Combinatorial Chemistry & High Throughput Screening - Volume 6, Issue 3, 2003

Fluorescence polarization was first observed in 1920 and during the next few decades the theoretical foundations of the phenomenon were clearly established. In the last two decades of the 20th century, fluorescence polarization became one of the most prevalent methods used in clinical and biomedical sciences. In this article we review the history of fluorescence polarization, its theoretical foundations and some of the more important practical developments, which helped to popularize the method. We also discuss important, but often misunderstood, practical considerations including the wavelength dependence of the limiting polarization and the effect of energy transfer on polarization. The present state of fluorescence polarization, both in pure research as well as in the applied biosciences is also reviewed. Finally, we speculate on possible future developments in the field, such as the use of multi-photon techniques.

A general survey of the analytical application of kinetic methodology in fluorescence polarization immunoassay (FPIA) is presented. Stopped-flow mixing technique (SF) allows the initial rate of the immunochemical reaction between the tracer and the antibody to be obtained, which is used as the analytical parameter instead of the equilibrium signal used in conventional FPIA. The instrumentation required is described and the features of the analytical methods proposed are compared with those obtained by conventional FPIA. The usefulness of SF-FPIA for routine screening in clinical, environmental and food analysis is discussed.

Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. This review will discuss fluorescence polarization assays in current use in drug discovery research as well as those in development that will likely be used in the near future. These assays include targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors.

Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP.

This article describes some recent developments in the field of fluorescence polarization (FP) as applied to enzyme assays and single nucleotide polymorphism (SNP) genotyping. First, we present our recent progress on the application of fluorescence polarization to high throughput screening (HTS). We show how the use of a 2-thiopyridinone-based, mixed disulfide biotinylation reagent can shorten the assay time of our recently reported kinase assay method based on thiophosphorylation and biotinylation from several hours to a few minutes. We also summarize our recent findings on a new approach for HTS of kinases, proteases and phosphatases based on the use of a cationic poly-amino acid such as polyarginine. We show how the careful selection of the polyarginine concentration and the ionic strength of the solution during the FP measurement allow one to expand the range of substrates that can be assayed. Both of these methods are valuable additions to the existing techniques for HTS. Most importantly, both of these methods can be applied to the assay of kinases without the need for any antibodies. In the area of genomics, we present some results from our studies on a new single nucleotide polymorphism typing approach based on the polymerase catalyzed extension of 3'fluorescein labeled primers. Contrary to our initial expectations, we observed that the enzymatic extension of these primers results in a significant decrease of the fluorescence polarization value. Possible explanations of this phenomenon are discussed.

Single nucleotide polymorphisms (SNPs) are the most abundant variations in the human genome and have become the primary markers for genetic studies for mapping and identifying susceptible genes for complex diseases. Methods that genotype SNPs quickly and economically are of high values for these studies because they require a large amount of genotyping. Fluorescence polarization (FP) is a robust technique that can detect products without separation and purification and it has been applied for SNP genotyping. In this article the applications of FP in SNP genotyping are reviewed and one of the methods, the FP-TDI assay, is discussed in details. It is hoped that readers could get useful information for the applications of FP in SNP genotyping and some insights of the FP-TDI assay.

The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46°C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl2 were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl2. Moreover, in the presence of 0.2 M MgCl2, there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.

Fluorescence Polarization Assays (FPAs) have been shown to have great utility in the detection of infectious diseases. Examples are presented of the use of O-polysaccharides (OPSs) for the detection of antibodies in serum, whole milk and whole blood to gram negative organisms (Brucella spp., Salmonella spp.). The use of proteins and peptides are also described for the detection of Mycobacterium bovis and Equine Infectious Anemia Virus. Fluorescence Polarization Inhibition Assays (FPIAs) are discussed for the specific and sensitive detection and quantitation of Salmonella spp. cells from culture. An example of the detection of enterohemorrhagic E. coli (EHECS) by Strand Displacement Amplification (SDA), coupled with FP, down to the single cell level, within thirty minutes, is described.

Antibodies raised against a given metal ion complex of a polyaminopolycarboxylate chelating agent can display specificity for the immunizing chelate and, when used in conjunction with a fluorophorelabeled analog of that chelate, can form the basis for highly sensitive and specific methods for detecting that metal ion by competitive inhibition fluorescence polarization immunoassay (FPIA). Chelate complexes of ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA) and of a hetrocyclic ring-substituted derivative of diethylenetriamine-N, N', N”-triacetic acid (DTTA) have been used to configure such assays for the heavy metal ions lead(II) and cadmium(II) respectively. Limits of detection for the 1:1 metal chelates under ideal conditions are 20 ppt for lead(II) and below 100 ppt for cadmium(II). Standard curves for 0 - 100 nM cadmium (II) chelate can be constructed in the presence of fixed 250 nM concentrations of the corresponding, potentially cross-reactive chelates of zinc(II), copper(II) and mercury(II). Cross-reactivity of the lead (II) FPIA with 15 non-target metals is below 0.2% in all cases except for mercury(II) (0.37%). These characteristics have allowed the development of FPIA methods for the quantitative analysis of lead in a variety of samples relevant to environmental monitoring, including soil, dust, solid wastes and drinking water. Although applied thus far to heavy metals that are of concern as toxic contaminants in the environment, anti-chelate FPIA methods are also in principle applicable to a wide variety of other metal ions, including precious metals and various transition and main group elements used or monitored in a range of industrial applications. As conventional methods for trace metal analysis based on atomic spectroscopy are relatively slow, expensive and cumbersome, anti-chelate FPIA methods have the potential to supplant many existing techniques and in so doing extend the use of immunoassay technology beyond the biomedical, veterinary and agricultural spheres in which it has historically found use.

Fluorescence polarization immunoassay methods for the detection of pesticides and their metabolites or degradation products are reviewed. Advantages and limitations for application to pesticide detection in environmental and food samples are discussed. The influence of the structure of fluorescentlabeled tracers and the affinity and specificity of antibodies on analytical performance is examined. The methods are simple, readily automated, and rapid (total time for assay of a water sample is about 1 min) with sensitivity of 1 - 10 ng / ml pesticide in 0.01 - 0.1 ml sample.

Successful use of fluorescence polarization assays (FPAs) in human clinical, infectious disease, and drug discovery fields has prompted us to extend its use to the grain mycotoxin field. An antibody specific to a mycotoxin and a mycotoxin-fluorophore conjugate are developed. Free toxin (extracted from the grains with a suitable solvent) competes with the toxin-fluorophore conjugate for the antibody and a change in FP relative to the quantity of free toxin occurs. This change is compared to a standard curve obtained by using known quantities of toxin. The use of FP and toxin-fluorophore conjugates for the quantification of fumonisins, deoxynivalenol and aflatoxins is described. These assays are field portable, simple to perform, rapid and require no washing steps.