Comparison between the surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) method in a structural analysis of human endothelin-1.
In this study, an automated surface plasmon resonance (SPR)-based biosensor was compared with a quartz crystal microbalance (QCM) biosensor. The two biosensor systems were used for characterizing a site-directed monoclonal antibody (mAb), raised against the C-terminal heptapeptide ET-1(15-21) of the human endothelin (ET-1). The mAb was characterized by its capacity for binding to ET-1, ET-3, Big.ET-1(22-38), the C-terminal (ET-1(15-21), ET-1(16-21), ET-1(17-21)), and six derivates of ET-1(16-21), each containing a substitution with alanine (Ala) of a single aminoacid from position 16-21, respectively. The mAb reacted well with ET-1 and its fragments ET-1(15-21), ET-1(16-21), ET-1(17-21), but showed only a partial cross-reaction with ET-3, and did not bind human Big.ET-1(22-38). The Ala substitution on position 16,17, or 19 of ET-1(16-21) did not affect the antibody binding capacity of the hexapaptide ET-1(16-21). On the contrary, Ala substitution or Asp(18), Ile(20) and particularly Trp(21), inhibited its immunoreactivity. Thus the C-terminal represents an immunodominant epitope in ET-1 and is important for antibody binding. The SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, we found a good correlation between results obtained using the BIA technology and the QCM.
Laricchia-Robbio
Leopoldo
L
Unit of Immunobiology and Cell Differentiation, Institute of Biomedical Technologies, C.N.R., Research Area, Via G. Moruzzi 1, 56100 Pisa, Italy.
Revoltella
Roberto P
RP
D000936:Antigen-Antibody Complex / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D019332:Endothelin-1 / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D021281:Quantitative Structure-Activity Relationship; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D014159:Transducers
D000936:Antigen-Antibody Complex; D019332:Endothelin-1; D011994:Recombinant Proteins