Layered Peptide Array for Multiplex Immunohistochemistry

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Layered Peptide Array for Multiplex Immunohistochemistry Gallya Gannot, Michael A. Tangrea, Heidi S. Erickson, Peter A. Pinto, Stephen M. Hewitt, Rodrigo F. Chuaqui, John W. Gillespie, Michael R. Emmert-Buck The Journal of Molecular Diagnostics Volume 9, Issue 3, Pages 297-304 (July 2007) DOI: 10.2353/jmoldx.2007.060143 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of the iLPA system. Each membrane is coated with a different peptide or antigen specific for an antibody of interest. An antibody set is applied to a tissue section and subsequently captured and analyzed by the appropriate membrane, while maintaining the two-dimensional information present in the sample. The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 iLPA analysis of prostate tissue. A whole-mount prostate tissue (A) was incubated with anti-PSA antibody and captured by the membranes coated with PSA peptide (numbers 1, 3, 5, and 7) but not by the control layers (numbers 2, 4, and 6) (B). Intensity measurements of annotated areas appear in panel C. The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of iLPA and IHC: prostate cancer. Three consecutive serial recuts of a whole-mount prostatectomy specimen were subjected to iLPA analysis, and these same sections were immunostained for PSA (A). A comparison of IHC and iLPA results showed that both techniques generate reproducible results (B). The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 iLPA analysis of a tissue microarray. Application of the iLPA system to a TMA showing specific capture of antibodies on their appropriate membranes. Panel A is a schematic representation of the different tissues on the TMA. Panel B represents the LPA membranes, and panel C is a magnification of the positive areas. The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Comparison of iLPA and IHC: tissue microarray. A comparison of IHC and iLPA shows that both methods generate reproducible staining patterns (A), with more detailed histological evaluation possible by IHC and a larger dynamic range provided by iLPA analysis (B). The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Multiplex iLPA: multiple tissues. The multiplex capability of the iLPA platform was evaluated by examining expression levels of six different proteins (A) in 12 different cellular phenotypes (B). The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 7 Multiplex iLPA: Sjögren's syndrome tissues and TMA. A Sjögren's syndrome sample and a TMA were analyzed by an iLPA capable of measuring seven analytes with the date analyzed in a prototype software system that allows alignment and visualization of the results (A). Quantitative signal intensity was measured by ImagePro (B). The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 8 Universal polymer iLPA system. The iLPA platform will be more versatile if universal shuttle polymers are used. A schematic of such a system is shown in A. Each polymer is bound with an antibody that is specific for one analysis membrane, and each polymer also contains an attachment site for a primary antibody (A1). The polymers are applied to a tissue section, and the primary antibody reacts with its antigen (A2). The polymer is then released from the tissue, binds to its specific membrane, and is measured (A3). B and C: Shuttle polymers can be successfully generated, bound to tissue, released, and analyzed by iLPA membranes. The Journal of Molecular Diagnostics 2007 9, 297-304DOI: (10.2353/jmoldx.2007.060143) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions