Soluble T cell receptors: detection and quantitative assay in fluid phase via ELISA or immuno-PCR.
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-TCR beta monoclonal antibody (H57), which detects all types of alpha beta TCR, regardless of their variable regions, and had a detection limit of about 6 ng/ml sTCR. The indirect sandwich ELISA employed anti-V beta 8 as capture antibody, and had a detection limit of 600 pg/ml. With the direct sandwich ELISA, that also employed anti-V beta 8, TCR concentrations as low as 100 pg/ml could be detected. The ELISA assays were specific for soluble alpha beta TCR, and showed no cross-reactivity when employing two control hamster anti-gamma delta TCR mAbs (GL3 and UC7), or with anti-TCR beta 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.
Sperl
J
J
Department of Medicine, Yale University School of Medicine, New Haven, CT 06520-8013, USA.
Paliwal
V
V
Ramabhadran
R
R
Nowak
B
B
Askenase
P W
PW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000302:isolation & purification; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000032:analysis; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D012995:Solubility; D018418:Th2 Cells / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins