Technologies considered:
KinExA - Kinetic Exclusion Assay
SPR - Surface Plasmon Resonance
KinExA is a technique for measuring unmodified biomolecules with cell surface receptors or other biomolecules in solution. The following document describes advantages KinExA has over SPR techniques.
Cell Measurements:
Over half of all existing drugs are soluble molecules that target a cell membrane protein (1). Membrane protein structure is partially dependent on the cell membrane so anytime the protein is isolated from the cell membrane and purified there is a risk that the binding epitope is modified, hidden, or eliminated. This is why there is such interest in measuring binding to the cells, rather than to purified soluble proteins.
KinExA - Measures binding of a drug (or candidate) to intact cells in any liquid matrix (media, buffer, serum, etc). KinExA has been demonstrated on both engineered over expressing cell lines (2,3) and on native cells expressing endogenous protein (unpublished). Measuring directly to the native cells is about as good as it can get in terms of biological relevance. KinExA has the sensitivity to measure sub pM Kd 's.
SPR - To our knowledge, no one has used a commercial SPR instrument with cells but there is literature on special apparatus. The first detects morphological changes in response to a binding event using cells grown on a gold substrate (4). No binding constant is reported. The second case used SPR microscopy to measure binding of wheat-germ agglutinin (a lectin) to a single cell and reports a Kd of 0.32µM (5). They also showed a noisy sensorgram for an antibody binding to a membrane protein, no Kd is given.
Solution Measurements:
KinExA - KinExA measures binding of the unmodified drug to the unmodified target in solution (6). The solute can be anything in any buffer (serum, cell lysate, buffer, etc.). KinExA has been demonstrated accurate for many single digit pM Kd
measurements and has measured Kd's as low as 12 fM (7).
SPR- These surface techniques require immobilization of one binding partner on the surface. The immobilization can cause conformational changes in the molecule leading to a change in Kd (8,9) or the surface itself can cause artifactual errors in the
measured Kd8. Examples using SPR to measure single digit pM Kd's can be found in the literature but most users don't trust it for Kd's below 100 pM, with many not trusting it below 1 nM.
References:
(1) Von Heijne, G.(2007) "The Membrane protein universe:what's out there and why bother?. Journal of Internal Medicine,
261:543-557. doi:10.1111/j.1365-2796.2007.01792.x
(2) Xie, L., R. Mark Jones, et al. (2005). "Measurement of the functional affinity constant of a monoclonal antibody for cell surface receptors using kinetic exclusion fluorescence immunoassay." J Immunol Methods 304(1-2): 1-14. doi: 10.1016/j.jim.2005.04.009
(3) Rathanaswami, P., J. Babcook, et al. (2008). "High-affinity binding measurements of antibodies to cell-surface-expressed antigens." Anal Biochem 373: 52-60. doi:10.1016/j.ab.2007.08.014.
(4) Chabot, V., C. M. Cuerrier, et al. (2009). "Biosensing based on surface plasmon resonance and living cells." Biosens Bioelectron 24(6): 1667-73. 10.1016/j.bios.2008.08.025.
(5) Wang, W., Y. Yang, et al. "Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells." Nat Chem 4(10): 846-53. i: 10.1038/nchem.1434.
(6) Bee, C., Y. N. Abdiche, et al. (2013). "Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum." PLoS One 8(11): e80501.doi: 10.1371/journal.pone.0080501
(7) Kovalchin J., et.al.(2018) "Preclinical Development of EBI-005: An IL-1 Receptor-1 Inhibitor for the Topical Ocular Treatment of Ocular Surface Inammatory Diseases." Eye & Contact Lens: Science & Clinical Practice: -Volume 44 - Issue 3 - p 170-181 doi: 10.1097/ICL.0000000000000414
(8) Drake, A. W., M. L. Tang, et al. (2012). "Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex." Anal Biochem 429(1): 58-69. doi:10.1016/j.ab.2012.06.024.
(9) Kamat V., et.al. (2020) "The impact of different human IgG capturemolecules on the kinetics analysis of antibody-antigen interaction." Anal Biochem. 593: 113580. doi/10.1016/j.ab.2020.113580.