Isolation of allele-specific, receptor-binding-defective low density lipoproteins from familial defective apolipoprotein B-100 subjects.

Abstract

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder apparently caused by a single amino acid substitution (Arg3500–>Gln) that disrupts the binding of low density lipoproteins (LDL) to the LDL receptor. The plasma of FDB heterozygotes contains a mixture of normal LDL and LDL that is defective in binding to the LDL receptor. In this study, the monoclonal antibody MB19 (which recognizes an immunogenetic polymorphism in apolipoprotein B-100) was used to determine the percentage of defective LDL in the plasma of FDB heterozygotes and to isolate allele-specific receptor-binding-defective LDL. Several FDB heterozygotes were identified who were heterozygous for the MB19 polymorphism: one apolipoprotein B allotype in each of these individuals bound with low affinity to MB19 and possessed the Arg3500–>Gln mutation, whereas the other apolipoprotein B allotype bound with high affinity to MB19 and normally to the LDL receptor. Using MB19 radio-immunoassay, we determined that an average of 73% (range 65-87) of the total LDL from FDB heterozygotes contained the Arg3500–>Gln mutation. Antibody MB19-Sepharose immuno-affinity chromatography was used to separate the receptor-binding-defective LDL from the normal LDL. The isolated LDL contained primarily the Arg3500–>Gln mutation and had only about 9% of normal LDL receptor-binding ability. Finally, the MB19-Sepharose chromatography procedure may be useful for isolating other allele-specific LDL that have functionally significant mutations.