1994
DOI: 10.1007/bf01901562
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Spectroscopic evidence for charge-transfer complexation in monoclonal antibodies that bind opiates

Abstract: Molecular complexes of four monoclonal anti-morphine antibodies (mAb) with the opiate ligands morphine, oxymorphone, and naloxone were studied using UV-VIS absorption spectroscopy. Although strong overlaps in the absorption spectra of the antibodies, ligands, and complexes were observed, a curve-fitting method was developed to correlate the absorbance with the concentration of the ligand-antibody complex. Using this technique, we determined the intrinsic association constants for the mAb with morphine to be in… Show more

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Cited by 7 publications
(9 citation statements)
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References 32 publications
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“…In doing so, we optimized a novel method that has the potential for broad-reaching applications within the field of immunology as a tool for measuring vaccine-induced unlabeled polyclonal antibody affinities to various unlabeled ligands. As expected, the K i value of ab1060 to morphine derived from MST is consistent both with the manufacturer reported K d value of 2 nM derived from UV-VIS [ 28 , 29 ] and with the K d value that we derived from ED-UPLC/MS/MS [ 13 ]. In contrast to ED-UPLC/MS/MS, the K i values of ab1060 to morphine do not significantly vary when different tracer and ab1060 concentrations are used.…”
Section: Discussionsupporting
confidence: 89%
See 1 more Smart Citation
“…In doing so, we optimized a novel method that has the potential for broad-reaching applications within the field of immunology as a tool for measuring vaccine-induced unlabeled polyclonal antibody affinities to various unlabeled ligands. As expected, the K i value of ab1060 to morphine derived from MST is consistent both with the manufacturer reported K d value of 2 nM derived from UV-VIS [ 28 , 29 ] and with the K d value that we derived from ED-UPLC/MS/MS [ 13 ]. In contrast to ED-UPLC/MS/MS, the K i values of ab1060 to morphine do not significantly vary when different tracer and ab1060 concentrations are used.…”
Section: Discussionsupporting
confidence: 89%
“…To confirm that MST can accurately measure antibody binding affinities, the K i of morphine to ab1060 was measured. This mouse mAb binds morphine with a manufacturer-reported K d value of 2 nM, as determined by a proprietary ultraviolet-visible (UV-VIS) absorption spectroscopy method [ 28 , 29 ] and by ED-UPLC/MS/MS [ 13 ]. Since neither ab1060 nor morphine contain strong fluorophores to allow the direct measurement of binding interactions by MST, and since the presence of a fluorophore on the antibody or the ligand had the potential to produce inaccurate binding data, our strategy was to measure the K i in a two-step process.…”
Section: Mst Of Morphine Monoclonal Antibodymentioning
confidence: 99%
“…Based on this information, the antibody concentration of the stock solution is ∼12.9 μM. ab1060 has a reported K d value of 2 nM, which was determined by proprietary ultraviolet-visible (UV-VIS) absorption spectroscopy method that correlated absorbance with the concentration of the ligand-antibody complex [ 26 ]. Samples for equilibrium dialysis were prepared by diluting the stock solution in DPBS to yield 2.4, 4.8, and 9.6 nM.…”
Section: Methodsmentioning
confidence: 99%
“…The emission is collected, as is routine in fluorescence spectroscopy, at right angles to the excitation path. The vertically and horizontally polarized emission intensities are measured separately and the polarization (P) and anisotropy (r) are calculated using the equations [23] and r ϭ…”
Section: Appendixmentioning
confidence: 99%
“…However, one may see double symbols used to identify the polarization of the excitation and emission paths distinctly. Equations [23] and [24] can be rewritten as P ϭ I vv Ϫ I vh I vv ϩ I vh [25] and r ϭ I vv Ϫ I vh I vv ϩ 2 ϫ I vh , [26] where the subscript symbols, from left to right, designate the excitation and emission paths, respectively. As a rule, optical parts of fluorometers possess unequal transmission (monochromators, lenses, filters) or varying sensitivities (photodetector) for vertically or horizontally polarized light.…”
Section: Appendixmentioning
confidence: 99%