Identification of human serum interferants in the recombinant P-selectin glycoprotein ligand-1 clinical ELISA using MALDI MS and RP-HPLC

A colorimetric enzyme-link-ed immunosorbent assay (ELISA) was developed to detect circulating levels of rPSGL to permit pharmacokinetic analysis of cl inical samples. The ELISA is an asymmetric sandwich utilizing a monoclonal antibody pair. Initial validation studies indicated that 57% of normal indi viduals scored above the limit of detection of the assay. Specificity exper iments indicated that the signal was not due to circulating endogenous P-se lectin glycoprotein ligand-1 (PSGL-1). Using matrix-assisted laser desorpti on ionization mass spectrometry (MALDI MS) and sampling within the individu al microplate wells, the interferant was detected in the vicinity of 6.6 kD a in lipemic and normal human sera, but not delipidized sera. These results were consistent with the ELISA data where 97.5% of known lipemic, 57% of n ormal, and 0% of delipidized sera scored above detectable limits in the ELI SA. Preparative isolations of the 6.6 kDa species were performed using reve rsed-phase high performance liquid chromatography (RP-HPLC) with UV and MS detection. Edman N-terminal sequencing identified the 6.6 kDa unknown as Ap olipoprotein C-I. Additional apolipoproteins were found by MALDI and RP-HPL C. Digestion of sera with liposome lipase and extraction of sera with anti- apolipoprotein C-I, C-II, and C-III antibody beads significantly reduced th e ELISA interference. These experiments combined with the MALDI detection o f phosphatidylcholine-type lipids from NHS eluate suggested that lipoprotei n particles or remnants were causing the interference. A method combining T riton-X 100 with sonication was developed to overcome this interference wit hout altering rPSGL recovery in the ELISA. (C) 2001 Elsevier Science B.V. A ll rights reserved.