Structure-Binding Relationships for the Interaction between a Vancomycin Monoclonal Antibody Fab Fragment and a Library of Vancomycin Analogues and Tracers

Abstract

A series of vancomycin analogues and tracers were synthesized, and their binding interactions with an anti-vancomycin Fab fragment were evaluated under mass transport limiting conditions using surface plasmon resonance detection. Differences observed in binding interactions were utilized to define the vancomycin structural elements critical for antibody recognition. Major structural regions of vancomycin shown to play an important role in anti-vancomycin Fab fragment recognition include two sugar moieties and one chlorinated phenyl ring. The N-methylleucyl residue, the carboxy terminal residue, and residues in the peptide-binding region of vancomycin have minimal impact on the anti-vancomycin Fab fragment/vancomycin binding interaction. The selection of an antibody with such binding properties plays a critical role in the development of a vancomycin immunoassay that employs stable calibrators and controls.

Surface Plasmon Resonance (SPR) as a Tool for Antibody Conjugate Analysis

“…In summary, our past studies have shown that SPR can be useful in determining the optimal structure of small molecule ligands for binding to a given antibody whether the ligand is in solution or bound to a solid support (4)(5)(6)(7)(8). While that was a significant advance in the design of immunoassay components, it also set the basis for monitoring lot to lot variations in the binding properties of the antibody.…”

“…Reactive sites remaining on the biosensor chip were blocked with ethanolamine (70 µL, 1.0 M). The binding capacities of the resulting biosensors were estimated using unlabeled anti-biotin mAb (4) or unlabeled anti-fluorescein mAb (5). Control surfaces for each sensor were generated at the same conditions, omitting the ligand immobilization step.…”

“…As commercialized on the BIAcore instrument, SPR allows for the real-time measurement of the kinetics of noncovalent interactions between a ligand and a binding partner ( − ). We have recently highlighted surface plasmon resonance on the BIAcore system as a valuable tool that can be used to characterize immunoreagents utilized in immunoassays ( − ). The previous reports have detailed the utility of SPR in evaluating how the structural variations in a library of small molecule haptens affected the kinetics and thermodynamics of binding to anti-hapten antibodies or Fab fragments.…”

“…In this direct SPR method, it was necessary to optimize the surface density of the ligand at a low level on the biosensor chip to minimize rebinding events and mass transport limitations. Other studies have taken a different approach in which a single biosensor was used to determine the affinity constants of each member of the library with the Fab in solution at equilibrium (5,8). The technique is based on the observation that under mass transport limited conditions the initial binding rate of the antibody with the biosensor is proportional to the concentration of the antibody and independent of the affinity of the antibody for the ligand on the biosensor chip (9).…”

Vancomycin architecture dependence on the capture efficiency of antibody-modified microbeads by magnetic nanoparticles

“…Adamczyk and coworkers have reported that the K d for a vancomycin derivative modified through the vancosamine nitrogen (as in SNP-3) can be 0.6-2900 times greater than the K d for the analogous derivative modified at the carboxylic acid (as in SNP-4). 17 However, because we employ a 1.7 6 10 5 times excess of nanoparticle over vAb-PS it could be possible that even very large differences in K d could be overcome with the extremely large excess of nanoparticle. We are currently exploring if a nanoparticle based system such as this could find use as an assay to qualitatively assess the relative position of the epitopes on vancomycin by exploring the interaction between SNP-3 and SNP-4 and PS beads modified with a variety of monoclonal vancomycin antibodies.…”