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J Immunol Methods. 2005 Oct 20;305(1):3-9. Epub 2005 Aug 18.

Development of a cluster of differentiation antibody-based protein microarray.

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CSIRO Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia.


Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (+/-30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP 'positive' samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.

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