Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.     

Fluorescence correlation spectroscopy of enzymatic DNA polymerization

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.     

Dynamics of fluorescence marker concentration as a probe of mobility

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.     

Characterization of scFv-421, a single-chain antibody targeted to p53

An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.     

Analyte and label binding assay read by flow cytometry

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.     

A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sj?gren's syndrome contain increased latent and active gelatinase-B levels

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys/IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly/IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sj?gren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 +/- 9.8 U/mg, controls 16.5 +/- 2.6 U/mg (p = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.     

Mass-sensing, multianalyte microarray immunoassay with imaging detection

Miniaturization of ligand binding assays may reduce costs by decreasing reagent consumption, but it is less apparent that miniaturized assays can simultaneously exceed the sensitivity of macroscopic techniques by analyte "harvesting" to exploit the total analyte mass available in a sample. Capture reagents (avidin or antibodies) immobilized in 200-microm diameter zones are shown to substantially deplete analyte from a liquid sample during a 1-3-h incubation, and the assays that result sense the total analyte mass in a sample rather than its concentration. Detection of as few as 10(5) molecules of analyte per zone is possible by fluorescence imaging in situ on the solid phase using a near-infrared dye label. Single and multianalyte mass-sensing sandwich array assays of the IgG subclasses show the sensitivity and specificity of ELISA methods but use less than 1/100 the capture antibody required by the 96-well plate format.     

Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic diseases

Olopatadine (AL-4943A; KW-4679) [(Z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2-acetic acid hydrochloride] is an antiallergic/antihistaminic drug under development for topical ocular use. The effects of the compound on release of proinflammatory mediators (histamine, tryptase and prostaglandin D2) from monodispersed human conjunctival mast cells were assessed. Histamine receptor subtype binding affinities and functional potencies were determined with ligand binding and phosphoinositide turnover assays, respectively. Olopatadine inhibited the release of histamine, tryptase and prostaglandin D2, in a concentration-dependent manner (IC50 = 559 microM). Evaluation of the interaction of olopatadine with histamine receptors revealed a relatively high affinity for the H1 receptor (Ki = 31.6 nM, pKi = 7.5 +/- 0.1, n = 7) but lower affinities for H2 receptors (Ki = 100 microM, pKi = 4.0 +/- 0.19, n = 7) and H3 receptors (Ki = 79.4 microM, pKi = 4.1 +/- 0.16, n = 7). The H1 selectivity of olopatadine was superior to that of other ocularly used antihistamines studied, such as ketotifen, levocabastine, antazoline and pheniramine. The profiling of olopatadine in 42 nonhistamine receptor binding assays revealed that olopatadine interacts with only two nonhistamine receptor/uptake sites to any significant degree (pIC50 < or = 5-6). Olopatadine inhibited histamine-induced phosphoinositide turnover in human conjunctival epithelial cells (IC50 = 10 nM, pIC50 = 8.0, n = 4) and in other human ocular cells (IC50 = 15.8-31.6 nM, pIC50 = 7.5-7.8) and exhibited apparent noncompetitive antagonist properties in these cells, with an estimated dissociation constant (Kb) of 19.9 nM (pKb = 7.7, n = 6). This combination of mast cell mediator release inhibition and selective H1 receptor antagonism suggests that olopatadine may be particularly useful in the treatment of ocular allergic diseases. Indeed, olopatadine has recently shown clinical efficacy in an allergic conjunctivitis model in human subjects.     

Detection of asymmetric PCR products in homogeneous solution by fluorescence correlation spectroscopy

The yield of the double-stranded DNA product (500 bp) of asymmetric PCR with a rhodamine-labeled primer (Rho-primer) was determined in a homogeneous solution using fluorescence correlation spectroscopy (FCS). FCS provides the average number of molecules in a focused volume and the diffusion constant that relates the molecular weight. Since FCS measures the fluctuation of fluorescence intensity in a very small sample volume, the reaction mixture was directly placed on the FCS optical field without any purification procedure after amplification. The result of changing the initial number of templates suggested that elongation of the Rho-primer could be detected by FCS in a PCR mixture containing a single copy of the target gene in the initial condition. Possible scientific applications and perspectives of the proposed approach are discussed.     

Will combinatorial chemistry deliver real medicines?

Over the next decade, the impact of library synthesis will play a major role in shortening the lead optimization phase of drug discovery. The prognosis for combinatorial chemistry to discover fundamentally different new classes of therapeutically active small molecules against some of the more difficult biological targets is less certain. Expectations are high because the technology potentially allows us to sample available drug space by synthesizing all possible small molecule ligands (variously estimated to be between 10(30)-10(50) compounds). Some caution is advised, however, since, despite recent increases in high-throughput screening of substantially greater numbers of synthetic compounds and natural products, we are not routinely finding a plethora of new structures. The outcome may be that combinational chemistry offers us the ability to work faster on finding ligands for well-established tractable targets, such as G-protein-coupled receptors, ion channels or proteases, rather than, say, the more complex protein-protein interactions which from the majority of targets in signal transduction pathways.     

The predictability of periodontal treatment as measured by tooth loss: a retrospective study

OBJECTIVE: To analyse the validity of the Micraltest and Microbumintest semi-quantitative methods for microalbuminuria screening in type 2 diabetes mellitus in primary care clinics. DESIGN: Crossover study to validate diagnostic tests. SETTING: Three general practices at an urban health centre. PATIENTS: Consecutive sample of 64 diabetics not dependent on insulin. MEASUREMENTS AND MAIN RESULTS: Each patient's own doctor performed in the consultation the semi-quantitative determinations in a simple sample of the morning's first urine. As standard, a rate of albumin excretion above 20 micrograms/min, determined by immunonephelometry in a 2-hour controlled time sample, was used. The prevalence of Microalbuminuria was 25% (C.I. 95%, 14.4-35.6). The sensitivity of Micraltest was 69% (CI, 42-88), and its specificity 52% (CI, 37-67), corresponding to the cut-off point of 10 mg/L. The sensitivity of Microbumintest was 63% (CI, 36-84), and its specificity 67% (CI, 52-79). The combination of both tests in one sample had 75% sensitivity (CI, 47-92) and 44% specificity (CI, 30-59). Negative predictive values ranged between 84 and 85%. CONCLUSIONS: The semi-quantitative microalbuminuria detection tests are of limited use in the primary care clinic. Their sensitivity and the negative predictive value obtained in an isolated sample do not seem acceptable for a screening method.     

CD10 is expressed on human thymic epithelial cell lines and modulates thymopentin-induced cell proliferation

Thymic hormones such as thymopoietin (TP) have been shown to regulate thymocyte differentiation and lymphocyte activation. However, it is not known whether thymopoietin affects thymic epithelial cell (TEC) functions. In this study we have examined the effect of a five amino acid active peptide (TP5), corresponding to amino acids 32-36 of TP, on the proliferation of nontransformed clones of human TEC. Our results indicate that TP5 induced reinitiation of DNA synthesis and potentiated fetal calf serum (FCS)-induced cell growth in postnatal and fetal-derived human TEC. We also found that TEC lines express high levels of endopeptidase 24.11, a cell-surface metallopeptidase also known as the CD10 antigen. We show that TP5 is cleaved by CD10 at the surface of TEC lines, indicating that this endopeptidase may regulate TP5-induced TEC proliferation. Phosphoramidon, a specific endopeptidase 24.11 inhibitor, consistently acts in synergy with TP5 to enhance FCS-induced TEC growth. Hence, we conclude that 1) TP5 alone or in combination with FCS supports the growth of TEC lines, and 2) TEC lines express high levels of CD10, which regulates TP5-induced TEC proliferation by acting as a thymic peptide degrading enzyme.     

Local drug delivery with porous balloons in the rabbit: assessment of vascular injury for an improvement of application parameters

OBJECTIVES: Sufficient intramural drug concentrations with the use of porous balloon catheters can be achieved with additional vascular trauma only. However, effective delivery of a potent drug even in deeper layers of the vessel wall might outweigh these traumatic side effects. Given the porous balloon catheter, the parameters of injection pressure and applied fluid volume will influence the interventional result. METHODS: We tested a 2.5-mm porous balloon (35 75-micron pores) in the right carotid artery of New Zealand rabbits and used injection pressures of 1, 2, and 5 atm and fluid volumes of 2 and 4 ml of low-molecular-weight heparin solution in combination with the different parameters (n = 5 animals/group). In 50 rabbits, an intimal fibromuscular plaque was induced by using the electrostimulation model. Balloon dilatation and then application of the porous balloon was performed in 30 animals, 10 animals were only electrostimulated, and 10 animals served as a control group with balloon dilatation only. The vessels were excised 7 d after intervention, stained, and analyzed histomorpologically. Anti-Xa assays revealed the extent of systemically escaped drug, and serial cuts allowed for exact determination of vessel wall injuries. RESULTS: Effective local drug delivery could not be achieved with an injection pressure of less than 2 atm. Specific pressure-driven effects such as jet injuries could be identified. When the pressure was high enough for disruptive drug delivery (> or = 2 atm), fluid volumes of 4 ml led to loose elastic membranes and local thickening within the media. CONCLUSIONS: Sufficient intramural drug distribution using porous balloon catheters can be achieved with low injection pressures. Different fluid volumes strongly determine the extent of additional vascular injury.     

Factors controlling haemopoiesis in ovine long-term bone marrow cultures

There is increasing interest in both standardization and simplification of methods for enumeration of CD34+ hematopoietic progenitor cells (HPC) to facilitate cellular therapies and to improve interinstitutional comparison of clinical and laboratory results. We evaluated a novel method for CD34+ cell enumeration based on microvolume fluorimetry (MVF) compared with our laboratory's routine flow cytometric method on samples of peripheral blood and leukapheresis products. The MVF method is semiautomated and uses a 633-nm light from a helium-neon laser to scan fluorochrome-labeled cells held in stasis in a capillary known volume. The performance of the MVF assay for enumeration of CD34+ cells was found to be comparable to our routine flow cytometric assay in linearity and accuracy in the range of 5-1500 cells per microliter. Precision of MVF for replicate assays on the same instrument was demonstrated by coefficient of variation (CV) values of 8.4% at a CD34+ cell concentration of 284/microliters for a sample volume of 0.8 microliters, and 15.7% at 12/microliters for a sample volume of 3.2 microliter. Precision among three different instruments was demonstrated, using sample volumes of 1.6 microliters, by CV values of 44% at 6 cells/microliters and 4.6% at 733 cells/microliters. In a field sample evaluation, precision of the entire assay system for paired measurements on 0.8-microliter sample volumes was demonstrated by CV values of 50%, 31%, and 15% for peripheral blood samples with concentrations of 0-10, 10-20, and 20-100 CD34+ cells/microliters, respectively, and 6.3%, 8.1% and 6.5% for leukapheresis samples with concentrations of 0-100, 100-1,000, and 1,000-2,500 CD34+ cells/microliters, respectively. The MVF assay was easy to perform, required minimal technical training time, and had a turnaround time of 40 min, of which less than 10 min was actual technical time. These observations suggest that the MVF method for CD34+ cell enumeration may prove useful to clinical laboratories providing support for HPC collection, processing, and transplantation services that require relatively simple, rapid assays for product quality control or to guide real-time clinical decisions.     

Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10(6) samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.     

Development and preliminary validation of a microtiter plate-based receptor binding assay for paralytic shellfish poisoning toxins

More than 20 countries have either established or proposed regulatory limits for one or more of the paralytic shellfish poisoning (PSP) toxins as they occur in seafood products. PSP toxin levels are generally estimated using the standard AOAC mouse bioassay, yet because of various limitations of this method [e.g. high variability (+/-20%), low sensitivity, limited sample throughput and use of live animals], there remains a need for alternative testing protocols. A sensitive and selective, high capacity assay was developed for the PSP toxins which exploits the highly specific interaction of these toxins with their biological receptor (i.e. voltage-dependent sodium channel) and is thus based on functional activity. This receptor binding assay provides a radioactive endpoint, and is performed in a microtiter filter plate format with results determined by standard liquid scintillation counting within 24 hr. The Ki for the assay is 3.66 +/- 0.86 nM saxitoxin, with a limit of detection of c. 5 ng saxitoxin/ml in a sample extract. Good quantitative agreement of the assay with both mouse bioassay and high-performance liquid chromatographic analysis of crude extracts of contaminated shellfish, as well as PSP toxin-producing algae, was observed. Our findings indicate that the receptor binding assay has a strong predictive value for toxicity determined by mouse bioassay, and that this approach warrants consideration as a rapid, reliable and cost-effective alternative to live animal testing for detection and estimation of PSP-related toxicity in seafood and toxic algae.     

Affinity capillary electrophoresis in biomolecular recognition

Affinity capillary electrophoresis is a new method for studies of biomolecular recognition. Applications reported in the literature include chiral separation of racemic biomolecules, measurement of binding constants, estimation of kinetic on- and off-rate constants, determination of binding stoichiometries (a useful tool in examining electrostatic interactions), estimation of effective charges and molecular weights of proteins, characterization of enzymatic activities and library screening for tight-binding drug candidates in solution. This technique demands only small amounts of sample (nanolitre injection volumes, picograms of proteins), involves no radiolabelled materials or chemically immobilized ligands, and does not require changes in spectroscopic characteristics upon binding. This paper reviews the most recent applications of affinity capillary electrophoresis and its use in the analysis of biomolecules.     

Maternal serum alpha-fetoprotein and dimeric inhibin A detect aneuploidies other than Down syndrome

OBJECTIVE: Our purpose was to determine whether the combination of maternal serum alpha-fetoprotein, free human chorionic gonadotropin-beta, dimeric inhibin A, and maternal age detects aneuploidies other than Down syndrome. STUDY DESIGN: We retrieved stored serum from pregnancies complicated by aneuploidies other than Down syndrome from 1988 to 1997 (n = 55, mean maternal age 35.2 +/- 5.6 years). Alpha-fetoprotein levels were obtained from our database, and free human chorionic gonadotropin-beta and dimeric inhibin A levels were measured in the thawed serum with use of commercial assays. Analyte values were used in both 3-analyte and 2-analyte multiple-marker screening tests; detection rates were determined at several different Down syndrome risk-positive cutoff values. RESULTS: In the 3-analyte test 58% (32/55) of all aneuploidies were detected with use of both the Down syndrome protocol at a screen-positive risk cutoff value of 1:300 (false-positive rate 17%) and a novel trisomy 18 screening algorithm. However, 67% (37/55) detection was obtained with use of the 2-analyte combination of alpha-fetoprotein and dimeric inhibin A, with both the Down syndrome protocol (screen positive cutoff value 1:300) and the trisomy 18 algorithm: 12 of 13 trisomy 18 (92%), 9 of 17 Turner's syndrome (53%), 10 of 17 other sex chromosome aneuploidies (59%), 1 of 1 trisomy 22 (100%), and 5 of 7 trisomy 13 (71%). CONCLUSIONS: The combination of maternal serum alpha-fetoprotein, dimeric inhibin A, and maternal age detects autosomal trisomies other than Down syndrome at a rate superior to that of the traditional analyte combination.     

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.