Methodology

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

LT Perlee^1* , J Christiansen¹ , R Dondero¹ , B Grimwade¹ , S Lejnine^1* , M Mullenix¹ , W Shao¹ , M Sorette¹ , VT Tchernev¹ , DD Patel² and SF Kingsmore^1,3*

¹  Molecular Staging Inc., 300 George St., New Haven, CT 06511 USA

²  Thurston Arthritis Research Center and Department of Medicine, University of North Carolina, 3330 Thurston Building, Chapel Hill, NC 27599 USA

³  National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, NM 87505 USA

author email corresponding author email* Contributed equally

Proteome Science 2004, 2:9doi:10.1186/1477-5956-2-9

Published:	15 December 2004

Abstract

Background

Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification.

Results

Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies.

Conclusion

The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.