Detection of multiple proteins in an antibody-based protein microarray system.

A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL) and high-throughput of microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format. Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase (HRP)-conjugated antibody(ies). The signals are finally visualized with ECL system. Experiments demonstrate that multiple cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array system. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics and discovery of new biomarkers.

[1]  S. Parthasarathy,et al.  Vitamin E Supplementation Decreases Autoantibodies to Oxidized Lipid-Protein Complexes , 1998 .

[2]  J. Bradbury Proteomics: the next step after genomics? , 2000, The Lancet.

[3]  S. Schreiber,et al.  Printing proteins as microarrays for high-throughput function determination. , 2000, Science.

[4]  T. Yip,et al.  Bactericidal domain of lactoferrin: detection, quantitation, and characterization of lactoferricin in serum by SELDI affinity mass spectrometry. , 1998, Biochemical and biophysical research communications.

[5]  F W Chu,et al.  Multianalyte microspot immunoassay--microanalytical "compact disk" of the future. , 1991, Clinical chemistry.

[6]  E. Petricoin,et al.  An approach to proteomic analysis of human tumors , 2000, Molecular carcinogenesis.

[7]  L. G. Mendoza,et al.  High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). , 1999, BioTechniques.

[8]  N. Anderson,et al.  Proteomics: applications in basic and applied biology. , 2000, Current opinion in biotechnology.

[9]  R. Service Protein Arrays Step Out of DNA's Shadow , 2000, Science.

[10]  Paul A. Haynes,et al.  Proteome Profiling—Pitfalls and Progress , 2000, Yeast.

[11]  Y. E. Chin,et al.  Stat1 as a Component of Tumor Necrosis Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NF-κB Activation , 2000, Molecular and Cellular Biology.

[12]  Dieter Stoll,et al.  A microarray enzyme‐linked immunosorbent assay for autoimmune diagnostics , 2000, Electrophoresis.

[13]  M. Gerstein,et al.  Analysis of yeast protein kinases using protein chips , 2000, Nature Genetics.

[14]  R. Ekins,et al.  Multianalyte microspot immunoassay. The microanalytical 'compact disk' of the future. , 1992, Annales de biologie clinique.

[15]  Immunoassay and other ligand assays: present status and future trends. , 1997, Journal of the International Federation of Clinical Chemistry.

[16]  G. Cagney,et al.  Large-scale functional analysis using peptide or protein arrays , 2000, Nature Biotechnology.

[17]  John W. Silzel,et al.  Mass-sensing, multianalyte microarray immunoassay with imaging detection. , 1998, Clinical chemistry.

[18]  P. Legrain,et al.  From the analysis of protein complexes to proteome-wide linkage maps. , 2000, Current opinion in biotechnology.

[19]  M J O'Hare,et al.  Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Mercola,et al.  Decreased Egr‐1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation , 1997, International journal of cancer.

[21]  R. Ekins,et al.  Ligand assays: from electrophoresis to miniaturized microarrays. , 1998, Clinical chemistry.

[22]  S. Gygi,et al.  Correlation between Protein and mRNA Abundance in Yeast , 1999, Molecular and Cellular Biology.

[23]  T. Ørntoft,et al.  Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions. , 1999, Cancer research.

[24]  I. Tomlinson,et al.  Antibody arrays for high-throughput screening of antibody–antigen interactions , 2000, Nature Biotechnology.

[25]  T. Yip,et al.  Quantitative analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry. , 1996, Rapid communications in mass spectrometry : RCM.

[26]  ティモシー ジェイ. ウーレイバー,,et al.  High-throughput microarray spotting system and method , 2002 .

[27]  H. Lehrach,et al.  Protein arrays for gene expression and molecular interaction screening. , 2000, Current opinion in microbiology.