Enhanced detection of autoantibodies on protein microarrays using a modified protein digestion technique.
High-throughput studies to determine differential immune (humoral) response to diseases are becoming of increasing interest because the information they provide can help in early diagnosis as well as monitoring of therapeutics. Protein microarrays are a high-throughput and convenient technology that can be applied to the study of the humoral response. Proteins can be arrayed on slides and then probed with serum from different classes of patients to observe differences that may exist among autoantibodies that reflect differences in disease states. However, such studies may be difficult to interpret due to the weak overall signal response of such protein microarrays. We propose that this weak signal response is due to the physical positioning of the disease proteins that renders them sterically hindered from binding partners in the serum. In this study, we hypothesize that reducing the complexity and size of the disease proteins by chemical digestion using cyanogen bromide (CNBr) may enhance the overall signal from the humoral response and facilitate visualization of disease-specific responses in various classes of serum. A modified protein microarray methodology using CNBr digestion is presented here. The new workflow was applied to a set of 10 serum samples from healthy subjects, 10 from patients with chronic pancreatitis and 10 from patients diagnosed with pancreatic cancer and the results were compared to results obtained in the absence of CNBr digestion. CNBr digestion allowed the identification of 10 additional autoantibodies that responded to serum, 5 of which were unique to pancreatitis and cancer sera. This new methodology may increase the sensitivity of microarray studies measuring autoantibodies in serum.
Patwa
Tasneem H
TH
Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656, USA.
Wang
Yanfei
Y
Simeone
Diane M
DM
Lubman
David M
DM
D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D001323:Autoantibodies / Q000032:analysis* / Q000097:blood / Q000276:immunology; D019748:Carbon-Carbon Double Bond Isomerases / Q000032:analysis / Q000276:immunology; D045744:Cell Line, Tumor; D003488:Cyanogen Bromide / Q000737:chemistry; D006801:Humans; D026901:Membrane Transport Proteins / Q000032:analysis / Q000276:immunology; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010190:Pancreatic Neoplasms / Q000097:blood / Q000276:immunology; D050500:Pancreatitis, Chronic / Q000097:blood / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000032:analysis / Q000276:immunology; D014357:Trypsin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis / Q000276:immunology
D000941:Antigens; D001323:Autoantibodies; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments; D011506:Proteins; C118122:TIMM8A protein, human; D014157:Transcription Factors; C471624:UBE2V1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D014357:Trypsin; D019748:Carbon-Carbon Double Bond Isomerases; C085575:delta(3,5),delta(2,4)-dienoyl-CoA isomerase; D003488:Cyanogen Bromide