Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.
We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.
Laboratory of Immunochemistry and Lipoproteins, Scientific Institute S. Raffaele, Milan, Italy.
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Marcovina
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Fantappie
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Zoppo
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Franceschini
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Catapano
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics / Q000276:immunology*; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D008075:Lipoproteins, HDL / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D010449:Peptide Mapping; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D000939:Epitopes; D008075:Lipoproteins, HDL; D010446:Peptide Fragments