Microarrays problems

Additional information on biomarker approaches and new technologies, such as microarray assays, developed to address complex pollution problems... 

Microarray data cannot be analyzed by purely brute force techniques to generate a causal model of a set of biological processes because the data represents gene expression patterns that are only correlated with temporal processes of interest in the organism. Lander (1999) comments on this problem as follows ... 

Kodadek, T. (2001), Protein microarrays prospects and problems , Chem. Biol., 8, 105 -115. 

Kusnezow W., Hoheisel J.D., Antibody microarrays promises and problems, Biotechniques 2002 33 14-23. 

Nitrocellulose and nylon membranes have been widely used in the production of macroarrays (arrays with probe sites of diameter 0.5 to 1 mm), but not so much in the production of microarrays (feature size of 25 to 200 xm) because of a lack of spot resolution (see Sect. 5.2, Spotting of Capture Probes). These membranes exhibit lateral wicking characteristics and the probe therefore tends to spread out from the point of apphcation. Casting of these membranes onto the surface of glass slides is a solution to this problem [28]. 

The protein microarray represents an emerging technology. While we have described its potential utility, several key problems remain to be overcome before this tool is fully adopted by the research and biopharmaceutical commxmities. The most likely first embodiment will be an antibody "protein-detecting" microarray. This is understandable based upon the availability and suitability of antibody libraries originally developed for ELISA. We have discussed many demonstrahons of antibody arrays in this chapter but commercial introductions (Pierce, Beckman Coulter) have been limited. 

In reality, these forays represent miniaturization of the standard sandwich ELISA to attain higher throughput assays by multiplexing a limited number (<50) of analytes, e.g., cytokine panels. Even at these low densities, quantification problems arise in part due to a lack of robustness in the printing process and also in the selection and stability of monoclonal antibodies that must be highly specific and of high binding affinity to be useful for microarrays. 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Young You can ask not only whether you have oscillation, but also whether the phase fits what you see in your microarray. And you can use the mutants to gain insight into the problem. You can ask what the response of the mutants is. As you gather additional pieces of information, your case gets either stronger or weaker. 

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