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Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays

  • Catherine Jozwik
  • Ofer Eidelman
  • Joshua Starr
  • Harvey B. Pollard
  • Meera SrivastavaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1513)

Abstract

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody’s titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Key words

Proteomics Reverse capture protein microarray Antibodies Body fluids Biomarkers Bioinformatics 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Catherine Jozwik
    • 1
  • Ofer Eidelman
    • 1
  • Joshua Starr
    • 1
  • Harvey B. Pollard
    • 1
  • Meera Srivastava
    • 1
    Email author
  1. 1.Department of Anatomy, Physiology and Genetics, Institute for Molecular Medicine, Center for Medical ProteomicsUniformed Services University School of MedicineBethesdaUSA

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