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

Apolipoprotein[a] phenotyping is a critically important method to explore t he role of kringle-l repeat number as a modulator of lipoprotein[a]-associa ted cardiovascular risk, The availability of a kringle-4 number-based refer ence standard is therefore necessary for a reliable and generally accepted classification of apo[a] phenotypes, We propose here a battery of recombina nt apo[a] isoforms that may be used as the reference standard in various ge l 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 electr ophoretic mobility of the recombinant apo[a] isoforms expressed by these ce lls in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotti ng 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 ver y good correlation (r = 0.97) was found with the genotype (pulsed-field gel electrophoresis of kpnI-digested restriction fragments of genomic DNA) and among electrophoretic methods. The proposed recombinant standard offers th e possibility to identify apo[a] isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic techniques a nd a nomenclature based on its molecular structure, i.e., the number of kri ngle-4 repeats.