Interaction standards for biophysics: anti-lysozyme nanobodies

Abstract

There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb’s or ‘nanobodies’ against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards.

Enhancement of Ligand Immobilization on Gold Surface by Optimizing Monolayer Concentration and Pre‐Concentration Studies for Surface Plasmon Resonance Analysis of Rutin‐Lysozyme Interaction

“…For example, (i) an SPR platform was designed to immobilize L ZM on the CM5 sensor surface and determine the affinity data of several nanobodies towards L ZM . According to the results, these nanobiostructures have shown a strong affinity for L ZM with low K D values in the range of 5–1300 nM [53] . (ii) In another study, binding kinetics of surface protein antigen (PAc)‐L ZM interaction were evaluated by SPR analysis.…”

“…According to the results, these nanobiostructures have shown a strong affinity for L ZM with low K D values in the range of 5-1300 nM. [53] (ii) In another study, binding kinetics of surface protein antigen (PAc)-L ZM interaction were evaluated by SPR analysis. In this study, PAc was immobilized on the CM5 sensor chip as a ligand and three concentrations of L ZM solutions were exposed serially to immobilized PAc.…”

Community-building and promotion of technological excellence in molecular biophysics: the ARBRE–MOBIEU network

“…This has already led to enhanced propagation of novel methodologies and collaboration between laboratories. The working group entitled 'Optimization of data quality' has been particularly popular and has focussed primarily on helping cross-laboratory teams (i) develop standard samples (Birchenough et al 2021) which will be used in future training workshops and benchmarking studies, (ii) establish standard operating procedures (SOP's), (iii) carry out benchmarking studies and iv) define procedures for the optimisation of sample quality. The network has already generated SOP's across many instruments and methods, including enzymatic Activity inhibition assays (Smirnovienė et al 2021), calibration of analytical ultracentrifuges (Zhao et al 2021), isothermal titration calorimetry (Bastos and Velazquez-Campoy 2021), fluorescent thermal shift assays (Kazlauskas et al 2021), microscale thermophoresis (MST) experiments (Sedivy 2021), Switchsense DRX instruments (Müller-Landau and Fernandez Varela 2021) and mass photometry (Wu and Piszczek 2021).…”