A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: quantitative detection of cytokine cDNA in activated and resting human cells.
Because of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10(-18) M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3delta amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3delta, TNF alpha or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.
Actor
J K
JK
Medical School, University of Texas-Houston, USA.
Kuffner
T
T
Dezzutti
C S
CS
Hunter
R L
RL
McNicholl
J M
JM
D000331:Aequorin; D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis*; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008163:Luminescent Measurements; D008934:Mitogens / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity
D016207:Cytokines; D018076:DNA, Complementary; D007202:Indicators and Reagents; D008934:Mitogens; D010835:Phytohemagglutinins; D012333:RNA, Messenger; D000331:Aequorin