title: Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Candidate 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.
  
  
  RESULTS
  Methods 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.
  
  
  CONCLUSIONS
  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. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody signatures for diagnostic purposes.
authors.affiliation: Molecular Medicine, Austrian Institute of Technology, Seibersdorf, Austria.
authors.forename: René
authors.initials: R
authors.lastname: Stempfer
authors.forename: Parvez
authors.initials: P
authors.lastname: Syed
authors.forename: Klemens
authors.initials: K
authors.lastname: Vierlinger
authors.forename: Rudolf
authors.initials: R
authors.lastname: Pichler
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.forename: Nicole
authors.initials: N
authors.lastname: Ludwig
authors.forename: Albert
authors.initials: A
authors.lastname: Kriegner
authors.forename: Christa
authors.initials: C
authors.lastname: Nöhammer
authors.forename: Andreas
authors.initials: A
authors.lastname: Weinhäusel
mesh_terms: D000912:Antibodies, Neoplasm / Q000276:immunology; D000951:Antigens, Neoplasm / Q000737:chemistry*; D001323:Autoantibodies / Q000737:chemistry*; D001324:Autoantigens / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001932:Brain Neoplasms / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D008175:Lung Neoplasms / Q000276:immunology* / Q000378:metabolism; D008403:Mass Screening / Q000379:methods*; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D001323:Autoantibodies; D001324:Autoantigens; D014408:Biomarkers, Tumor; D011994:Recombinant Proteins