Selective binding of polychlorinated biphenyl congeners by a monoclonal antibody: analysis by kinetic exclusion fluorescence immunoassay

A previously described monoclonal antibody, S2B1, was highly selective for coplanar (non-ortho-chlorinated) PCB congeners in enzyme immunoassays that measured binding at equilibrium. In the present study, kinetic exclusion fluoroimmunoassay (KinExA) was used to determine the dissociation constants (Kd) and on and off rates (k(on), k(off)) for binding of various PCB congeners to affinity-purified S2B1 IgG and Fab fragments in solution. This method revealed that mono- and di-ortho-chlorinated PCBs were bound by S2B1, but the on rates were slower, and the off rates faster by 6-60-fold, than with congeners that had no ortho chlorines. Although the sensitivity of immunoassays may be improved by using competing haptens that S2B1 binds more weakly than the parent PCB, the KinExA results demonstrate that congener specificity is an intrinsic property of S2B1 and does not require weaker binding haptens. KinExA also provided new information on the percentage of active binding sites, valence, and effects of buffer, solvent, and biotinylation on S2B1. The advantages and drawbacks of KinExA for measuring antibody-ligand binding are described.     

Combinational use of antibody affinities in an immunoassay for extension of dynamic range and detection of multiple analytes

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.     

Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.     

Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin using a uniform-labeled scFv immunoreagent

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.     

Human tear protein analysis enabled by an alkaline microfluidic homogeneous immunoassay

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sj?gren's syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.     

Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance.

Small molecules are difficult to directly detect using commercially available surface plasmon resonance (SPR) instruments. This is because low molecular weight compounds do not have sufficient mass to cause a measurable change in refractive index. Refractive index is sensitive, however, to other properties besides the mass of the analyte. Recently the detection of substantial conformational changes for immobilized proteins using SPR has been reported. However, this property has not yet been exploited for the detection of low molecular weight ligand binding to immobilized protein receptors. Here we demonstrate that ligand-induced conformational changes can be used to monitor the binding of small molecules to immobilized maltose-binding protein and tissue transglutaminase. Ligand binding to a receptor that decreases in hydrodynamic radius yielded a net decrease in refractive index. A net positive change in refractive index was observed for a receptor that increases in hydrodynamic radius. Refractive index changes could not be explained by addition of analyte molecular mass to the surface. These SPR responses were a result of specific receptor-ligand interactions, as judged by the reversibility of the response and the similarities between the SPR-determined equilibrium dissociation constants and reported dissociation constants. Additionally, this technique proved to be effective at detecting specific ligands from a panel of small molecules. This SPR method required no alterations in widely used and commercially available instrumentation yet allowed direct detection of very small molecules such as calcium ions (40 Da). Use of receptor conformation to detect low molecular weight analytes has potential applications in the high-throughput screening of small molecule drug libraries and the development of biosensors.     

Detection of chemicals by a reporter immunoassay: application to fluoride

This report describes a concept in which an immunoassay is used indirectly to quantify a nonantigenic very low molecular weight compound participating in a chemical reaction with a haptenic reporter. The detection limit of each reagent is, therefore, governed only by the affinity of the antibodies toward the reporter. Fluoride was used as a model, and silylated estradiol was used as a reporter. Upon silylation with N-O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-O-bis(dimethylterbutylsilyl) trifluoroacetamide (MTBSTFA), estradiol is no longer recognized by antibodies specific to estradiol. After reaction with hydrofluoric acid (HF) or fluoride salts (KF, CsF, NaF), its immunoreactivity is restored, and native estradiol is formed and is detected by immunoassay. The level of synthesized estradiol is dependent on the concentration of fluoride. A fluoride detection limit of 0.3 microg/L (15 nM) is obtained. Potential interference with other acids has been eliminated by choosing the silyl group (trimethylsilyl vs tert-butyldimethylsilyl) and by selecting optimal reaction conditions for the desilylation. The method has been applied to the detection of fluoride salts in natural waters (range 0.28-9.0 mg/L) and in an atmosphere artificially contaminated with HF between 8 and 160 microg/m(3) in the parts-per-billion range. This indirect immunoassay combines simplicity and high sensitivity and, therefore, can be used in field monitoring. Finally, the extension of the concept to other chemicals is discussed.     

Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: application to an enzyme immunometric assay

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly to monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2, 45-min irradiation) and 312 nm (40 J/cm2, 160-min irradiation). The irradiation fully denatured both individual molecules (i.e., 17 beta-estradiol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17 beta-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.     

DNA-encoding to improve performance and allow parallel evaluation of the binding characteristics of multiple antibodies in a surface-bound immunoassay format

High affinity capture agents against protein targets are essential components for immunoassays, regardless of specific analysis format. Here, we describe the use of DNA-encoded antibodies for rapidly screening the kinetic and equilibrium binding properties of twelve commercial antibodies in a parallel analysis format using a multiplexed array of microring optical resonators. We show that DNA-encoding offers advantages in terms of antigen binding capacity, compared to covalently tethered antibodies; we also demonstrate that this linkage modality facilitates the rapid self-assembly of multiplexed arrays on account of complementarity between the DNA sequences on the antibodies and sensor array, respectively. Furthermore, DNA-encoded antibodies also allow for sensor array regeneration and reprogramming, as chaotropic agents can be used to disrupt the DNA-DNA duplexes that link the capture agents to the sensor without harming the underlying DNA on the surface, which can subsequently be reloaded with antibodies either targeting the same or different antigens.     

Improving the response of a wheel speed sensor by using frequency-domain adaptive filtering

In this paper, a frequency-domain least-mean-square adaptive filter is used to cancel noise in a wheel speed sensor embedded in a car under performance tests. In this case the relevant signal is buried in a broad-band noise background, where we have little or no prior knowledge of the signal or noise characteristics. The results of the experiments show that the signal of interest and the noise (all forms of interference, deterministic, as well as stochastic) share the same frequency band and that the filter used significantly reduced the noise corrupting the information from the sensor while it left the true signal unchanged from a practical point of view. In this paper, a signal-to-noise ratio improvement higher than 40 dB is achieved. The results of the experiment show the importance of using digital signal processing when dealing with a signal corrupted by noise.     

Hall sensor response to an inhomogeneous magnetic field

The response of a Hall-effect sensor to a spatially dependent magnetic field is of importance for many applications such as magnetic microscopy and nondestructive testing. Using the analytical expression of the response of a Greek cross Hall sensor response to an ideal field dot published a few years ago, we have calculated its sensitivity and its full width at half maximum for the field produced by a magnetic dipole and by two coplanar lines. The experimental results are in good agreement with theory. They show that the spatial resolution is roughly equal to the dimension of the central part of the Greek cross and that a flux-meter approximation is not appropriate for modeling such Hall-effect sensors for very close field sources.     

Application of the generalized mixing kinematic model to the calculation of crushing response of complex prismatic sections

Impact behaviour of vehicles can be conveniently studied by means of hybrid numerical models using equivalent elements such as bars and non-linear springs. Wierzbicki and Abramowicz proposed a kinematic model for calculating the average and instantaneous crushing force for these macro elements. An equally simple model for the pre-collapse phase was developed by Huang and Wierzbicki. Our obective in the present paper is to determine the entire crushing characteristic of prismatic members consisting of plates with different wall thickness. Several examples are worked out in which comparisons with FE calculations and experimental results are made.     

Analysing an accident related to orphan sources including dose assessment

This paper discusses an accident, which occurred in one of the radiation application centres in Iran, follow-up investigations as well as lessons learnt. In January 2004 the Regulatory Authority was informed through a university radiation protection officer of an accident regarding orphan sources. Investigations revealed that one Am-Be and three (137)Cs sources in the container were subject to extensive heat due to burning of the container and melting of the paraffin content of the container; consequently, sources were stuck to the side wall of the container, but they were still undamaged and no radioactive leaking had occurred. Further investigations showed that the container had been given to the above mentioned centre a long time before by a foreign well-logging company without notifying the Regulatory Authority. Follow-up measurements and assessments indicated that the collective effective dose due to the accident is unlikely to be more than 21 mSv; consequently, no severe deterministic effect to individuals was expected. The findings showed that the main reasons for the accident were as follows: (1) violation of obligation under radiation protection regulations by the owner of the sources; (2) leaving the sources in an improper storage condition; (3) unauthorised access to the radiation sources at the owner centre; and (4) lack of an integrated national registration system in the Regulatory Authority.     

Damage induced constitutive response of a thermoplastic related to composites and adhesive bonding

This paper addresses the volume averaged constitutive behavior of a polymer sustaining void growth under dilational deformation. Polyvinylacetate is deformed between two aluminum beams in a double cantilever geometry and the deformations, recorded with the aid of optical interferometry, are used to deduce the stress-strain behavior of the material as it passes from small strain behavior through the voiding process to near-failure under exclusion of rate-sensitivity considerations. Observations regarding failure in composites where high spatial constraint limits the development of volume controlled failure processes are included, as well as remarks relevant to computational efforts delineating the effect of void development in plastically deforming solids.

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Résumé L'étude concerne le comportement de base, rapporté à un volume moyen, d'un polymère qui est le siège d'une croissance de cavités sous l'effet d'une dilatation.Du polyvinylacetate est déformé entre deux barreaux d'aluminium selon une disposition en double cantilever; les déformations sont enregistrées par interférométrie optique, et sont utilisées pour obtenir le comportement contraintes-dilatations du matériau lorsqu'il passe d'un comportement sous failbles déformations, qui correspond au processus de création des lacunes, à un état voisin de la rupture, à l'exclusion de toutes considérations de sensibilité à la vitesse de sollicitation.Des observations sont faites sur la rupture dans les composites, dans lesquels des astreintes importantes d'espace limitent le développement des processus de rupture régis par une notion de volume. On formule également des remarques sur les travaux de calculs nécessaires pour délimiter l'effect d'un développment de lacunes dans des solides sous déformations plastiques.

     

Improving holographic data storage by use of an optimized phase mask.

A method for improving the spectral distribution and for reducing the reconstruction error in optical holographic data storage is proposed. By use of an optimized phase mask in the input plane, the uniformity of the spectral distribution is optimized and the reconstruction error minimized. The phase mask is designed by use of amplitude-phase retrieval algorithms. Simulation results show the merits of the proposed method.     

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 μg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.     

High-energy picosecond near-vacuum ultraviolet pulses generated by sum-frequency mixing of an amplified Ti:sapphire laser

We demonstrate high-energy picosecond near-vacuum ultraviolet laser pulse generation. Frequency quadrupling is achieved by noncollinear sum-frequency mixing of the fundamental and the third harmonic of a two-stage Ti:sapphire amplifier in beta-BaB(2)O(4) crystal. UV pulses with energies of approximately 10 mJ tunable from 195 to 210 nm at a 10 Hz repetition rate are obtained.     

Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding: an example with [3H]estradiol

A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [3H]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (Ka) of estradiol for human serum albumin (HSA). The Ka was estimated at 8.9 x 10(4) M(-1), which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [3H]estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods.     

Using polymeric materials to generate an amplified response to molecular recognition events

Clinical and field-portable diagnostic devices require the detection of atto- to zeptomoles of biological molecules rapidly, easily and at low cost, with stringent requirements in terms of robustness and reliability. Though a number of creative approaches to this difficult problem have been reported, numerous unmet needs remain in the marketplace, particularly in resource-poor settings. Using rational materials design, we investigated harnessing the amplification inherent in a radical chain polymerization reaction to detect molecular recognition. Polymerization-based amplification is shown to yield a macroscopically observable polymer, easily visible to the unaided eye, as a result of as few as approximately 1,000 recognition events (10 zeptomoles). Design and synthesis of a dual-functional macromolecule that is capable both of selective recognition and of initiating a polymerization reaction was central to obtaining high sensitivity and eliminating the need for any detection equipment. Herein, we detail the design criteria that were used and compare our findings with those obtained using enzymatic amplification. Most excitingly, this new approach is general in that it is readily adaptable to facile detection at very low levels of specific biological interactions of any kind.