Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay.
We used a multiplexedfluorescent microsphere immunoassay to develop a sandwich capture assay to assess simultaneously the production of thymus helper (TH) 1- and TH2-type cytokines in tissue culture supernatant obtained from stimulated peripheral blood mononuclear cells. The assay then was used to assess the cytokine production of patients with hyperimmunoglobulinemia E syndrome and in cord blood from neonates. The multiplexed assay has a reportable range of less than 10 to 50,000 pg/mL. For linearity and recovery studies, R2 values for the 6 cytokines ranged from 0.988 to 0.999 for samples spiked with known concentrations of recombinant cytokine standards and for patient samples. The assay showed good specificity, with little cross-reactivity between cytokines. Results from supernatants of Staphylococcus aureus-stimulated peripheral blood mononuclear cells obtainedfrom 6 patients with hyperimmunoglobulinemia E syndrome showed significantly less interferon (IFN)-gamma production than cells from healthy control subjects. Cord blood cells from neonates produced significantly less interleukin 12 and IFN-gamma than cells from adults in group B streptococci-stimulated mononuclear cells. The fluorescent multiplexed microsphere immunoassay can be used to quantitate multiple cytokines from 1 sample and should be useful for further understanding of the cytokine role in disease.
Martins
Thomas B
TB
Associated Regional and University Pathologists, Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
Pasi
Brian M
BM
Pickering
Jerry W
JW
Jaskowski
Troy D
TD
Litwin
Christine M
CM
Hill
Harry R
HR
D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D005312:Fetal Blood / Q000276:immunology; D005453:Fluorescence; D006801:Humans; D006942:Hypergammaglobulinemia / Q000276:immunology; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007073:Immunoglobulin E; D007153:Immunologic Deficiency Syndromes / Q000276:immunology*; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000502:physiology; D008863:Microspheres; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology*; D018418:Th2 Cells / Q000276:immunology*
D016207:Cytokines; D007073:Immunoglobulin E