Biomedical subjects

A W Kusterbeck

Publications and source records attributed to A W Kusterbeck.

14 recordsLinked to original sources

PubMed2001

Explosives detection in soil using a field-portable continuous flow immunosensor.

A field method for quantitative analysis of explosives in contaminated soil samples is described. The method is based on a displacement immunoassay performed in a commercial instrument, the FAST 2000, engineered by Research International Inc. The method can be used on-site to measure 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) within 5min. For this study, replicate analyses were performed on soil extracts prepared from each field sample as well as appropriate controls, blanks, and laboratory standards. Statistical analyses were done to assess accuracy, bias, and predictability of the method. The results demonstrated that the immunosensor could be used effectively to screen environmental samples for the presence or absence of explosives. In most samples, the method also provided quantitative values that were in good agreement with standard laboratory analyses using HPLC. A limited number of sample matrices interfered with the immunoassay and produced results that varied significantly from the laboratory data. In each case, the compounds causing the problem have been identified and efforts are being made to minimize these matrix interferences in future field evaluations.

Chromatography, High Pressure Liquid↗

PubMed2000

Continuous flow displacement immunosensors: a computational study.

Numerical modeling has been used to investigate the disparity in performance and sensitivity that has been reported for flow displacement immunosensors based on bead-packed columns, membranes, and capillary tubes. The simulations strongly suggest that the high surface areas in the porous media systems may actually be detrimental to sensor performance because of large numbers of free antibody binding sites. Since the free antibody sites are created during the wash step in which the baseline is established, wash protocols are critical in optimizing the sensitivity for a given displacement sensor.

Binding Sites, Antibody↗

PubMed2000

Trace detection of explosives using a membrane-based displacement immunoassay.

A compact membrane-based displacement immunoassay has been designed for rapid detection of explosive compounds 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at high femtomole levels. The system consists of activated porous membranes, onto which either TNT or RDX antibodies are immobilized, that are inserted into microreactor columns, incorporated into a flow system. The assay is prepared by saturating the immobilized antibody binding sites with labeled antigen. Target analyte is introduced upstream of the microreactor, while the displacement of labeled antigen is monitored downstream using a fluorometer. The concentration of displaced labeled antigen detected is proportional to the concentration of the target analyte introduced into the system. This system provides a reusable and reagentless sensor, suitable for continuous monitoring of explosives, with an operating lifetime of over 50 positive samples. Multiple assays were performed in approximately 5 min at different flow rates, using membranes saturated with varying antibody concentrations. The membrane-based format exhibited a detection limit of approximately 450 fmol for TNT and RDX (100 microl of 1 ng/ml solution) in laboratory samples.

Antibodies, Monoclonal↗

PubMed1999

Trace level detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by microimmunosensor.

Reported in this paper is the development and characterization of a highly sensitive microcapillary immunosensor for the detection of the explosive, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The immunosensor exploits antibodies as recognition elements for target antigens, fluorescence dye conjugates for reporter molecules and fused silica microcapillaries for its high surface-to-volume ratio. Detection of RDX with the microcapillary immunosensor requires covalent immobilization of anti-RDX antibodies on the inner core of the microcapillaries via heterobifunctional cross-linker chemistry. Subsequent saturation of all antibody binding domains follows with a synthetically prepared fluorescent analog of RDX. Displacement immunoassays were performed with the microcapillary immunosensor with the injection of unlabeled RDX at concentration levels from 1 part-per-trillion (pptr) to 1000 part-per-billion (ppb). As unlabeled RDX reaches the binding domain of the antibody, fluorescent RDX analog is displaced from the antibody, flows downstream and is measured by a spectrofluorometer. Fluorescence measurements of the displaced fluorescent RDX analog were equated to a standard calibration curve to quantify sample concentration. Complete evaluation of the RDX microcapillary immunosensor for selectivity and sensitivity was performed based on the following criteria: variable flow rates, antibody cross-reactivity, reproducibility and cross-linker (carbon spacer) comparison. Results indicate the lowest detectable limit (LDL) for RDX is 10 pptr (ng/l) with a linear dynamic range from 0.1 to 1000 ppb (ug/l).

Biosensing Techniques↗

PubMed1998

A membrane-based displacement flow immunoassay.

The use of a membrane-based continuous flow displacement immunoassay for detection of nanomolar quantities of explosives is demonstrated, and the kinetics of this system are characterized through experimentation. Antibodies of 2,4,6-trinitrotoluene (TNT) are immobilized onto a porous membrane with surface reactive sites designed to facilitate the covalent binding of the antibody. After saturating the immobilized antibody binding sites with labeled antigen, target analyte is introduced in flow, and the displacement reactions are monitored using a fluorometer. The displaced labeled antigen detected is proportional to the concentration of the analyte introduced to the antibody-labeled antigen complex. Multiple assays were performed at flow rates of 2.0, 1.0, 0.50, and 0.25 mL/min using membranes saturated with varying TNT antibody concentrations. The signal intensity (i.e. the concentration of displaced labeled antigen) was independent of antibody concentration at 1.0 mL/min, but proportional to antibody concentration at 0.25 mL/min. Our data suggests that the lower flow rate created a longer interaction time between the injected analyte and the antibody-labeled antigen complex, resulting in greater displacement of the labeled antigen and higher signal intensities than seen at higher flow rates.

Animals↗

PubMed1998

Multianalyte detection using a capillary-based flow immunosensor.

A highly sensitive, dual-analyte detection system using capillary-based immunosensors has been designed for explosive detection. This model system consists of two capillaries, one coated with antibodies specific for 2,4,6-trinitrotoluene (TNT) and the other specific for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) combined into a single device. The fused silica capillaries are prepared by coating anti-TNT and anti-RDX antibodies onto the silanized inner walls using a hetero-bifunctional crosslinker. After immobilization, the antibodies are saturated with a suitable fluorophorelabeled antigen. A "T" connector is used to continuously flow the buffer solution through the individual capillaries. To perform the assay, an aliquot of TNT or RDX or a mixture of the two analytes is injected into the continuous flow stream. In each capillary, the target analyte displaces the fluorophore-labeled antigen from the binding pocket of the antibody. The labeled antigen displaced from either capillary is detected downstream using two portable spectrofluorometers. The limits of detection for TNT and RDX in the multi-analyte formate are 44 fmol (100 microliters of 0.1 ng/ml TNT solution) and 224 fmol (100 microliters of 0.5 ng/ml RDX solution), respectively. The entire assay for both analytes can be performed in less than 3 min.

Antibodies↗

PubMed1997

Assessment of heterogeneity in antibody-antigen displacement reactions.

The intrinsic binding characteristics of monoclonal antibodies are modified upon immobilization onto a solid-phase matrix. Factors such as the distribution in affinity must therefore be taken into consideration in order to predict the kinetics of antibody binding at solid-liquid interfaces. A mathematical analysis is presented herein that allows the assessment of heterogeneity in the affinity of monoclonal antibodies immobilized onto a solid support. This model is based on a modified version of the Sips distribution function adapted to the conditions of a solid-phase displacement assay in flow. An assay for trinitrotoluene (TNT) provides the data to evaluate the extent of heterogeneity introduced by immobilization of antibodies in a flow immunoassay. We determined the index of antibody heterogeneity on two solid supports, controlled-pore glass beads and agarose beads, coated with a monoclonal anti-TNT antibody at varying densities. The data confirm that the threshold for crossover from homogeneous to heterogeneous forms of the reaction isotherm is different in displacement reactions than in association-dissociation reactions. Our analysis shows that the measured displacement isotherm is consistent with a homogeneous or only moderately heterogenous distribution of relative affinities.

Antibodies, Monoclonal↗

PubMed1996

Use of the USDT flow immunosensor for quantitation of benzoylecgonine in urine.

The flow immunosensor works on a principle somewhat distinct from other immunoassay systems in that it performs a displacement immunoassay. Antibody-coated matrices are saturated with fluorescently labeled benzoylecgonine (BE), which is released and measured in the presence of BE-containing urine and measured downstream from the matrix. The same antibody matrix can be used for many samples. A flow immunosensor instrument, built by US Drug Testing, Inc., has recently received "Premarket Notification' (510(K)) from the Food and Drug Administration (FDA) to screen urine for the presence of the cocaine metabolite BE. The performance of the flow immunosensor for screening BE in urine was validated by comparison with results of a blind study using the Syva EMIT, the Abbott TDx and gas chromatography-mass spectroscopy (GC-MS). Potentially interfering drugs were also spiked into urine and evaluated using the flow immunosensor. While the FDA approval is for determining whether the BE concentration is above or below the 300 micrograms/l cutoff recommended by the National Institute of Drug Abuse, we have also shown that the flow immunosensor can be adapted to produce quantitative determinations of the amount of BE in the urine samples. The reliability of the quantitation was confirmed by testing 100 urine samples containing unknown amounts of BE using the flow immunosensor. GC-MS and the Abbott TDx system. Comparison of quantitative data obtained using the immunosensor and GC-MS showed a 97% correlation, compared with a much lower value for data from the TDx and GC-MS.

Antibody Specificity↗

PubMed1994

Effect of antibody density on the displacement kinetics of a flow immunoassay.

This study investigates the effect of antibody density on the kinetics of a solid-phase displacement immunoassay. Conducted in flow under nonequilibrium conditions, the assay utilizes a monoclonal antibody to the cocaine metabolite benzoylecgonine, which has been immobilized onto Sepharose beads and saturated with fluorophore labeled antigen. Displacement of antibody-bound labeled antigen by non-labeled antigen occurs when sample is introduced in the buffer flow. Comparison of matrices coated with two different antibody densities revealed that the displacement efficiency is a function of the density of antibody-bound labeled antigen. A higher density of antibody provides a higher amount of displaced labeled antigen, but the displacement efficiency of the assay is decreased. The effect of antibody density on the immunoassay kinetics was analyzed using a mathematical formulation developed to characterize antibody-antigen interactions at solid-liquid interfaces. Higher antibody density proved to be associated with a lower apparent dissociation rate constant. The implications of these results on the design of immunoassays in flow are discussed.

Animals↗

PubMed1992

Kinetics of antibody binding at solid-liquid interfaces in flow.

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.

Antibodies, Monoclonal↗

PubMed1991

Novel trifunctional carrier molecule for the fluorescent labeling of haptens.

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

A continuous flow immunoassay for rapid and sensitive detection of small molecules.

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins-125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 microliters) using DNP-Ins-125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Animals↗

PubMed1986

Effect of covalent attachment of immunoglobulin fragments on liposomal integrity.

Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.

Animals↗

PubMedDate not supplied

Synthesis of a fluorescent analog of polychlorinated biphenyls for use in a continuous flow immunosensor assay.

A synthetic scheme has been developed for the preparation of a dye-labeled analog of polychlorinated biphenyls. The reaction of 2,3,5-trichlorophenol with 3-bromopropylamine hydrobromide under basic conditions was used to introduce a free primary amine group into the parent compound by formation of a stable ether linkage. Reaction of this amine with the succinimidyl ester of a sulfoindocyanine dye resulted in amide bond formation to produce a fluorescently-labeled product. The dye conjugate was used to charge a column containing immobilized antibodies against polychlorinated biphenyls. Upon application of samples containing various concentrations of polychlorinated biphenyls, the fluorescent analog was displaced from the column in amounts proportional to the concentration of analyte. Concentrations of polychlorinated biphenyl as low as 1 ppm were measurable using this system.

Antibodies↗