Immunoprecipitation of lubricin from human serum, plasma and synovial fluid.

Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.