Article on Methodology

A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping

Correspondence to: E. Anglés-Cano

Apolipoprotein[a] phenotyping is a critically important method to explore the role of kringle-4 repeat number as a modulator of lipoprotein[a]-associated cardiovascular risk. The availability of a kringle-4 number-based reference standard is therefore necessary for a reliable and generally accepted classification of apo[a] phenotypes. We propose here a battery of recombinant apo[a] isoforms that may be used as the reference standard in various gel systems. Five plasmids encoding for r-apo[a] containing a known number (n = 9, 13, 17, 25, 33) of plasminogen-like kringle-4 copies were constructed, and transfected into the human embryonic kidney cell line 293. The electrophoretic mobility of the recombinant apo[a] isoforms expressed by these cells in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotting using an antibody specific for human apo[a]. The equation of the linear relationship between log r-apo[a] kringle number and relative migration was used to determine the isoform size of apo[a] in normal human plasma. A very good correlation (r = 0.97) was found with the genotype (pulsed-field gel eletrophoresis of kpnI-digested restriction fragments of genomic DNA) and among electrophoretic methods. The proposed recombinant standard offers the possibility to identify apo[a] isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic techniques and a nomenclature based on its molecular structure, i.e., the number of kringle-4 repeats.—Anglés-Cano, E., S. Loyau, G. Cardoso-Saldaña, R. Couderc, and P. Gillery. A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping. J. Lipid Res. 1999. 40: 354–359.

Supplementary key words: apo[a] kringle-4 type 2, plasminogen kringle 4, apo[a] genotype, apo[a] isoform heterogeneity, apo[a] electrophoretic mobility, SDS electrophoresis, immunoblot

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