Autonomous capillary system for one-step immunoassays

Autonomous capillary systems (CSs), in which liquids are transported owing to capillary forces, have been previously used to perform fast and sensitive multi-step immunoassays using minute volumes of analyte. CSs for diagnostic applications have different requirements than CSs that are used as a research tool in life sciences, where a high flexibility and performance prevail over ease of use and portability. In this paper a proof-of-concept for one-step immunoassays based on CSs is demonstrated. This proof-of-concept is based on preloading detection antibodies (dAbs) on the flow path of analytes and redissolving these antibodies to form dAb-analyte complexes, which can be detected using fluorescence downstream on accurately patterned capture antibodies. A user therefore only needs to load a sample at the beginning of a CS and obtains the result of the assay using a conventional fluorescence microscope or scanner. As an example, C-reactive protein was detected in human serum at clinical concentrations within 10 min and using only of sample.     

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne's disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a large degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD-negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested using commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads using laser-induced fluorescence. Antigen-coated magnetic beads treated with the bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples with the microfluidic system were compared to those analyzed with the flow cytometer, a high level of correlation (linear regression, r(2)=0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD.     

Development of a new antibody to the human inhibin/activin betaB subunit and its application to improved inhibin B ELISAs

Inhibin B has emerged as a clinically useful analyte for studies of reproductive function in both men and women. The antibody to the betaB subunit (C5) used in current commercially available assays (DSL and OBI) was raised in this laboratory to a synthetic peptide from the betaB subunit. These assays require pre-treatment of samples with hydrogen peroxide to oxidise two methionines in the epitope to the sulfoxide for full immunoreactivity. It was also claimed that this antibody cross-reacted significantly with inhibin A leading to a 0.5% cross-reaction of inhibin A in the current generation of immunoassays. Both of the above immunoassays required overnight incubation with sample. The development of improved antibodies to the betaB subunit has proved difficult due to the conservation of the betaB subunit between species. We describe the development of new monoclonal antibodies to the betaB subunit, by immunisation of mice with recombinant X. laevis activin B using the RIMMS method of immunisation. The result has been the development of highly specific antibodies in a short time period. One of these antibodies 46A/F is shown to be a highly effective capture antibody in a human inhibin B ELISA, without any sample pre-treatment. The results of the validation of an improved inhibin B assay using 46A/F as the capture antibody are shown, with comparison to one of the commercially available inhibin B assays. Overall, the inhibin B assay is simplified and the performance improved by using this new antibody 46A/F. It was further shown that the cross-reaction of inhibin A in both the OBI and DSL inhibin B ELISAs is ten fold less than previously reported. This can be attributed to the poor quality of recombinant inhibin B available for use as standard in 1996. Although the present generation of inhibin B assays has proved adequate to enable the physiological function of inhibin to be determined and novel clinical applications found, the simplification of the assay made possible by the improved antibody should make possible a new generation of more rapid, sensitive, convenient and robust tools for routine use.     

A bead-based immunogold-silver staining assay on capillary-driven microfluidics

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in “bead lanes”, which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using “classical” enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL^?1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL^?1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.     

Modeling of a Microfluidic Channel in the Presence of an Electrostatic Induced Cross-Flow

Amongst the processes that have been implemented in microfluidic devices, electrophoretic transport of charged molecules, along microfluidic channels, is one of the most commonly found. However, less work has been done about continuous, pressure gradient driven flow systems where an electric field is applied orthogonally with respect to the microchannel walls. The perspective applications of this technique, include continuous flow separation and concentration of analyte molecules, and the kinetic control of surface reactions. In order to dimensioning and optimizing such a device, a mathematical model has been formulated and analyzed both with numeric and analytic methods. The given solutions let the designer of microfluidic devices able to estimate the concentration profiles along the microchannel length, as a function of the main system parameters. As a practical example of application which could be of great interest in biotechnology applications, the results relative to the simulation of the electrostatic induced cross flow of single strand DNA oligonucleotides of about 20 bases has been reported.     

Design and Use of a General Capturing Surface in Optical Biosensor Analysis of Sulphamethazine in Milk

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 microg kg -1 and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cow's milk. The advantages of the new assay format include analyte-independent immobilisation and regeneration. Furthermore, the assay enables measurements with covalent interactions between analyte and detecting molecule. The main disadvantage is the requirement of a conjugate between analyte and the hapten H1. Moreover, it is likely that the antibody surface will have a shorter life span than a surface with the antimicrobial immobilised.     

Cytometric bead array: a multiplexed assay platform with applications in various areas of biology

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency.     

Detection of pathogenic bacteria by magneto-immunoassays: a review

Magnetic particle-based immunoassays are widely used in microbiology-related assays for both microbial capture, separation, analysis, and detection. Besides facilitating sample operation, the implementation of micro-to-nanometer scale magnetic beads as a solid support potentially shortens the incubation time (for magnetic immuno capture) from several hours to less than an hour. Analytical technologies based on magnetic beads offer a rapid, effective and inexpensive way to separate and concentrate the target analytes prior to detection. Magneto-immuno separation uses magnetic particles coated with specific antibodies to capture target microorganisms, bear the corresponding antigens, and subsequently separate them from the sample matrix in a magnetic field. The method has been proven effective in separating various types of pathogenic bacteria from environmental water samples and in eliminating background interferences. Magnetic particles are often used to capture target cells (pathogenic bacteria) from samples. In most commercially available assays, the actual identification and quantitation of the captured cells is then performed by classical microbiological assays. This review highlights the most sensitive analytic methods (i.e., long-range surface plasmon resonance and electrochemical impedance spectroscopy) to detect magnetically tagged bacteria in conjunction with magnetic actuation.     

Enzyme immunoassay techniques an overview

In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups ‘analyte-observed’ and ‘ reagent-observed’ assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee ‘one step’ assays of only a few minutes.

Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10–1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2–10.

Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzymes complex to analyte-containing immune complexes are discussed.

Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules.

Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.     

Multispot, multianalyte, immunoassay

Consideration of the basic principles of immunoassay design reveals that highly sensitive assays can, in principle, be developed using amounts of "sensor" antibody far smaller than are currently conventional in this field. Furthermore, when using such amounts, the fractional occupancy of antibody binding sites by analyte is independent of both sample volume and antibody concentration. Labelling of both the sensor-antibody and a developing antibody (designed to recognize either occupied or unoccupied sensor-antibody binding sites) permits the development of "ratiometric" immunoassays relying on measurement of the ratio of signals emitted by the two labelled antibodies. Furthermore, the sensor-antibody can be located within a "microspot" a few microns 2 in area. By labelling both sensor and developing antibodies with fluorescent labels, and scanning the microspot using a highly focussed laser beam, microspot immunoassays at least comparable in sensitivity with conventional "macroscopic" immunoassays are made possible. This in turn permits the development of immunoassay "arrays" capable in principle of measuring very large numbers of different substances within small samples (such as a drop of blood). The general principles and theory underlying these concepts are discussed, and preliminary experimental data using currently available instrumentation reported.     

Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines

Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.     

Immunoassay. Development and directions in antibody technology

Improvements and new applications for the technique of immunoassay have been extensive, owing in part to improvements in specific binding reagents used. Early reports advocated application of naturally occurring antibodies, specific serum-binding proteins, and receptors as binding reagents in immunoassays. Each is still used today, although applications are limited because affinities are generally low. Polyclonal antibodies produced in laboratory animals in response to injection of specific antigens have been the cornerstone of immunoassay because of their high affinities and wide range of specificities. Recently, monoclonal antibodies have begun to replace polyclonal antibodies because of improved specificity, ease of use, production of an unlimited supply, and advantages of worldwide assay standardization. Although only 50 analytes are currently measured by Food and Drug Administration--approved reagents, widespread use in the future is predicted.     

Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.     

Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 1. Kinetic characteristics and comparison with other Elisa formats

Solid‐phase two‐site enzyme immunoassays were developed using a number of monoclonal antibodies with differing specificities for wheat grain gluten proteins. While polystyrene microwells performed well in the assay, very high levels of non‐specific binding occured with PVC (Polyvinylchloride) microwells in the absence of ‘capture’ (solid‐phase bound) antibody. This was due to binding of complexes of antigen and labelled antibody, and could only be reduced by the use of rather stringent blocking conditions. Assays involving either the simultaneous or sequential addition of antigen and labelled antibody were developed, the latter providing superior results. A number of successful two‐site assays were constructed using single monoclonal antibodies, suggesting that repeating epitopes, known to occur in gluten proteins, were commonly recognized. Pairs of monoclonal antibodies with dissimilar antigenic specificites did not function in the two‐site assay. A comparison of monoclonal antibody based two‐site and antigencompetition immunoassays for gliadin was made. Several antibodies performed considerably better in one format than the other; thus, both assay formats will be of use in quantification of specific gluten protein subclasses.     

Multiplexed particle-based flow cytometric assays

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, 'no wash' assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.     

Theoretical considerations of the ability of monoclonal antibodies to detect antigenic differences between closely related variants, with particular reference to heterospecific reactions

In theory monoclonal antibodies can be used to analyse antigenic determinants in great detail by correlating differences in antibody affinity for variant antigens with their amino acid differences. In particular, heteroclitic antibodies should be detected, which would normally be masked in a polyclonal antiserum. Recognition of such antibodies may be important for our understanding of the scope of antibody repertoires particularly when the immunogen is closely related to a component of the immunised animal. In practice the immunoassays commonly used to measure affinity differences between different antigens fall short of these capabilities. Mathematical studies were carried out to identify factors controlling the sensitivity of 4 types of assay to differences in affinities for different antigens. The most important factors controlling assay sensitivity were found to be the ratio of antibody affinity (K) to epitope density in direct binding assays, the ratio of K to antibody concentration in liquid phase competition assays, and the ratio of solid phase to liquid phase values of K for solid-phase competition assays. It is predicted that a combination of solid-phase competition assay with high epitope density and direct binding assay with low epitope density would result in optimal detection of heteroclitic antibodies and small differences in antibody affinity for cross-reactive antigens.     

Specificity of two-site immunoassays

When cross-reaction in two-site immunoassays was investigated both theoretically and experimentally it was found that such systems do not always result in enhanced specificity. Computer simulation studies indicated that substances which display negligible cross-reaction in a radioimmunoassay could produce an assay response identical to that of the analyte in a two-site immunoassay using excess antibody. Cross-reactivity in two differing two-site immunoassays was compared to that obtained in radioimmunoassays using the same monoclonal antibodies for human chorionic gonadotrophin. In addition to the effects of excess antibody, cross-reactivity was observed in one of the two-site immunoassays which could not have been predicted from the specificity of the antibodies or cross-reactivity in the radioimmunoassays. The unexpected cross-reaction of the beta subunit of human chorionic gonadotrophin in the assay resulted from an apparent alteration in the specificity of one of the antibodies following binding of the beta subunit to the second antibody. These studies emphasise the complexity of binding reactions in two-site immunoassays.     

Use of multivariate statistical methods to identify immunochemical cross‐reactants

Quantitative competition immunoassays with appropriate combinations of antibodies give consistent dose‐response patterns which may be used to identify and estimate amounts of cross‐reacting compounds. Previously reported methods of analyzing cross‐reaction patterns include multiple regression, principal components analysis and minimum estimates of variance (MEV). Four other techniques which are preferable in theory have been surveyed: discriminant analysis (DA), maximum likelihood estimates (MLE), classification and regression trees (CART), and computational neural networks (NN). MLE and simple back‐propagation neural networks can estimate the concentration, as well as the identity, of individual compounds. These four methods worked well with unfitted, unscaled data from monoclonal assays of triazines, phenylureas and avermectins. Immunoassays must be properly designed to provide adequate data for pattern recognition. Cross‐reactivity pattern analysis will make multi‐analyte, multi‐antibody immunoassays feasible for many applications in toxicology and hazard assessment.     

Microspheres, nanospheres and flow cytometry: from cellular to molecular analysis

Micro- and nanospheres are tightly associated with the development of flow cytometry. They are indispensable tools to optimize diffraction and fluorescence signals as well as for fluorescence calibration and cellular purification (magnetic micro- and nanospheres). They are also usefull to evaluate phagocytosis and to detect slightly expressed antigens. Recently, developments of microspheres-based flow cytometric assays have raised to quantify soluble analytes in biological fluids, cellular and tissue samples. The technology utilizes spectrally distinct fluorescent microspheres as a solid support for a conventional immunoassay, affinity assay or DNA hybridisation assay which is subsequently analyzed on a flow cytometer. Several multiplexed bead systems are now available facilitating the development of multiplexed assays that simultaneously measure many different analytes in few microliters of sample. Some recent applications with fluorescent microspheres coated with antibodies or oligonucleotides include cytokines and PCR products quantitation and single nucleotide polymorphism genotyping. Thus, multiplex assays using microspheres and flow cytometry technologies are exciting techniques which have the potential to contribute to the development of efficient diagnostic and research methods.     

Sequential analysis of multiple analytes using a surface plasmon resonance (SPR) biosensor

A sequential analysis method for the analysis of two analytes was developed using a surface plasmon resonance (SPR) biosensor. A sample with both analytes was introduced into the single sensing region and then each analyte was analyzed sequentially. Two detection models were devised for the samples with the following composition: (1) one target analyte resulting in a sensor response without any label and the other analyte with only additional label, (2) both target analytes requiring additional labels for detection. A standard curve for each model was prepared and applied for sequential analysis of anti-bovine serum albumin (anti-BSA) antibodies and horseradish peroxidase (HRP). The errors of the sequential analysis of Models 1 and 2 were found to be less than 6%, and this method was therefore acceptable for application. No cross-reaction arising from non-specific binding among the participating antigens and antibodies was shown to occur in Models 1 and 2. For optimization of the analyte binding capacity of immunoaffinity (IA), the concentration ratio of the molecular recognition element at the immobilization step was adjusted. Subsequently, from the measurement of the maximum sensor response (R(max)), optimization of the analyte binding capacity could be made. Using Model 2, the feasibility of sequential analysis was demonstrated by detecting levels of human chorionic gonadotropin (hCG) and human albumin (hA) in healthy human urine, since both proteins are known to be related to abortion and preterm delivery during early pregnancy.