A quantitative immunoassay for the lysine-binding function of lipoprotein(a). Application to recombinant apo(a) and lipoprotein(a) in plasma.
Apo(a), the unique apoprotein of lipoprotein(a) (Lp[a]), can express lysine-binding sites(s) (LBS). However, the LBS activity of Lp(a) is variable, and this heterogeneity may influence its pathogenetic properties. An LBS-Lp(a) immunoassay has been developed to quantitatively assess the LBS function of Lp(a). Lp(a) within a sample is captured with an immobilized monoclonal antibody specific for apo(a), and the captured Lp(a) is reacted with an antibody specific for functional LBS. The binding of this LBS-specific antibody is then quantified by using an alkaline phosphatase-conjugated disclosing antibody. The critical LBS-specific antibody was raised to kringle 4 of plasminogen. When applied to plasma samples, the LBS activity of Lp(a) ranged from 0% to 100% of an isolated reference Lp(a); the signal corresponded to the percent retention of Lp(a) on a lysine-Sepharose but did not correlate well with total Lp(a) levels in plasma. Mutation of residues in the putative LBS in the carboxy-terminal kringle 4 repeat (K4-37) in an eight-kringle apo(a) construct resulted in marked but not complete loss of activity in the LBS-Lp(a) immunoassay. These data suggest that this kringle is the major but not the sole source of LBS activity in apo(a). The LBS-Lp(a) immunoassay should prove to be a useful tool in establishing the role of the LBS in the pathogenicity of Lp(a).
Hoover-Plow
J L
JL
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
Boonmark
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N
Skocir
P
P
Lawn
R
R
Plow
E F
EF
D000595:Amino Acid Sequence; D001054:Apolipoproteins A / Q000097:blood* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007106:Immune Sera; D007118:Immunoassay; D017270:Lipoprotein(a) / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes / Q000235:genetics; D011994:Recombinant Proteins
D001054:Apolipoproteins A; D007106:Immune Sera; D017270:Lipoprotein(a); D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D008239:Lysine