Identification of apolipoprotein B100 polymorphisms that affect low-density lipoprotein metabolism: description of a new approach involving monoclonal antibodies and dynamic light scattering.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL. Because each LDL contains a single apoB, a maximum of two LDLs may be bound by the bivalent monoclonal antibody. Thus, at the appropriate concentration, an equivalent amount of MB19 will promote substantial dimer formation of LDL containing the strongly binding apoB(Ile71), but little dimer formation of LDL containing the weakly binding apoB(Thr71). For LDL isolated from heterozygous individuals, the amount of dimer formed, determined by dynamic light scattering, yields an estimate of the allelic ratio of the two forms of LDL. For such individuals, not only the effect of the polymorphism recognized by MB19 but also the effects of other polymorphisms on the LDL allelic ratio can be determined. Examination of six normolipemic MB19 heterozygotes gave percent allelic ratios between 48:52 and 51:49 tight:weak-binding LDL, not significantly different from a 50:50 ratio. These individuals were also heterozygous for six common apoB polymorphisms, allowing calculation of the odds that each of these polymorphisms caused significant alterations in lipid levels. In contrast, the rare mutation at residue 3500 causing defective binding to the LDL receptor and familial defective apoB100 (FDB) resulted in substantial changes (26:74 and 13:87) in LDL allelic ratio in both of two FDB individuals examined.

Apolipoprotein B: the Ag(a1/d) immunogenetic polymorphism coincides with a T-to-C substitution at nucleotide 1981, creating an Alu I restriction site.

Short cDNA fragments covering the entire coding region for apolipoprotein (apo) B have been cloned in the pING expression vector. A monoclonal antibody specific for the Ag(d) epitope on apo B has been used, together with the expressed apo B peptides, to locate this epitope to a stretch of 26 amino acids. Sequencing of this region from several genomic DNAs of known Ag(a1/d) genotype showed a single T-to-C substitution at nucleotide 1981, creating an Alu I restriction site and resulting in a val to ala residue change in the corresponding peptide (at position 591 in the mature protein). Southern blots, using the Alu I restriction endonuclease and a short probe for this region of the cDNA, showed perfect correspondence between the restriction fragment length polymorphism and the Ag(a1/d) immunochemical polymorphism in all 17 persons examined.

Two monoclonal antibodies that discriminate between allelic variants of human low density lipoprotein.

Human low density lipoprotein shows a genetic polymorphism, the so-called Ag-system. it consists of 5 pairs of allelic epitopes, x/y, al/d, c/g, t/z, and h/i, which are localized on apolipoprotein B. We have generated a large number of monoclonal antibodies against low density lipoprotein. Two of them, D2E1 and H11G3, recognize epitopes related to this genetic polymorphism. Direct ELISA and ELISA inhibition experiments with different low density lipoproteins of known phenotype showed that D2E1 is directed against the allelic epitope c and H11G3 against d. The two antibodies were used for the characterization of low density lipoprotein in sera from different blood donors and the results compared to those obtained by passive hemagglutination using human allotypic anti-sera. Sera from homo- or heterozygous donors (which display the relevant epitope) could be distinguished from the sera of homozygous donors (which lack the epitope) with the monoclonal antibodies described.

[A simple model for the calculation of stability (author's transl)]. / Ein einfaches Modell zur Haltbarkeitsberechnung.

A simple model for stability calculation is demonstrated, taking into consideration the velocity constant, its variance, the analytical error by given loss tolerance and the rejection probability. The procedure is explained in detail and exemplified by creatine kinase and acid phosphatase, both relative instable components in control samples.