Development of an assay to measure in vivo cytokine production in the mouse.
The short in vivo lifespan of many cytokines can make measurement of in vivo cytokine production difficult. A method was developed to measure in vivo IL-4 and IFN-gamma production that eliminates this problem. Mice are injected with a biotin-labeled neutralizing IgG anti-IL-4 or anti-IFN-gamma mAb and bled 2-24 h later. Secreted cytokine is captured by the biotin-labeled mAb to produce a complex that has a relatively long in vivo half-life and consequently accumulates in serum. Serum concentrations of the complex are determined by ELISA, using wells coated with an antibody to a second epitope on the same cytokine to capture the complex. This technique is specific and increases sensitivity of detection of secreted IL-4 at least 1000-fold. The amount of cytokine measured is directly proportional to the amount produced and relatively independent of the site of cytokine production. Furthermore, because mice are injected with small quantities of biotin-labeled anti-cytokine mAb, which sample, rather than neutralize, all secreted cytokines, cytokine-dependent responses are not inhibited. The in vivo half-lives of the cytokine-anti-cytokine mAb complexes are sufficiently short to allow cytokine production to be measured every 2-3 days in the same mice. Thus, use of this assay provides a practical and relatively simple and inexpensive way to measure ongoing in vivo cytokine production. Furthermore, the techniques that have been developed to measure in vivo production of IL-4 and IFN-gamma can be applied to in vivo measurement of other molecules that have a short in vivo lifespan, including other cytokines.
Finkelman
F D
FD
Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Morris
S C
SC
D000818:Animals; D000911:Antibodies, Monoclonal; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D007158:Immunologic Techniques; D016898:Interferon-alpha / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology
D000911:Antibodies, Monoclonal; D016207:Cytokines; D016898:Interferon-alpha; D015847:Interleukin-4