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Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor.
L Laricchia Robbio1, P Uboldi, S Marcovina
1Institute of Mutagenesis and Differentiation, CNR, Area della Ricerca San Cataldo, 56100 Via Moruzzi, Ghezzano-Pisa, Italy. lel@imd.pi.cnr.it
Biosensors & Bioelectronics
|October 27, 2001
Summary
Surface Plasmon Resonance (SPR) biosensors effectively map distinct epitopes on apolipoprotein B-100 (apoB-100) from human low-density lipoprotein (LDL). This label-free method provides detailed epitope identification and is time-efficient compared to traditional immunoassays.
Area of Science:
- Biochemistry
- Immunology
- Biotechnology
Background:
- Apolipoprotein B-100 (apoB-100) is the primary protein in human low-density lipoprotein (LDL), crucial for LDL receptor interaction.
- Understanding apoB-100 epitopes is vital for characterizing LDL structure and function.
- Existing methods for epitope mapping can be time-consuming and require labels.
Purpose of the Study:
- To investigate the interaction of monoclonal antibodies (mAbs) with distinct domains of apoB-100.
- To characterize epitopes on apoB-100 relevant to LDL-receptor interaction using SPR.
- To evaluate the utility of SPR-based biosensor technology (BIA-technology) for epitope mapping.
Main Methods:
- Utilized a Surface Plasmon Resonance (SPR)-based biosensor (BIA-technology).
- Immobilized anti-apoB-100 monoclonal antibodies (mAbs) via a rabbit anti-mouse IgG(1)-Fc antibody.
- Performed multisite binding analysis by sequentially flowing different mAbs over immobilized apoB-100.
Main Results:
- Ten murine IgG(1) mAbs identified distinct epitopes on apoB-100 in the amino-terminal and middle regions.
- SPR real-time monitoring allowed detailed characterization of mAb binding to apoB-100.
- Results were comparable to western immunoblotting but offered more precise epitope identification.
Conclusions:
- SPR-based BIA-technology is a powerful, label-free tool for mapping distinct epitopes on apoB-100.
- This method provides detailed epitope identification in solution.
- SPR biosensing offers a significant time-saving advantage over conventional immunoassays for epitope mapping.