Soluble T cell receptors: detection and quantitative assay in fluid phase via ELISA or immuno-PCR

Abstract

To establish the concentration range in which soluble murine T cell receptors (sTCR), derived from the Th2 clone D10, exhibited biological activity, and to follow production and purification of D10 sTCR, we devised four quantitative immunoassays: three ELISA systems, and an immuno-PCR assay. The direct ELISA, employed hamster anti-TCRβ monoclonal antibody (H57), which detects all types of αβ TCR, regardless of their variable regions, and had a detection limit of about 6 ng/ml sTCR. The indirect sandwich ELISA employed anti-Vβ8 as capture antibody, and had a detection limit of 600 pg/ml. With the direct sandwich ELISA, that also employed anti-Vβ8, TCR concentrations as low as 100 pg/ml could be detected. The ELISA assays were specific for soluble αβ TCR, and showed no cross-reactivity when employing two control hamster anti-γδ TCR mAbs (GL3 and UC7), or with anti-TCRβ and monoclonal hamster IgG as a control antigen. Further, we demonstrated that in some assays where use of passive binding ELISA plates resulted in a high background, replacement with covalent binding ELISA plates resulted in an acceptable low background value. With the immuno-PCR assay, concentrations of sTCR as little as 0.8 pg/ml could be detected. In summary, the assays described here may prove valuable in investigating the occurrence and amount of sTCR in vitro and in vivo.