Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a).
Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes. The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA). It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do. It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).
Seo
Y K
YK
Cardiovascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon, Republic of Korea.
You
K H
KH
Kwak
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001054:Apolipoproteins A / Q000737:chemistry* / Q000276:immunology; D010450:Endopeptidases / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D006825:Hybridomas; D018082:Kringles / Q000276:immunology*; D017270:Lipoprotein(a) / Q000097:blood / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D001054:Apolipoproteins A; D017270:Lipoprotein(a); D010450:Endopeptidases