Serum profiling using protein microarrays to identify disease related antigens

Abstract

Disease related antigens are of great importance in the clinic. They are used as markers to screen patients for various forms of cancer, to monitor response to therapy, or to serve as therapeutic targets (Chapman et al., Ann Oncol 18(5):868-873, 2007; Soussi et al., Cancer Res 60:1777-1788, 2000; Anderson and LaBaer, J Proteome Res 4:1123-1133, 2005; Levenson, Biochim Biophy Acta 1770:847-856, 2007). In cancer endogenous levels of protein expression may be disrupted or proteins may be expressed in an aberrant fashion resulting in an immune response that bypasses self tolerance (Soussi et al., Cancer Res 60:1777-1788, 2000; Disis et al., J Clin Oncol 15(11):3363-3367, 1997; Molina et al., Breast Cancer Res Treat 51:109-119, 1998). Protein microarrays, which represent a large fraction of the human proteome, have been used to identify antigens in multiple diseases including cancer (Anderson and LaBaer, J Proteome Res 4:1123-1133, 2005; Disis et al., J Clin Oncol 15(11):3363-3367, 1997; Hudson et al., Proc Natl Acad Sci U S A 104(44):17494-17499, 2007; Beyer et al., J Neuroimmunol 242:26-32, 2012). Typically, arrays are probed with immunoglobulin (Ig) samples from patients as well as healthy controls, then compared to determine which antigens (Ag's) are more reactive within the patient group (Hudson et al., Proc Natl Acad Sci U S A 104(44):17494-17499).

Fig. 1

Cross-sectional diagram of a typical protein array that has been probed with an Ig sample and fluorescently labeled secondary antibodies to the Ig and common epitopes of the arrayed proteins. The glass surface of the array is shown in blue and the nitrocellulose surface coating in gray. Proteins are represented in black. Bound Ig's are depicted in brown, while the anti-Ig detection antibodies are green and the anti-epitope detection antibodies in red. There is a serum response to the protein on the left, while the protein to the right is not reactive with Ig's in the sample. Both proteins are recognized by the anti-epitope detection antibody as both contain the same fusion tag (e.g., GST)

Fig. 2

Panel a shows a protein microarray with 9,000+ full length GST tagged human proteins spotted in duplicate that was probed with sera from a patient with multiple myeloma. Human IgG signals were detected with an Alexa Fluor 555 anti-human IgG secondary antibody and GST-protein content was detected with an Alexa Fluor 647 anti-GST antibody. IgG signal is shown in green and GST in red. The yellow highlighted block is shown in panel b. IgG and GST reactive protein antigens are visible and dilution series of human IgG and anti- human IgG capture antibodies are shown in red blocks. Reactivity profiles of the antigens in the white block are shown in panel c. Spot reactivity is shown in order from left to right. Panel d shows the same block from a corresponding array that was probed with sera from a healthy individual. An antigen that shows differential reactivity from the myeloma serum probing is shown in the blue block