Optimization and characterization of capture ELISA methodology for Lp(a) lipoprotein quantification

In order to better characterize and optimize a typical capture ELISA system for Lp(a) lipoprotein, we have analysed kinetic details of the reaction. Plate coating with polyclonal antibody, recognition of captured analyte with monoclonal antibody, and detection of monoclonal antibody with alkaline phosphatase-labeled antiglobulin were essentially complete after one hour, probably being driven forward by a relative excess of reagent. However, complete capture of the Lp(a) analyte required about 6 h at low input concentrations. Shorter time periods for capture might therefore result in decreased sensitivity and reproducibility. Deviations from linearity in the assay dose response were associated with incomplete capture of Lp(a) and significant depletion of the monoclonal recognition antibody. With the final reaction conditions described, no significant differences in immunochemical reactivity between samples were found by analysis of dose response slopes. Finally, interferences from plasminogen, -20 degrees C storage, anticoagulants, LDL, haemolysis, and bilirubin were minimal.