Fluorescence polarization immunoassay. Theory and experimental method

Dandliker, Walter B.; Kelly, Robert J.; Dandliker, J; Farquahar, J; J, Levin

doi:10.1016/0019-2791(73)90198-5

Cited by 194 publications

(53 citation statements)

References 10 publications

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“…FP has been employed in applications such as competitive immunoassay for antibody-antigen binding [18], DNA hybridization and detection [19], DNA-protein binding [20], and protein-ligand binding and enzyme assays [21]. When combined with separation techniques such as CE, FP measurements of separate complexes provide additional information on binding interactions [22][23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser‐induced fluorescence polarization using aptamers as probes

Guthrie

2006

Electrophoresis

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Binding stoichiometries between four DNA aptamers (RT12, RT26, RTlt49, and ODN93) and the reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1) were studied using affinity CE (ACE) coupled with LIF polarization and fluorescence polarization (FP). The ACE/LIF study showed evidence of two binding stoichiometries between the HIV-1 RT protein and aptamers RT12, RT26, and ODN93, suggesting that these aptamers can bind to both the p66 and p51 subunits of the HIV-1 RT. Only one binding stoichiometry for aptamer RTlt49 was found. The affinity complexes were easily separated from the unbound aptamers; however, the different stoichiometries were not well resolved. A complementary technique, FP, was able to provide additional information about the binding and supporting evidence for the ACE/LIF results. The ACE/LIFP study also revealed that the FP values of the 1:1 complexes of the HIV-1 RT protein with aptamers RT12, RT26, and ODN93 were always much greater than those of the 1:2 complexes. This was initially surprising because the larger molecular size of the 1:2 complexes was expected to result in higher FP values than the corresponding 1:1 complexes. This phenomenon was probably a result of fluorescence resonance energy transfer between the two fluorescent molecules bound to the HIV-1 RT protein.

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Section: Introductionmentioning

confidence: 99%

Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser‐induced fluorescence polarization using aptamers as probes

Guthrie

2006

Electrophoresis

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“…Eq. 4 shows that the correlation result, pl/2, will increase by 21/2 when NbIj = Nf. Because the x axis is given by Nf/NbIb, the corresponding x axis value is Ib.…”

Section: Resultsmentioning

confidence: 99%

“…Several fluorescence-based immunoassay techniques are currently in use or are undergoing clinical evaluation (1)(2)(3)(4). This paper introduces an approach that relies on the existence of long-lived correlations of fluorescently labelled complexes in solution.…”

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confidence: 99%

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Fluorescence immunoassay based on long time correlations of number fluctuations.

Nicoli¹,

Briggs²,

Elings³

1980

Proc. Natl. Acad. Sci. U.S.A.

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We report the development of a fluorescencebased immunoassay technique relying on the physical phenomena of random number fluctuations and diffusion, which we review. By determining the autocorrelation of the fluctuations in the fluorescent intensity, this method is able to measure the amount of labeled antigen or antibody that is bound to micrometer-sized carrier particles in solution. The principal advantage of this technique is its insensitivity to small, fast-diffusing sources. It also discriminates against weakly fluorescent contaminants of size comparable to the carrier particles. We demonstrate these attributes by using two model systems: a human IgG assay and an idealized system consisting of polystyrene fluorescent spheres and rhodamine dye. and diffusion of particles, in which the diffusion coefficient is inversely related to particle size. To distinguish between large, slowly diffusing sources of fluorescence (due to Ag*-Ab or Ab-Ab* attached to carrier particles) and small, rapidly diffusing ones, we simply keep track of the fluctuations in fluorescence intensity that occur for a given small volume element in the solution. Clearly, any fluctuation in intensity that occurs due to a fluctuation in the number of particles present in the volume will, on average, persist for a time which depends inversely on the rate of diffusion of the particles into or out of the sampling volume. For the large tagged carrier particles these fluctuations will be very long lived. For any small, freely diffusing fluorescing molecules there will effectively be no "memory" of a fluctuation having occurred at a finite time earlier, provided that the sampling volume is made appropriately small. the root mean square magnitude of the fluctuations is N1'2. A convenient way to monitor these fluctuations is to evaluate the familiar intensity autocorrelation function, [1] in which I(t') is the fluorescent intensity originating from OV at time t' and the symbol ( ... )tt indicates an average of the intensity product over a large number of sampling times t'.We now periodically sample the fluorescent intensity at hundreds of locations within the solution, using in each case a volume of the same size (bV 10-6 cm3). The large number of samples increases the signal/noise ratio of function C(t). When time t equals an integral multiple of the repetition period T, the two intensities that form the product in Eq. 1 refer to the same volume element, for a given t'. The usefulness of the autocorrelation function in our application depends upon the fact that a number fluctuation in WV has a finite lifetime, r. Fig. la is an idealized trace of the intensity I(t') showing two fluctuations, each of which repeats after the period T (» >> T). In reality, the intensity fluctuations do not resemble the regular, symmetric pattern of Fig. la; rather, they appear as "random" noise (as illustrated in Fig. 2). This idealization was chosen to permit a simple algebraic computation of C(t) as discussed below. We assume a mean number of particles N per...

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“…[5][6][7][8] Fluoroimmunoassays are suited for adaptation to microfluidic biosensors due to the availability of standard protocols for fluorescent tagging of biomolecules as well as miniaturized optical detectors for sensing the fluorescence. Some of the limitations in these techniques, especially in a miniaturized device, include background noise from the unbound analyte as well as from the environment, limited ability to reuse the device with surfacebound receptors, and in many cases the inability to detect more than one analyte simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Phase sensitive enhancement for biochemical detection using rotating paramagnetic particle chains

Vuppu

García

Hayes

et al. 2004

Journal of Applied Physics

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Paramagnetic particle suspensions placed in a rotating unidirectional magnetic field form magnetic chains that rotate with the same frequency as the field. The motion of the fluid and particles surrounding the chain differs in phase and frequency from the chain rotation, a phenomenon that forms the basis of a sensitive detection scheme. Fluorescent particles that bind to the paramagnetic particles through their surface chemistry are used to demonstrate the concept. Epifluorescence video microscopy is used to capture images of the rotating chains. View windows placed over sequential images of rotating chains allows for measurement of the fluorescence brightness in the window, which is composed of periodic signal from the steady rotation of the chain plus the background. A lock-in reference synchronized to the chain rotation is used to enhance the fluorescence signal from chain and improve signal to noise. Two different modes of chain rotation and signal collection are demonstrated. This technique can be used to develop a fast and sensitive, homogenous microdevice based solid-phase immunoassay.

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Fluorescence polarization immunoassay. Theory and experimental method

Cited by 194 publications

References 10 publications

Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser‐induced fluorescence polarization using aptamers as probes

Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser‐induced fluorescence polarization using aptamers as probes

Fluorescence immunoassay based on long time correlations of number fluctuations.

Phase sensitive enhancement for biochemical detection using rotating paramagnetic particle chains

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