Validation of serum protein profiles by a dual antibody array approach

• Published in: Journal of proteomics Vol. 73; no. 2; pp. 252 - 266
• Main Authors: Rimini, Rebecca, Schwenk, Jochen M., Sundberg, Mårten, Sjöberg, Ronald, Klevebring, Daniel, Gry, Marcus, Uhlén, Mathias, Nilsson, Peter
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.12.2009; Elsevier
• Subjects: Analysis of complex biological substances; Analytical, structural and metabolic biochemistry; Antibodies; Antibody microarray; Antibody proteomics; Biological and medical sciences; Biotinylation; Blood and biological fluids; Blood Proteins - analysis; Diverse techniques; Fundamental and applied biological sciences. Psychology; generation; Humans; identification; immunoassays; Methods; microarray analysis; Molecular and cellular biology; normalization; pancreatic-cancer; Planar microarray; plasma-proteome; profiling; Protein Array Analysis - methods; Proteomics - methods; Serum; Serum profiling; specimens; support; Suspension bead array; systematic variation; Validation Studies as Topic; Serum profiling; Suspension bead array; Planar microarray; Antibody proteomics; NHS; HPA; 3-D; Antibody microarray; EC; PBS-T; Human; Validation; Proteomics; Serum; Microarray; Serum protein
• ISSN: 1874-3919; 1876-7737; 1876-7737
• DOI: 10.1016/j.jprot.2009.09.009

In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.