Nonregeneration Protocol for Surface Plasmon Resonance: Study of High-affinity Interaction with High-density Biosensors
Authors
Affiliations
Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.
Biosensors Based on the Binding Events of Nitrilotriacetic Acid-Metal Complexes.
Zhu L, Chang Y, Li Y, Qiao M, Liu L Biosensors (Basel). 2023; 13(5).
PMID: 37232868 PMC: 10216662. DOI: 10.3390/bios13050507.
2D Nanomaterial-Based Surface Plasmon Resonance Sensors for Biosensing Applications.
Singh S, Singh P, Umar A, Lohia P, Albargi H, Castaneda L Micromachines (Basel). 2020; 11(8).
PMID: 32824184 PMC: 7463818. DOI: 10.3390/mi11080779.
NLRP6 self-assembles into a linear molecular platform following LPS binding and ATP stimulation.
Leng F, Yin H, Qin S, Zhang K, Guan Y, Fang R Sci Rep. 2020; 10(1):198.
PMID: 31932628 PMC: 6957519. DOI: 10.1038/s41598-019-57043-0.
Chiu N, Kuo C, Chen C Int J Nanomedicine. 2019; 14:4833-4847.
PMID: 31308661 PMC: 6613200. DOI: 10.2147/IJN.S208292.
Structural insights into SorCS2-Nerve Growth Factor complex formation.
Leloup N, Chataigner L, Janssen B Nat Commun. 2018; 9(1):2979.
PMID: 30061605 PMC: 6065357. DOI: 10.1038/s41467-018-05405-z.