Integral Protein Microarrays for the Identification of Lung Cancer Antigens in Sera That Induce a Humoral Immune Response*S

The identification of biomarkers (both molecules and profiles) in patient sera offers enormous interest for the diagnosis of cancers. In this context, the detection of antibodies to tumor cell autologous antigens possesses great potential. The humoral immune response represents a form of biological amplification of signals that are otherwise weak because of very low concentrations of antigen, especially in the early stages of cancers. Herein we present the use of integral microarrays spotted with tumor-derived proteins to investigate the antibody repertoire in the sera of lung cancer patients and controls. The use of two-dimensional liquid chromatography allowed us to separate proteins from the lung adenocarcinoma cell line A549 into 1760 fractions, which were printed in duplicate, along with various controls, onto nitrocellulose coated slides. The sensitivity and specificity of the microarrays to detect singular antibodies in fluids were first validated through the recognition of fractions containing a lung marker antigen by antibody probing. Twenty fractions were initially selected as highly reactive against the anti-PGP9.5 antibody, and subsequent mass spectrometry analyses confirmed the identity of PGP9.5 protein in four of them. As a result, the importance of neighboring fractions in microarray detection was revealed due to the spreading of proteins during the separation process. Next, the microarrays were individually incubated with 14 serum samples from patients with lung cancer patients, 14 sera from colon cancer patients, and 14 control sera from normal subjects. The reactivity of the selected fractions was analyzed, and the level of immunoglobulin bound to each fraction by each serum sample was quantified. Eight of the 20 fractions offered p values < 0.01 and were recognized by an average of four reacting patients, whereas no serum from normal individuals was positive for those fractions. Protein microarrays from tumor-derived fractions hold the diagnostic potential of uncovering antigens that induce an immune response in patients with certain types of cancers.

[1]  Emanuel F Petricoin,et al.  Technology Insight: pharmacoproteomics for cancer—promises of patient-tailored medicine using protein microarrays , 2006, Nature Clinical Practice Oncology.

[2]  Weiliang Qiu,et al.  Development of a “reverse capture” autoantibody microarray for studies of antigen‐autoantibody profiling , 2006, Proteomics.

[3]  S. Fields,et al.  A pooling-deconvolution strategy for biological network elucidation , 2006, Nature Methods.

[4]  G. Nolan,et al.  Mapping normal and cancer cell signalling networks: towards single-cell proteomics , 2006, Nature Reviews Cancer.

[5]  H. Lehrach,et al.  Recent advances of protein microarrays , 2005, Current Opinion in Chemical Biology.

[6]  Paul Bertone,et al.  Advances in functional protein microarray technology , 2005, The FEBS journal.

[7]  Axel Kowald,et al.  Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology* , 2005, Molecular & Cellular Proteomics.

[8]  Fuchu He,et al.  Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project , 2005, Proteomics.

[9]  Lloyd J. Old,et al.  Cancer/testis antigens, gametogenesis and cancer , 2005, Nature Reviews Cancer.

[10]  E. Kopf,et al.  Panorama™ Ab Microarray Cell Signaling kit: A unique tool for protein expression analysis , 2005, Proteomics.

[11]  Bhupinder Bhullar,et al.  Self-Assembling Protein Microarrays , 2004, Science.

[12]  G. Hu,et al.  Parallel detection of autoantibodies with microarrays in rheumatoid diseases. , 2004, Clinical chemistry.

[13]  M. Snyder,et al.  Analyzing antibody specificity with whole proteome microarrays , 2003, Nature Biotechnology.

[14]  Cecelia E Schmalbach,et al.  Molecular profiling of the immune response in colon cancer using protein microarrays: Occurrence of autoantibodies to ubiquitin C‐terminal hydrolase L3 , 2003, Proteomics.

[15]  S. Hanash,et al.  Multi-dimensional liquid phase based separations in proteomics. , 2003, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[16]  P. Cutler.  Protein arrays: The current state‐of‐the‐art , 2003, Proteomics.

[17]  David Sidransky,et al.  Emerging molecular markers of cancer , 2002, Nature Reviews Cancer.

[18]  P. Brown,et al.  Autoantigen microarrays for multiplex characterization of autoantibody responses , 2002, Nature Medicine.

[19]  S. Hanash,et al.  Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer. , 2001, Cancer research.

[20]  S M Hanash,et al.  Protein based microarrays: A tool for probing the proteome of cancer cells and tissues , 2001, Proteomics.

[21]  E. Petricoin,et al.  Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front , 2001, Oncogene.

[22]  P. Brown,et al.  Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions , 2001, Genome Biology.

[23]  A. Nakao,et al.  PGP9.5 as a prognostic factor in pancreatic cancer. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

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

[25]  M. Mann,et al.  Proteomics to study genes and genomes , 2000, Nature.

[26]  David E. Misek,et al.  Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. , 2000, Analytical chemistry.

[27]  S. Goodman,et al.  PGP9.5 as a candidate tumor marker for non-small-cell lung cancer. , 1999, The American journal of pathology.

[28]  H. Lehrach,et al.  Protein microarrays for gene expression and antibody screening. , 1999, Analytical biochemistry.

[29]  R. Souhami,et al.  Favourable prognosis in Lambert-Eaton myasthenic syndrome and small-cell lung carcinoma , 1999, The Lancet.

[30]  A. Dickinson,et al.  Prophylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies , 1999, The Lancet.

[31]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[32]  A. Yamamoto,et al.  Detection of auto‐antibodies against L‐myc oncogene products in sera from lung cancer patients , 1996, International journal of cancer.

[33]  J. Minna,et al.  Antibodies against autologous tumor cell proteins in patients with small-cell lung cancer: association with improved survival. , 1993, Journal of the National Cancer Institute.

[34]  R. Darnell,et al.  Regression of small-cell lung carcinoma in patients with paraneoplastic neuronal antibodies , 1993, The Lancet.

[35]  K D Wilkinson,et al.  The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. , 1989, Science.

[36]  J. Polak,et al.  Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung , 1987, The Journal of pathology.

[37]  S. Hanash,et al.  Disease proteomics , 2003, Nature.

[38]  Kim-Anh Do,et al.  Fingerprinting the circulating repertoire of antibodies from cancer patients , 2003, Nature Biotechnology.

[39]  A. Nakao,et al.  PGP9.5 as a marker for invasive colorectal cancer. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[40]  J. Pollard,et al.  Two-dimensional polyacrylamide gel electrophoresis of proteins. , 1984, Methods in molecular biology.

[41]  J. Pollard.  Two-dimensional polyacrylamide gel electrophoresis of proteins. , 1984, Methods in molecular biology.