Indirect Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Plasma Apolipoprotein E

Abstract

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1.7 g/L, 1250 mumol/L and 13.0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo.E = 1.04 Immunonephelometric apo-E + 16; r2 = 0.954, P < 0.0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 2.0%, within-run coefficients of variation (CV) ranged from 3.7 to 6.0% and between-run CV from 6.1 to 15.1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1.25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.