  Thus accommodating rare and abundant proteins will probably require separate arrays (7,8,9).
            Multiple antibodies with varying affinities for the target may be positioned at different areas of the array however studies have shown that only 20% of arrayed antibodies provide measurements of proteins at low concentrations (33). 



Next: Protein Production for Protein Arrays

References for Protein and Antibody Microarrays

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Introduction and Background to Protein Chips and Antibody Chips.

Types of Antibody and Protein Chips



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