Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor.
Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG(1)), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1-1297) and in the middle region (mAb 6B: aa 1480-1693; mAbs 2A, 3B: aa 2152-2377; mAbs 9A, L2 and L4: aa 2657-3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG(1)-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).
Laricchia Robbio
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Institute of Mutagenesis and Differentiation, CNR, Area della Ricerca San Cataldo, 56100 Via Moruzzi, Ghezzano-Pisa, Italy. lel@imd.pi.cnr.it
Uboldi
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Marcovina
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Revoltella
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RP
Catapano
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D000818:Animals; D000911:Antibodies, Monoclonal; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D011973:Receptors, LDL / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D000911:Antibodies, Monoclonal; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008024:Ligands; D011973:Receptors, LDL