Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), w e have studied the interaction of ten different murine monoclonal antibodie s (mAbs, all I-G,), raised against the main protein constituent of human lo w density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). Thes e mAbs identify distinct domains on apoB-100, relevant to LDL-receptor inte raction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: a a 1-1297) and in the middle region (mAb 6B: aa 1480-1693; mAbs 2A, 3B: aa 2 152-2377; mAbs 9A, L2 and L4: aa 2657-3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recogn ized 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 wi th this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over th e 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 immunoblotti ng using the same reagents. However, SPR ensures a more detailed epitope id entification, demonstrating that BIA-technology can be successfully used fo r mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoa ssays, it is significantly time saving (CNR-P.F. MADESS 2). (C) 2001 Elsevi er Science B.V. All rights reserved.