A WESTERN-BLOT APPROACH TO DETECTION OF HUMAN PLASMA-PROTEIN CONJUGATES DERIVED FROM D-PENICILLAMINE

Objectives-To develop and apply an immunochemical approach to the stud y of drug-plasma protein conjugates derived from the anti-arthritic dr ug D-penicillamine (DP). Methods-An antiserum with specificity for pro tein-conjugated DP was raised in a rabbit. Plasma samples from patient s receiving DP or from incubations of isolated normal plasma with DP w ere analysed for DP-derived conjugates by Western blotting using the a nti-drug antibody. Results-A single DP-positive protein band was detec ted in plasma samples from 15/16 patients with rheumatoid arthritis re ceiving DP but in none of 20 patients of similar disease status who ha d not taken DP. The positive band appeared in patients' plasma during the course of treatment with DP. It was seen under nonreducing but not reducing conditions indicating that the drug is disulphide linked to the protein. The drug-modified protein migrated to a position intermed iate between the trailing edge of albumin and the leading edge of tran sferrin (both non-reduced) suggesting a molecular weight of between 66 and 77 kDa. Incubations of normal human plasma, but not purified albu min or transferrin, with low concentrations of DP generated the same d istinct band plus several less intense DP-positive bands. Conclusions- Drug-plasma protein conjugates derived from DP in vivo and in vitro ca n be detected immunochemically Liverpool, by the Western blot method. Theories of Liverpool, DP immunotoxicity have implicated antigenicity of the drug, but this is the first immunochemical demonstration of a p otential DP-derived antigen in human plasma. The method we describe ma y have application to studies of the relationship between DP antigenic ity and toxicity.