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  Reference Spotlight

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.

Kusano-Arai 0., et al. 2016. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus. Anal Biochem. 10.1016/j.ab.2016.04.004. https://www.ncbi.nlm.nih.gov/pubmed/27095060

KinExA References

Affinity & Kinetic Measurements:

KinExA technology overview:

KinExA’s role in drug discovery:

Significance of “solution phase” measurements to unmodified molecules:

  • Kusano-Arai 0., et al. 2016. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus. Anal Biochem. 10.1016/j.ab.2016.04.004. https://www.ncbi.nlm.nih.gov/pubmed/27095060
  • Blake R.C., Li X., Blake D.A. 2007. Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Biochemistry 46: 1573-1586. http://www.ncbi.nlm.nih.gov/pubmed/17279622

Comparison to SPR:

Sensitivity to measure tight binders:

Reverse assay techniques:

Whole cell binding techniques:

  • Bedinger, D., et al. 2015. Differential pathway coupling of activated insulin receptor drives signaling selectivity by XmetA, an allosteric partial agonist antibody. J Pharmacol Exp Ther 353(1):35-43. http://www.ncbi.nlm.nih.gov/pubmed/25613982
  • Rathanaswami P., Babcook J., Gallo M. 2008. High-affinity binding measurements of antibodies to cell-surface-expressed antigens. Anal Biochem 373: 52-60. http://www.ncbi.nlm.nih.gov/pubmed/17910940
  • Xie L., 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: 1-14. http://www.ncbi.nlm.nih.gov/pubmed/16098983

Unpurified antigens:

Other interesting studies:

  • Li X., Kaattari S.L., Vogelbein M.A., Vadas G.G., Unger M.A., 2016. A highly sensitive monoclonal antibody based biosensor for quantifying 3-5 ring polycyclic aromatic hydrocarbons (PAHs) in aqueous environmental samples. Sens Biosensing Res. 7:115-120. https://www.ncbi.nlm.nih.gov/pubmed/26925369
  • Lou J., et al. 2010. Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating. Protein Eng Des Sel 23(4): 311-319. http://www.ncbi.nlm.nih.gov/pubmed/20156888
  • Kahle K.M., Steger H.K., Root M.J. 2009. Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding. PLOS Path 5(11): 1-11. http://www.ncbi.nlm.nih.gov/pubmed/19956769
  • Nowakowski A., et al. 2002. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proc Natl Acad Sci 99: 11346-11350. http://www.ncbi.nlm.nih.gov/pubmed/12177434

Immunoassay Techniques:

  • Wani T.A., et al. 2016. Analytical Application of Flow Immunosensor in Detection of Thyroxine and Triiodothyronine in Serum. Assay Drug Dev Technol.14(9):535-542. https://www.ncbi.nlm.nih.gov/pubmed/27801595
  • Darwish I.A., et al. 2013. Kinetic-exclusion analysis-based immunosensors versus enzyme-linked immunosorbent assays for measurement of cancer markers in biological specimens. Talanta 111: 13-19. http://www.ncbi.nlm.nih.gov/pubmed/23622520
  • Prieto-Simon B., Miyachi H., Karube I., Saiki H. 2010. High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples. Biosens Bioelectron 25: 1395-1401. http://www.ncbi.nlm.nih.gov/pubmed/19939663
  • Sasaki K., Oguma S., Namiki Y., Ohmura N. 2009. Monoclonal antibody to trivalent chromium chelate complex and its application to measurement of the total chromium concentration. Anal Chem 81: 4005-4009. http://www.ncbi.nlm.nih.gov/pubmed/19438265
  • Glass T.R., Ohmura N., Saiki H. 2007. Least detectable concentration and dynamic range of three immunoassay systems using the same antibody. Anal Chem 79: 1954-1960. http://www.ncbi.nlm.nih.gov/pubmed/17256970
  • Bromage E.S., et al. 2007. The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments. Biosens Bioelectron 22: 2532-2538. http://www.ncbi.nlm.nih.gov/pubmed/17088054
  • Sasaki K., Glass T.R., Ohmura N. 2005. Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method. Anal Chem 77: 1933-1939. http://www.ncbi.nlm.nih.gov/pubmed/15801721
  • Glass T.R., et al. 2004. Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects. Anal Chem 76: 767-772. http://www.ncbi.nlm.nih.gov/pubmed/14750874
  • Ohmura N., Lackie S., Saiki H. 2001. An immunoassay for small analytes with theoretical detection limits. Anal Chem 73: 3392-3399. http://www.ncbi.nlm.nih.gov/pubmed/11476240
 

  Referenz Mittelpunkt

In einer PLoS Publikation, von 2012, untersuchten die Forscher den dynamischen Bereich des Kinetischen Exklusions Ansatzes (KinExA) zur Charakterisierung von Antigen-Antikörper Interaktionen. Aufgrund der Vielseitigkeit des KinExA konnten Experimente mit rekombinanten oder nicht-aufgereinigten Antikörpern durchgefüht werden. Durch die Verwendung vom Standard- oder Revers-Ansatz-Format konnte das erforderliche Material minimiert werden. Anti-Idiotypen wurden, wenn möglich, eingesetzt, um Materialmenge und Kosten zu reduzieren. Die Spannweite der Kd-Werte, für die getesteten Systeme, umfasste sechs Grössenordnungen, von ungefähr 100fM bis 100nM.

Bee Christine, et al. 2012. Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions. PLOS ONE 7(4): e36261 http://www.ncbi.nlm.nih.gov/pubmed/22558410

KinExA Referenzen

Affinitäts- & Kinetik-Messungen:

KinExA Technologie, Überblick:

KinExAs Rolle in der Pharmaforschung:

Signifikanz von Messungen in „biologischen Lösungen“ für unmodifizierte Moleküle:

  • Kusano-Arai 0., et al. 2016. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus. Anal Biochem. 10.1016/j.ab.2016.04.004. https://www.ncbi.nlm.nih.gov/pubmed/27095060
  • Blake R.C., Li X., Blake D.A. 2007. Covalent and noncovalent modifications induce allosteric binding behavior in a monoclonal antibody. Biochemistry 46: 1573-1586. http://www.ncbi.nlm.nih.gov/pubmed/17279622

Sensitivität in der Messung fester Bindungspartner:

Reversansatz-Techniken:

Messungen in Zellsystemen:

  • Bedinger, D., et al. 2015. Differential pathway coupling of activated insulin receptor drives signaling selectivity by XmetA, an allosteric partial agonist antibody. J Pharmacol Exp Ther 353(1):35-43. http://www.ncbi.nlm.nih.gov/pubmed/25613982
  • Rathanaswami P., Babcook J., Gallo M. 2008. High-affinity binding measurements of antibodies to cell-surface-expressed antigens. Anal Biochem 373: 52-60. http://www.ncbi.nlm.nih.gov/pubmed/17910940
  • Xie L., 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: 1-14. http://www.ncbi.nlm.nih.gov/pubmed/16098983

Nicht aufgereinigte Antigene:

Andere interessante Studien:

  • Li X., Kaattari S.L., Vogelbein M.A., Vadas G.G., Unger M.A., 2016. A highly sensitive monoclonal antibody based biosensor for quantifying 3-5 ring polycyclic aromatic hydrocarbons (PAHs) in aqueous environmental samples. Sens Biosensing Res. 7:115-120. https://www.ncbi.nlm.nih.gov/pubmed/26925369
  • Lou J., et al. 2010. Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating. Protein Eng Des Sel 23(4): 311-319. http://www.ncbi.nlm.nih.gov/pubmed/20156888
  • Kahle K.M., Steger H.K., Root M.J. 2009. Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding. PLOS Path 5(11): 1-11. http://www.ncbi.nlm.nih.gov/pubmed/19956769
  • Nowakowski A., et al. 2002. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proc Natl Acad Sci 99: 11346-11350. http://www.ncbi.nlm.nih.gov/pubmed/12177434

Immunoassay Techniken:

  • Darwish I.A., et al. 2013. Kinetic-exclusion analysis-based immunosensors versus enzyme-linked immunosorbent assays for measurement of cancer markers in biological specimens. Talanta 111: 13-19. http://www.ncbi.nlm.nih.gov/pubmed/23622520
  • Prieto-Simon B., Miyachi H., Karube I., Saiki H. 2010. High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples. Biosens Bioelectron 25: 1395-1401. http://www.ncbi.nlm.nih.gov/pubmed/19939663
  • Sasaki K., Oguma S., Namiki Y., Ohmura N. 2009. Monoclonal antibody to trivalent chromium chelate complex and its application to measurement of the total chromium concentration. Anal Chem 81: 4005-4009. http://www.ncbi.nlm.nih.gov/pubmed/19438265
  • Glass T.R., Ohmura N., Saiki H. 2007. Least detectable concentration and dynamic range of three immunoassay systems using the same antibody. Anal Chem 79: 1954-1960. http://www.ncbi.nlm.nih.gov/pubmed/17256970
  • Bromage E.S., et al. 2007. The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments. Biosens Bioelectron 22: 2532-2538. http://www.ncbi.nlm.nih.gov/pubmed/17088054
  • Sasaki K., Glass T.R., Ohmura N. 2005. Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method. Anal Chem 77: 1933-1939. http://www.ncbi.nlm.nih.gov/pubmed/15801721
  • Glass T.R., et al. 2004. Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects. Anal Chem 76: 767-772. http://www.ncbi.nlm.nih.gov/pubmed/14750874
  • Ohmura N., Lackie S., Saiki H. 2001. An immunoassay for small analytes with theoretical detection limits. Anal Chem 73: 3392-3399. http://www.ncbi.nlm.nih.gov/pubmed/11476240