Development and validation of protein microarray technology for simultaneous inflammatory mediator detection in human sera.
Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA) guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL-) 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the "gold standard" ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays.
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK ; Roche Products Limited, 6 Falcon Way, Shire Park, Welwyn Garden City AL7 1TW, UK.
Senthooran
0000-0002-5088-4727
S
Selvarajah
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK ; Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt.
Ola H
OH
Negm
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK ; Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt.
Mohamed R
MR
Hamed
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Carolyn
C
Tubby
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Ian
I
Todd
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Patrick J
PJ
Tighe
Nottingham Respiratory Research, University of Nottingham Clinical Sciences Building, City Hospital Campus, Nottingham NG5 1PB, UK.
Tim
T
Harrison
School of Life Sciences, The University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Lucy C
LC
Fairclough
D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000032:analysis; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D018836:Inflammation Mediators / Q000097:blood*; D057230:Limit of Detection; D040081:Protein Array Analysis / Q000379:methods*
D015415:Biomarkers; D001798:Blood Proteins; D016207:Cytokines; D018836:Inflammation Mediators