This disclosure provides a method for quantifying total soluble Programmed Death-1 (sPD-1) in a sample solution comprising the steps of performing a “sandwich” immunoassay on the sample solution and a series of reference solutions containing known quantities of sPD-1, wherein the immunoassay is performed using (a) a sPD-1 reference antigen in monomeric form; (b) a capture antibody that is capable of binding to sPD-1 in both monomeric and dimeric forms, which binding is essentially unaffected by the presence of the PD-1 ligands, PDL-1, PDL-2, and/or a therapeutic anti-PD-1 antibody; and (c) an electrochemiluminescent-labeled detection antibody that is capable of binding to sPD-1 in both monomeric and dimeric forms, which binding is to a different epitope on sPD-1 than the epitope bound by the capture Ab and is essentially unaffected by the presence of PDL-1, PDL-2 and/or a therapeutic anti-PD-1 Ab.
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