DEVELOPMENT OF A SANDWICH IMMUNOASSAY FOR THE DETECTION OF SOLUBLE OVINE IL-2R-ALPHA CHAIN

Following T cell activation with antigen or mitogens, there is an up-r egulation of interleukin-2 receptor alpha (IL-2R alpha) chain expressi on. A high proportion of the IL-2R alpha chain is shed from the surfac e of the T cell in a soluble form following proteolytic cleavage, and thus determination of soluble IL-2R alpha (sIL-2R alpha) chain is an e xcellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2R alpha chain has been developed. Three mo noclonal antibodies (mAbs) with specificity for the IL-2R alpha chain, demonstrated by immunoprecipitation of a 50 kDa protein from an ovine IL-2R alpha chain cDNA transfected Chinese hamster ovarian (CHO IL-2R ) cell line, were analysed for additive and competitive binding to CHO IL-2R cells and Concanavalin A (Con A) activated ovine lymphocytes, r espectively. Two non-competitive ovine IL-2R alpha chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2R alph a chain in the supernatant (SN) of Con A activated ovine lymphocytes a nd recombinant sIL-2R alpha chain in the SN of CHO IL-2R cells. Solubl e IL-ZR alpha chain could also be detected in complex biological fluid . In the efferent lymph of a cannulated ovine popliteal lymph node (LN ), an increase in the level of sIL-2R alpha chain following local allo antigen LN activation was observed. This increase correlated with an i ncrease in the output of activated T cell blasts.