Development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis.
Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs.
Knight
Paul R
PR
Department of Anesthesiology, SUNY at Buffalo, Buffalo, New York, USA.
Sreekumar
Arun
A
Siddiqui
Javed
J
Laxman
Bharathi
B
Copeland
Shannon
S
Chinnaiyan
Arul
A
Remick
Daniel G
DG
D000906:Antibodies / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D003101:Collodion / Q000737:chemistry; D016207:Cytokines / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007249:Inflammation; D040081:Protein Array Analysis / Q000379:methods*; D020543:Proteome; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
D000906:Antibodies; D016207:Cytokines; D020543:Proteome; D001710:Biotin; D003101:Collodion; D019809:Streptavidin