Biomedical subjects

F S Ligler

Publications and source records attributed to F S Ligler.

At least 19 recordsLinked to original sources

PubMed2001

Kinetics of antigen binding to arrays of antibodies in different sized spots.

A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 x 10(5) M(-1) s(-1). Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 microm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.

Animals↗

PubMed2001

Fluidics cube for biosensor miniaturization.

To create a small, portable, fully automated biosensor, a compact means of fluid handling is required. We designed, manufactured, and tested a "fluidics cube" for such a purpose. This cube, made of thermoplastic, contains reservoirs and channels for liquid samples and reagents and operates without the use of any internal valves or meters; it is a passive fluid circuit that relies on pressure relief vents to control fluid movement. We demonstrate the ability of pressure relief vents to control fluid movement and show how to simply manufacture or modify the cube. Combined with the planar array biosensor developed at the Naval Research Laboratory, it brings us one step closer to realizing our goal of a handheld biosensor capable of analyzing multiple samples for multiple analytes.

Biosensing Techniques↗

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↗

PubMed2000

A ganglioside-based assay for cholera toxin using an array biosensor.

A rapid assay for cholera toxin (CT) has been developed using a fluorescence-based biosensor. This sensor was capable of analyzing six samples simultaneously for CT in 20 min with few manipulations required by the operator. The biochemical assays utilized a ganglioside-"capture" format: ganglioside GM1, utilized for capture of analyte, was immobilized in discrete locations on the surface of the optical waveguide. Binding of CT to immobilized GM1 was demonstrated with direct assays (using fluorescently labeled CT) and "sandwich" immunoassays (using fluorescently labeled tracer antibodies). Limits of detection for CT were 200 ng/ml in direct assays and 40 ng/ml and 1 microg/ml in sandwich-type assays performed using rabbit and goat tracer antibodies. Binding of CT to other glycolipid capture reagents was also observed. While significant CT binding was observed to loci patterned with GD1b, Gb3, and Gb4, CT did not bind significantly to immobilized GT1b at the concentrations tested. This is the first description of such a non-antibody-based recognition system in a multi-specific planar array sensor.

Antibodies, Monoclonal↗

PubMed2000

A liquid crystal pixel array for signal discrimination in array biosensors.

A new optical design uses a liquid crystal pixel array (LCPA) to discriminate multiple fluorescence signals on a two-dimensional biosensor array. The LCPA can selectively control the transmission of fluorescence generated from multiple biosensing elements on a planar waveguide. This device sequentially acquires the fluorescence data from the substrate by making multiple individual measurements of the sensing elements on the waveguide. The biosensing elements are patterned according to the pixel layout of the LCPA and optically aligned so that each electronically driven pixel can either transmit or filter out the fluorescence signal as specified by the user. The primary advantage of this system is that a single detection channel (i.e. photomultiplier tube (PMT)) can be used to measure multiple fluorescence signals from a two-dimensional substrate while the LCPA provides for spatial resolution. We evaluate the performance of the LCPA by testing the optical homogeneity of the liquid crystal pixels and linear dynamic range for transmitting light. The LCPA is also used with well-developed biosensing chemistry modified for this optical format.

Biosensing Techniques↗

PubMed2000

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor.

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.

Bacteria↗

PubMed2000

Array biosensor for detection of biohazards.

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

Biosensing Techniques↗

PubMed1999

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.

Antibodies↗

PubMed1999

An array immunosensor for simultaneous detection of clinical analytes.

A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.

Antigens, Bacterial↗

PubMed1999

Detecting staphylococcal enterotoxin B using an automated fiber optic biosensor.

The Man-portable Analyte Identification System (MANTIS), the first fully automated, self-contained, portable fiber optic biosensor, was utilized for the detection of Staphylococcal Enterotoxin B (SEB), a bacterial toxin produced by Staphylococcus aureus that commonly causes food poisoning. Because of its remarkable toxicity and stability, SEB is considered a prime threat as a biological weapon of mass destruction. The assay for SEB was used to evaluate the MANTIS' ability to function in the presence of various environmental interferents. The sensor could reliably detect SEB spiked into liquid samples containing a variety of smoke particles. However, substantial interference occurred when SEB was mixed into matrices capable of adsorbing SEB, such as 1% solutions of clay, topsoil, or pollen. Of equal importance, none of the interferents produced false positives in the MANTIS. The MANTIS demonstrated the capability to perform simultaneous immunoassays rapidly in the field with little or no user intervention.

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

Detection of multiple toxic agents using a planar array immunosensor.

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

Biosensing Techniques↗

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↗

PubMed1998

Quantifying serum antiplague antibody with a fiber-optic biosensor.

The fiber-optic biosensor, originally developed to detect hazardous biological agents such as protein toxins or bacterial cells, has been utilized to quantify the concentration of serum antiplague antibodies. This biosensor has been used to detect and quantify the plague fraction 1 antigen in serum, plasma, and whole-blood samples, but its ability to quantify serum antibodies has not been demonstrated. By using a competitive assay, the concentration of serum antiplague antibodies was ascertained in the range of 2 to 15 microgram/ml. By making simple dilutions, concentrations for 11 serum samples whose antiplague antibody concentrations were unknown were determined and were found to be in good agreement with enzyme-linked immunosorbent assay results. The competitive assay method could be used to effectively determine the exposure to plague of animals or humans or could be applied to other diseases, such as hepatitis or AIDS, where the presence of antibodies is used to diagnose infection.

Animals↗

PubMed1998

Rapid detection of D-dimer using a fiber optic biosensor.

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min. Samples from septic patients were assayed using ELISA and the fiber optic method; concentrations determined by fiber optic assay were strongly correlated with those determined by ELISA (r = 0.918); intra- and inter-assay errors were comparable to those from ELISAs. Given its accuracy and rapid response time, this fiber optic biosensor shows great potential for development as a diagnostic tool.

Biosensing Techniques↗

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↗

PubMed1997

Effectiveness of protein A for antibody immobilization for a fiber optic biosensor.

The fiber optic biosensor performs fluoroimmunoassays at the surface of multimode optical fibers. The effectiveness of protein A, an immunoglobulin binding protein, for antibody immobilization on the surface of these fiber probes has been investigated. No difference was observed in the binding of fluorescently-labeled goat-IgG by rabbit anti-goat IgG regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal as probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes since antibody-antigen complexes have been successfully eluted from protein A under low pH conditions. Protein A probes coated with rabbit anti-goat IgG obtained nearly identical signal levels at 500 and 5000 ng/ml of Cy5.5 goat IgG five consecutive times following regeneration with glycine-HCl, 2% acetic acid, pH 2.5.

Antigen-Antibody Reactions↗