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Protein Microarray Capture Molecules and Their Limitations

Protein and Antibody Microarray Chips

Capture Molecules and Their Limitations - Protein Chips

Capture Molecules and their Limitations
            The most common form of analytical protein arrays are antibody microarrays in which antibodies (or antibody mimics) that bind specific antigens are arrayed on a glass slide at high density. A lysate is passed over the array and the bound antigen is detected after washing. The biggest challenge with these methods is producing reagents that identify the protein of interest and with high enough specificity in a high-throughput fashion.  Although antibodies are the traditional reagent of choice for detecting proteins in complex mixtures, polyclonal sera are often not specific and are expensive to produce. Also, the conventional hybridoma method of producing highly specific monoclonal antibodies is time-consuming, laborious and costly (8).
           
Several studies using antibodies have recently been conducted despite the obstacles in obtaining specific antibodies.  In one of the largest studies to date, Sreekumar et al. spotted 146 distinct antibodies on glass to monitor the alternations of protein quantity in LoVo colon carcinoma cells. Their results revealed radiation-induced up-regulation of many interesting proteins, including p53, DNA fragmentation factor 40 and 45, tumour necrosis factor-related ligand, as well as down-regulated proteins (58).

To date, most antibody microarrays were produced with several dozen or a few hundred commercially available poly- or mono-clonal antibodies.  Although tens of thousands of antibodies are commercially available, this number is insufficient because for most proteins there are no available antibodies.  The fact that many antibodies are glycosylated and contain large protein-based supporting structures means that they often cross-react with more than one target protein.  This can contribute to a large number of false positives (9).  Thus another problem has been obtaining high-specificity antibodies.    

            One of the greatest problem with antibody arrays is specificity. Proteins are often present in a very large dynamic range (106); thus, reagents that might have high affinity for one protein, but are low affinity for another will still exhibit detection of the lower affinity protein if it is much more prevalent (9). One group investigated the ability of 115 well-characterized antibody–antigen pairs to react in high-density microarrays on modified glass slides. 30% of the pairs showed the expected linear relationships, indicating that a fraction of the antibodies were suitable for quantitative analysis (33). Many groups have been using sandwich assays to avoid this problem.  A sandwich assay is performed by spotting the first antibody on the array and then detecting the using a second antibody that recognizes a different part of the proteins. This approach dramatically increases the specificity of the antigen detection, but required that a least two high-quality antibodies exist for each antigen to be detected (8).

Next: Antibody Microarrays: Problems and Solutions

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