USE OF THE PROXIMITY LIGATION ASSAY TO DETECT IMMUNE COMPLEXES IN HISTOLOGIC SECTIONS

Abstract

The Proximity Ligation Assay (PLA) combines elements of immunohistology and molecular biology to allow for in‐situ detection of specific proteins, protein modifications, and protein‐protein interactions. PLA is often used to study the interaction of signaling cascade phosphoproteins on cytospin preparations of cultured cells. We demonstrate that the PLA can be adapted to detect immune complexes in archived tissue sections. We established immune complexes in sections of H929 xenografts, which constitutively express human antibody kappa light chains, by incubating the sections with either mouse‐anti‐human light chain monoclonal or anti‐human IgG polyclonal antibodies. We then used multicolor immunofluorescence to confirm that both mouse and human antibodies specifically co‐localized to the H929 cells in the xenograft. Although multi‐color immunofluorescence indicates whether two proteins are located in the same general vicinity, it does not prove that the two proteins are bound to one another. In contrast, the PLA generates a fluorescent signal via rolling circle DNA amplification only when two proteins are bound to one another. Using the PLA, we confirmed that the mouse antibodies formed immune complexes with the constitutively expressed human antibodies in the cytoplasm of the H929 xenograft cells. PLA may be useful to detect the presence of immune complexes in both preclinical and clinical samples.