Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for (125)I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-alpha-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.     

Single-label time-resolved luminescence assay for estrogen receptor--ligand binding

Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR–FRET) assays provide high sensitivity due to low background signal. The TR–FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)–ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu–E₂) is quenched using soluble quencher molecules. The luminescence signal of Eu–E₂ on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu–E₂ from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8–9.2) were clearly higher than the S/B values for the FP assay (1.3–1.5). A K_d value of 30 nM was calculated for binding of Eu–E₂ to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.     

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.     

Development of a time-resolved fluorescent assay for measuring tyrosine-phosphorylated proteins in cells

We have developed a time-resolved fluorescent assay using Wallac's DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein-tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells.     

A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen-antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-alpha-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.     

A time-resolved fluorescence resonance energy transfer-based HTS assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone required for the stability and function of a number of client proteins, many of which are involved in cancer development. The natural products geldanamycin (GM) and radicicol (RD) are known inhibitors of Hsp90, and their derivatives are being developed for the treatment of various cancers. To identify novel Hsp90 inhibitors, a highly robust time-resolved fluorescence resonance energy transfer (TR-FRET)-based HTS assay that measures the binding of biotinylated geldanamycin (biotin-GM) to the His-tagged human Hsp90 N-terminal ATP-binding domain (Hsp90N) was developed. This assay was optimized in 1536-well plates and was used as the primary assay to screen 10(6) compounds. Identified "hits" were then confirmed in a scintillation proximity assay (SPA) and a DEAE membrane-based assay for [(3)H]AAG binding to Hsp90. In addition, a surface plasmon resonance (SPR) assay that measures the direct interaction of Hsp90 with its inhibitors was developed and used to further characterize the identified inhibitors. Several potent and reversible inhibitors of human Hsp90 with K(d) values measured in the high nanomolar range were identified.     

A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand–PXR interactions. Here we report the characterization of BODIPY FL–vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a K_d value of 673 nM. We provide evidence that BODIPY FL–vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL–vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL–vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Analysis of glycoprotein hormone receptor extracellular domain interactions using a solid-phase capture assay

The receptors for the glycoprotein hormones are unique in having a large extracellular domain that is responsible for mediating ligand binding. We describe the characterization, validation, and application of a solid-phase radioligand binding assay that can be used to assess the interaction of peptides and small molecules at the extracellular domain (ECD) of the follicle-stimulating hormone receptor (FSHR). The assay utilizes a C-terminal tag on the FSHR-ECD, which is used to capture the ECD and position it in a sterically favorable orientation on a solid-phase platform. Competition experiments with the cognate ligand, FSH, indicated that the interaction at the FSHR-ECD using the solid-phase assay was comparable to the full-length receptor assayed using a standard filtration assay. The utility of the assay was evaluated by competing several peptides and a small molecule for both the full-length FSHR and the FSHR-ECD. The solid-phase capture format allowed for the establishment of an assay to specifically evaluate compounds that interact at the ECD or require the full-length receptor, thereby facilitating structure-activity studies. This assay format should be applicable to the other receptors of this family.     

A solid-phase assay for identification of modulators of prion protein interactions

The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K-resistant form of the prion protein, which has been converted from the endogenous, proteinase K-sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high-throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and the protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a nonisotopic labeling strategy for large-scale screens.     

A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity.     

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K_d) of 6.5 μM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z′ factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.     

CA19-9时间分辨荧光免疫层析检测方法的建立

本研究旨在建立一种定量检测血清中CA19-9含量的时间分辨荧光免疫层析检测方法。采用双抗体夹心法与荧光免疫层析技术,以羧基荧光微球和NC膜为载体将CA19-9配对抗体进行标记和包被,制备CA19-9检测试纸条。通过标记、包被抗体量对工艺进行优化,并通过线性范围、最低检出限、精密性等性能指标对CA19-9时间分辨荧光层析检测方法进行评价。最终确定20μL荧光微球的标记抗体量为80μg,检测线包被抗体浓度为1.5 mg/mL时,检测时间为15 min,线性范围为12.5–800 U/mL,最低检出限为6.32 U/mL,批内精密性与批间精密性均小于15%,平均回收率为101%,与罗氏电化学发光检测试剂盒平行检测50份临床样本,两者相关系数为0.980 6。初步建立了定量检测血清中CA19-9的荧光免疫层析检测方法,有较好的临床应用前景。     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (K_d) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

Synthesis and lodination of human (phenylalanine13, tyrosine19) melanin-concentrating hormone for radioreceptor assay

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr¹³ and Val¹⁹ of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe¹³, Tyr¹⁹] -MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [¹²⁵I]-[Phe¹³, Tyr¹⁹]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD ) was 1.18 × 10^?10 M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as α-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.     

A serological assay for the detection of cell surface receptors of nerve growth factor

When single-cell suspensions prepared from embroyonic day 8 (E8) chick sensory ganglia are incubated with nerve growth factor (NGF), anti-NGF antiserum, and complement, an NGF-dependent cytotoxic kill of 20 (±3)% of the ganglia cells is observed. This percentage is increased by a factor of two when only the neuronal cells are tested. No kill is observed on the nonneuronal cell population representing 50% of the ganglia dissociate. When E8 sensory ganglia cells are cultured in the presence of NGF following cytotoxic kill, the large, phase-bright NGF-reponsive neurons are missing from the culture. These results indicate that the cells recognized in the cytotoxicity assay have to carry NGF-binding sites of type I, which is the one with the higher affinity of the two types of NGF-binding sites (I and II) present on sensory ganglia cells. This conclusion is further supported by the following data: (a) half maximal cytotoxicity is reached already at a concentration of NGF which is below the K_D of binding site I; (b) a washing step which removes all NGF bound to type II receptors while leaving a high percentage of type I receptors occupied has no effect on the percentage of ganglia cells killed. Using the cytotoxicity assay the presence of high-affinity binding sites of type I can be demonstrated on sensory ganglia cells from E8 chick embryos but not from E4 embryos and not on liver and heart cells from E8 embryos. Further, type I receptor-bearing cells were detectable in the brain using this assay. At E8, NGF receptors could be detected on cells of the forebrain and the tectum but not on brain stem cells. Cytotoxic kill of forebrain cells was found to be especially high at E8 and E9, and decreased by E10.     

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.