Characterization of monoclonal antibodies against apolipoproteins A-I and A-II. Epitope expression in LpA-I and LpA-I:A-II particles.
Two monoclonal antibodies (MAbs) against apolipoprotein A-I (apo A-I), 6B9 and FF9B10, and one MAb against apolipoprotein A-II (apo A-II), 3F5, were characterized. To establish the epitope of apo A-I recognized by these antibodies, different experimental approaches were performed. First, competition between MAbs and the related epitopes on the same antigen was performed using double-determinant tests with previously characterized MAbs. Second, competition of different synthetic peptides of apo A-I in solution with apo A-I immobilized to solid phase was carried out. The MAbs against apo A-I (6B9 and FF9B10) appear to recognize discontinuous epitopes located in the amino-terminal region of the apo A-I. In competition experiments MAb 3F5 did not recognize central- or carboxy-terminal peptides of apo A-II. Furthermore, apo A-II was stronger recognized when it was included in HDL or LpA-I:A-II than in its purified form. So the epitope for 3F5 is better expressed in the lipoprotein structure. Finally, to establish the epitopes expression in different antigens in solution, competition of purified apo A-I, apo A-II, LpA-I, and LpA-I:A-II particles or HDL3, with apo A-I or HDL immobilized to solid phase, was carried out. The results showed that both MAbs against apo A-I reacted with poor affinity against free apo A-I, with high and similar affinities against Lp A-I and Lp A-I:A-II lipoparticles and with the highest affinity against HDL3. The MAb 3F5 against apo A-II recognized only LpA-I:A-II and not LpA-I lipoparticles.
Ulloa
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N
Departamento de Bioquímica Clínica e Immunología, Facultad de Farmacia, Universidad de Concepción, Chile. nulloa@mail.udec.cl
Bustos
P
P
Castro
G
G
Fruchart
J
J
Vera
M
M
Fruchart
J C
JC
Calvo
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D016633:Apolipoprotein A-II / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D000939:Epitopes; D008075:Lipoproteins, HDL; D010455:Peptides