Related Experiment Videos
Development of a sandwich immunoassay for the detection of soluble ovine IL-2R alpha chain
A M Verhagen1, W G Kimpton, A D Nash
1Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Vic., Australia.
Following T cell activation with antigen or mitogens, there is an up-regulation of interleukin-2 receptor alpha (IL-2R alpha) chain expression. A high proportion of the IL-2R alpha chain is shed from the surface 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 excellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2R alpha chain has been developed. Three monoclonal 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, respectively. Two non-competitive ovine IL-2R alpha chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2R alpha chain in the supernatant (SN) of Con A activated ovine lymphocytes and recombinant sIL-2R alpha chain in the SN of CHO IL-2R cells. Soluble IL-2R 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 alloantigen LN activation was observed. This increase correlated with an increase in the output of activated T cell blasts.
Following T cell activation with antigen or mitogens, there is an up-regulation of interleukin-2 receptor alpha (IL-2R alpha) chain expression. A high proportion of the IL-2R alpha chain is shed from the surface 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 excellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2R alpha chain has been developed. Three monoclonal 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, respectively. Two non-competitive ovine IL-2R alpha chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2R alpha chain in the supernatant (SN) of Con A activated ovine lymphocytes and recombinant sIL-2R alpha chain in the SN of CHO IL-2R cells. Soluble IL-2R 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 alloantigen LN activation was observed. This increase correlated with an increase in the output of activated T cell blasts.