GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.     

A novel method for monitoring monoclonal antibody production during cell culture

We describe a new format for surface-based fluoroimmunoassays that allows detection of biomolecule interactions without separation steps. The bioactive layer was immobilized on the surface of a glass substrate covered with silver islands that provide optical amplification of the distinctive fluorescence signal from bound probes when compared to unbound probes. The technique used was phase-modulation fluorometry that allows sensitive detection of bound probes with a very short lifetime in the presence of excess free probes in solution. The new method was applied to assay monoclonal antibody production during cell culture. Excellent agreement was found between the new method and ELISA analysis of hybridoma cell culture samples. It is predicted that the near real time monitoring of protein products during bioprocessing will be possible with the described technology.     

Development of a homogeneous high throughput fluorescence polarization assay for G protein-coupled receptor binding

Traditional methods that follow receptor ligand interactions are competitive assays in which the test compound displaces a radiolabeled molecule. These assays require either a time-consuming step for separation of free ligands from bound ligands or immobilization of receptors and the scintillant on a solid-phase support. In this report, we describe the development of a homogeneous binding assay for a G protein-coupled receptor in the fluorescence polarization format. This homogeneous fluorescence polarization binding assay format is superior to the traditional binding methods because no radioisotope, separation step, or solid-phase support is required. The elimination of the separation step enhances detection of low-affinity ligands and enables a real-time, continuous readout of the binding activity in a high throughput 384-well microplate format.     

A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surface plasmon resonance

The opportunistic food pathogen Listeria monocytogenes is of great concern to the food industry and its rapid detection is of major importance. This paper describes the detection of L. monocytogenes with a polyclonal antibody by means of a new subtractive inhibition assay using a BIAcore 3000 biosensor. Incubating L. monocytogenes cells and antibody for a short period of time, followed by subsequent separation of free unbound antibody with a stepwise centrifugation process, allowed the detection of 1 x 10(5) L. monocytogenes cells/ml in less than 30 min. Free antibody was passed over an anti-Fab ligand-coated sensor chip surface with the generated response being inversely proportional to the inhibiting cell concentration. The method was simple, rapid and needed minimum sample preparation. This assay format has the potential for the quick and sensitive detection of pathogens with limited sample handling and preparation.     

New approaches for the analysis of molecular recognition using the IAsys evanescent wave biosensor

Trends in the analysis of molecular recognition using the IAsys evanescent wave biosensor are outlined. Diversification of sensor surface chemistry, an open cuvette format and the advent of robotics controlled by intelligent software are widening the range and throughput of applications. Analyses of binding and dissociation are now carried out across a wide spectrum of biomolecules, including protein, nucleic acid, carbohydrate and lipid. Determinations are obtained from a range of experimental formats. These include qualitative 'yes/no' screening assays, through semi quantitative ranking of kinetic association, dissociation and equilibrium constants for a family of binding partners, to deriving constants comparable with those which would be obtained in free solution. A dependence of the initial rate of biomolecular association on concentration allows analyte concentration to be measured--an increasingly common application class. This is often employed in situations where a rapid determination is required The ability to recover bound analyte from the sensor surface in sufficient amounts for subsequent characterization is opening up new routes to the parallel analysis of structure and function.     

Comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays

Using both experimental assays and fluid-dynamic finite element simulation models, we directly compared the achievable performance limits of four distinct assay configurations for label-free detection of an analyte from a test sample on a biosensor surface. The assay configurations studied in this work included a biosensor incorporated into the bottom surface of a microplate well and a microfluidic channel. For each configuration, we compared assay performance for the scenario in which the entire bottom surface of the fluid-handling vessel is coated with capture ligands with assay performance for the scenario in which the capture ligands are applied in the form of localized spots. As a model system, we used detection of the protein biomarker tumor necrosis factor-alpha (TNF-α) using immobilized TNF-α capture antibody. Results show that the microfluidic assay format dramatically reduces the time required to establish a stable equilibrium. Spot-based assays are advantageous for microplate-based detection for reducing the time required for equilibrium sensor response. The results derived are generally applicable to any label-free biosensor technology and any ligand-analyte system with adjustable variables that include sensor mass density sensitivity, analyte-ligand adsorption/desorption rate constants, immobilized ligand density, flow channel geometry, flow rate, and spot size.     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.     

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.     

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.     

A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin.

Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.     

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.     

Enzymes as versatile labels and signal amplifiers for monitoring immunochemical reactions

Summary Enzymes have proven to be sensitive and versatile labels for immunochemical assays. The sensitivity of an enzyme label stems from its extraordinary catalytic power which in turn provides a great amplification of signals. Its versatility, however, stems from the fact that enzyme activity can be modulated by a number of substances.Enzyme labeled immunoassays can be divided into two categories: (a) heterogeneous and (b) homogeneous (non-separation). In the heterogeneous systems, the quantitation of the antibody bound and unbound fractions requires a physical separation of these two fractions, whereas the homogeneous or non-separation systems do not require such a separation. In the homogeneous systems, the unbound and antibody bound fractions can be distinguished functionally.A total of 11 unique principles used in the development of enzyme labeled immunoassays are described. The advantages, disadvantages and limitations of them are considered, as well as the future paths for research and developments.     

A lectin immunosensor technique for determination of alpha(1)-acid glycoprotein fucosylation

The fucosylation of alpha(1)-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.     

Subnanoliter enzymatic assays on microarrays

Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.     

A label-free optical technique for detecting small molecule interactions

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.     

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.     

A novel and robust homogeneous fluorescence-based assay using nanoparticles for pharmaceutical screening and diagnostics

We have established a new type of homogeneous immunoassay based on nanoparticles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA). This method allows the characterization of single fluorescently labeled molecules or particles with respect to their molecular brightness and concentration. Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the complex scales with the number of bound conjugates. The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present various examples of NPIAs where capture antibodies were linked to the nanoparticles, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quantitate free antigen through competition experiments, and second, an antigen-directed antibody was labeled to establish an assay similar to a sandwich ELISA setup. The major advantages of a NPIA are the robustness and high signal-to-noise ratio at short measurement times, as demonstrated with a miniaturized experiment in a Nanocarriertrade mark holding a volume of 1 microl/well. In addition to the good data quality, NPIAs are straightforward to perform because they require no washing steps. NPIAs open new dimensions for high throughput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is applicable to most types of assays.