Abstract
BackgroundThe simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate.MethodsCandidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients.ResultMethods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly.ConclusionProtein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody signatures for diagnostic purposes.
Highlights
The simplicity and potential of minimal invasive testing using serum from patients make autoantibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease
Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins
Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing
Summary
The simplicity and potential of minimal invasive testing using serum from patients make autoantibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Proteomics techniques have shifted biomarker identification and validation research to the level of the main actual biological agents of health and disease, the proteins. The development of specific capturing agents for the candidate markers requires a costly production process and thorough validation. This ensures high avidity for the target while minimizing the risk of unspecific binding
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