Journal of Lipid Research, Vol 30, 23-37, Copyright © 1989 by Lipid Research, Inc.

ARTICLES

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity

HC Guo, VW Armstrong, G Luc, C Billardon, S Goulinet, R Nustede, D Seidel and MJ Chapman
Groupe de Recherches INSERM sur les Lipoproteines, Hopital de la Pitie, Paris, France.

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB- 100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50- fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP- 1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Gonbert, B. Saint-Jore, P. Giral, C. Doucet, J. Chapman, and J. Thillet Molecular Analysis of Apo(a) Fragmentation in Polygenic Hypercholesterolemia: Characterization of a New Plasma Fragment Pattern Arterioscler. Thromb. Vasc. Biol., August 1, 2001; 21(8): 1353 - 1358. [Abstract] [Full Text] [PDF]

				C. DOUCET, V. MOOSER, S. GONBERT, F. RAYMOND, J. CHAPMAN, C. JACOBS, and J. THILLET Lipoprotein(a) in the Nephrotic Syndrome: Molecular Analysis ofLipoprotein(a) and Apolipoprotein(a) Fragments in Plasma and Urine J. Am. Soc. Nephrol., March 1, 2000; 11(3): 507 - 513. [Abstract] [Full Text] [PDF]

				F. van Kooten, J. van Krimpen, D. W.J. Dippel, N. Hoogerbrugge, and P. J. Koudstaal Lipoprotein(a) in Patients With Acute Cerebral Ischemia Stroke, July 1, 1996; 27(7): 1231 - 1235. [Abstract] [Full Text]

				G Utermann The mysteries of lipoprotein(a) Science, November 17, 1989; 246(4932): 904 - 910. [Abstract] [PDF]

				B. Garner, A. H. Merry, L. Royle, D. J. Harvey, P. M. Rudd, and J. Thillet Structural Elucidation of the N- and O-Glycans of Human Apolipoprotein(a). ROLE OF O-GLYCANS IN CONFERRING PROTEASE RESISTANCE J. Biol. Chem., June 15, 2001; 276(25): 22200 - 22208. [Abstract] [Full Text] [PDF]