Fluorescence polarization in immunochemistry

Dandliker, Walter B.; Saussure, V.A de

doi:10.1016/0019-2791(70)90221-1

Cited by 113 publications

(48 citation statements)

References 79 publications

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“…Alternatively, a correction factor, G, can be introduced to the measurement of fluorescence polarization, P, according to [10,[12][13][14].…”

Section: Determination Of Fluorescence Polarizationmentioning

confidence: 99%

“…Fluorescence polarization is governed by the orientation of a molecule at excited state relative to the electric vector of the excitation [10,[12][13][14]. Depolarization occurs in solution due to the loss of molecular orientation resulted from rotational diffusion.…”

Section: Introductionmentioning

confidence: 99%

“…Depolarization occurs in solution due to the loss of molecular orientation resulted from rotational diffusion. The magnitude of fluorescence polarization in solution depends on the fluorescence lifetime and the rotational relaxation time of the molecule [12][13][14]. To the first order of approximation, spherical molecules rotational relaxation time, r, is related to parameters of both the fluorescent molecules and the solvent by r = 3ZV/RT (1) where Z and T are the viscosity and temperature of the solution, respectively, V is the molar volume of the fluorescent analyte, and R is the gas constant.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence polarization detection for affinity capillary electrophoresis

Wan

Lam

2002

Electrophoresis

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Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.

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“…Alternatively, a correction factor, G, can be introduced to the measurement of fluorescence polarization, P, according to [10,[12][13][14].…”

Section: Determination Of Fluorescence Polarizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Fluorescence polarization detection for affinity capillary electrophoresis

Wan

Lam

2002

Electrophoresis

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“…Mixing of one hydrocarbon with another occurs readily, despite the fact that the process is endothermic (AH mixing is positive) or has a negligible AH, at best [1]- [3]. This process is driven by the increased randomness or disorder occuring upon mixing two pure compounds; this makes AS mixing positive.…”

Section: Thermodynamic Considerationsmentioning

confidence: 99%

Principles governing biomolecule interactions at foreign interfaces

Hoffman

1974

J. Biomed. Mater. Res.

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SummaryThe adsorption, conformational changes, and desorption of biological molecules at foreign interfaces are effected by both kinetic and thermodynamic factors. The thermodynamic factors which drive the biomolecule/foreign interface system towards equilibrium include the enthalpy and entropy changes occurring during each step in the adsorption process. The principles governing interaction of proteins with nonwettable (nonpolar) and wettable (polar) foreign interfaces are discussed in terms of these thermodynamic parameters. Some examples to illustrate these arguments are presented for plasma-protein interactions on Silastic and radiation-grafted hydrogel/Silastic interfaces.

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“…The steady-ciation (7-9), fluorescence polarization to measure state anisotropy of Ru-HSA increased approximately changes in the rotational correlation time, and fluoreseightfold upon binding to the antibody. These spectral cence quenching or enhancement (10)(11)(12)(13)(14). In all of effects were observed both in the direct association of these methods the assays are based on the use of or- stability.…”

mentioning

confidence: 99%

Fluorescence Energy Transfer Immunoassay Based on a Long-Lifetime Luminescent Metal–Ligand Complex

Youn

Terpetschnig

Szmacinski

et al. 1995

Analytical Biochemistry

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We describe an immunoassay based on fluorescence resonance energy transfer (FRET). The antigen was human serum albumin (HSA), which was labeled with a ruthenium-ligand complex, [Ru(bpy)2(phen-ITC)]2+. The antibody (IgG) to HSA was labeled with a nonfluorescent absorber, Reactive Blue 4. Association of the Ru-labeled HSA with the antibody was detected by three spectral parameters, a decreased quantum yield of Ru-HSA, a decrease in its fluorescence lifetime, and an increase in its fluorescence anisotropy. The steady-state anisotropy of Ru-HSA increased approximately eightfold upon binding to the antibody. These spectral effects were observed both in the direct association of the Ru-HSA with Reactive Blue 4-labeled antibody, and in a competitive assay format wherein unlabeled HSA competed with Ru-HSA for the binding sites on the antibody. Some nonspecific interactions of HSA may have occurred with Reactive Blue 4-labeled AHA, a difficulty which can be avoided with a different acceptor. The use of FRET provides a reliable means to alter the spectral properties upon antigen-antibody binding. The advantages of a ruthenium-ligand fluorophore include its long-wavelength absorption and emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluorescence from biological samples, and long life-times allow off-gating of the prompt autofluorescence.

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Fluorescence polarization in immunochemistry

Cited by 113 publications

References 79 publications

Fluorescence polarization detection for affinity capillary electrophoresis

Fluorescence polarization detection for affinity capillary electrophoresis

Principles governing biomolecule interactions at foreign interfaces

Fluorescence Energy Transfer Immunoassay Based on a Long-Lifetime Luminescent Metal–Ligand Complex

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