Monoclonal antibodies to human A-I apolipoprotein and characterisation of cyanogen bromide fragments of APOA-I

Abstract

Several monoclonal antibodies to human A-I apolipoprotein were produced after immunising mice with pure delipidated apoA-I. These monoclonal antibodies were characterised for their ability to react with whole lipoproteins, apolipoproteins and fragments of apoA-I generated by cleavage with cyanogen bromide. The data suggest that production of monoclonal antibodies using apoA-I as antigen was influenced by two major epitopes subsequently localised to cyanogen bromide fragments 1 and 3, and have been designated antibodies 1 → 5 A-I B and 6 → 10 A-I B, respectively. Cyanogen bromide fragments were first purified to homogeneity before screening by competitive displacement or immunoblotting procedures. Definitive characterisation of one antibody series (1 → 5 A-I B) depended ultimately on Western blotting following isoelectric focusing of purified apoA-I fragments. This technique identified the epitope for these antibodies to fragment 1, an identification not fully concluded from competitive displacement studies. These studies have also revealed the presence of microheterogeneity in fragment 1 (as well as in fragment 4) of apoA-I, suggesting that structural variations in several regions may account for the polymorphism observed in this apolipoprotein.