Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice.
Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.
Bobrowski
Walter F
WF
Pfizer Global Research and Development, 2800 Plymouth Road, 35/183, Ann Arbor, MI 48105, USA. walter.bobrowski@pfizer.com
McDuffie
J Eric
JE
Sobocinski
Gregg
G
Chupka
Jonathan
J
Olle
Eric
E
Bowman
Amy
A
Albassam
Mudher
M
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D016207:Cytokines / Q000097:blood*; D004195:Disease Models, Animal; D018450:Disease Progression; D019446:Endotoxemia / Q000097:blood* / Q000139:chemically induced; D005260:Female; D008070:Lipopolysaccharides; D051379:Mice; D040081:Protein Array Analysis
D000906:Antibodies; D016207:Cytokines; D008070:Lipopolysaccharides