Capillary electrophoretic immunoassays

Capillary electrophoretic immunoassay (CEIA) has recently emerged as a new analytical technique. CEIA, when combined with sensitive detection methods such as laser induced fluorescence (LIF), offers several advantages over conventional immunoassays. CEIA can perform rapid separations with high mass sensitivity, simultaneously determine multiple analytes and is compatible with automation. The objective of this review is to describe the applications of CE in antibody related studies, focussing especially on recent developments of CEIA technique. The principles for competitive and non-competitive CEIA are described with examples. Several detection methods and various applications are summarized and future developments in CEIA are speculated. CEIA has many potential applications, especially if the throughput is improved by using either multicapillary array or microchips with multiple channels.     

Application of bead-based assays for flow cytometry analysis

To date microsphere-based assays in flow cytometry have focused on the detection of antibody or antigen. Most studies have been research based to evaluate the performance of the technique relative to conventional techniques. However, there have not been any carefully controlled studies of the sensitivity and specificity, as well as analytic sensitivity of the FMIA technique. As such, it is difficult to document advantages of this tecnique clearly. The data suggest that FMIA is considerably more sensitive than conventional techniques, and the ability to analyze for multiple analytes in one sample dilution is attractive. This ability to simultaneously analyze for multiple samples is primarily dependent upon the size difference as sensed by FALS of the microspheres. However, it is also possible to use microspheres of the same size but that differ in either fluorescence or RALS signal. If microspheres of the same size are used but one fluoresces red and the signal in the assay uses a green fluorochrome, then the two microspheres can be separated by their red fluorescence. Using this technique, one can increase the number of microspheres that can be used in an assay. It is also possible to use microspheres of the same size but with different abilities to scatter the incident light at right angles. The use of these microspheres is then similar to the nonfluorescent versus red microspheres. By the judicious combination of microsphere size, it is possible to easily differentiate eight different microspheres. With the addition of a fluorescebt dye and/or differences in right-angle light-scatter capabilities, the number of different microspheres that can be used simultaneously becomes quite large. In practice, the number of microspheres that can be differentiated is no doubt greater than the number of analytes that need to be assayed in one assay.Although the apparent increase in sensitivity and the ability to simultaneously detect and quantitate numerous analytes are important attributes of FMIA, there are drawbacks to this method. Although the FMIA lends itself well to one-step no-wash procedures, when wash steps are necessary they are time-consuming and ineffecient. Most wash steps in FMIA use centrifugation of the microspheres to remove them from the reagent. There is a significant loss of microspheres in these wash steps, which are time-consuming. There are studies ussing vacuum filtration of the suspension to separate the microspheres from the reagents. A number of different groups are pursuing an automated or semiautomated method for the efficient washing and reagent delivery system for FMIA. Commercial systems are being developed that may allow for the easier handling of these reagents.Numerous groups are investigating the use of microspheres and flow cytometry primarily in immunoassay development. The procedure has the advantages of the simultaneous yet discrete analysis of multiple analytes and the inherent increase in sensitivity using fluorescence over other signals. There will no doubt be wider applications     

Direct hapten-linked multiplexed immunoassays on polycarbonate surface

Direct hapten-linked multiplexed immunoassay is developed on the polycarbonate surface of standard Digital Versatile Discs (DVDs) for six compounds of environmental concern, as proof of concept. Carboxylated haptens are directly linked to the aminated polycarbonate surface through carbodiimide/succinimide coupling. The modified DVD surface maintained its physical and optical properties. Multiplexed assay reached detection limits down to 0.1 μg/L for chlorpyrifos, 2,4,5-trichlorophenoxypropionic acid, sulfathiazole and sulfasalazine and down to 1.0 μg/L for fenthion and malathion. This approach presents advantages such as the improvement in sensitivity in comparison to protein-hapten conjugate format for all the studied analytes and the absence of cross-interference effects, allowing high throughput multianalysis on the same surface. Also, a comparison of the performance of two sensing strategies indicated that DVD disc and drive approach turned out in a simpler mode, the assays being more reproducible and with higher signal to noise ratios.     

Implementation of force differentiation in the immunoassay

A technique has been developed to apply force to the antibody-antigen complex in a solid-phase immunoassay. Force was applied to the immunochemical complex by labeling the secondary antibody with a magnetically susceptible, micrometer-size particle and placing the assay chamber in a magnetic field of defined magnitude and orientation. The force was strong enough to displace weakly bound particles but was not strong enough to rupture the immunochemical complex. The number of particles bound to the surface after applying the differentiation force was related to the analyte concentration, thus an optical detection scheme was developed for counting the number of particles on the surface. The sensitivity of the force differentiation assay was demonstrated to be one to two orders of magnitude higher than conventional solid-phase immunoassay techniques for model protein, virus, and bacterial analytes, with 99% specificity. The enhanced sensitivity of this assay appears to result from lowering the assay background through the identification of weakly adhesive, nonspecific interactions.     

Bead-based microfluidic immunoassays: the next generation

Microfluidic devices possess many advantages like high throughput, short analysis time, small volume and high sensitivity that fulfill all the important criteria of an immunoassay used for clinical diagnoses, environmental analyses and biochemical studies. These devices can be made from a few different materials, with polymers presently emerging as the most popular choice. Other than being optically clear, non-toxic and cheap, polymers can also be easily fabricated with a variety of techniques. In addition, there are many polymer surface modification methods available to improve the efficiency of these devices. Unfortunately, current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays. Flow cytometry employ the use of encoded microbeads in contrast with normal or paramagnetic microbeads applied in current microfluidic devices. The encoded microbead is the key in providing multiplexing capability to the assay by allowing multi-analyte analysis. Using several unique sets of code, different analytes can be detected in a single assay by tracing the identity of individual beads. The same principle could be applied to microfluidic immunoassays in order to retain all the advantages of a fluidic device and significantly improve multiplexing capability.     

A flow immunoassay for studies of human exposure and toxicity in biological samples

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.     

Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA

The measurement of soluble cytokines and other analytes in serum and plasma is becoming increasingly important in the study and management of many diseases. As a result, there is a growing demand for rapid, precise, and cost-effective measurement of such analytes in both clinical and research laboratories. Multiplex bead array assays provide quantitative measurement of large numbers of analytes using an automated 96-well plate format. Enzyme-linked immunosorbent assay (ELISAs) have long been the standard for quantitative analysis of cytokines and other biomarkers, but are not well suited for high throughput multiplex analyses. However, prior to replacement of ELISA assays with multiplex bead array assays, there is a need to know how comparable these two methods are for quantitative analyses. A number of published studies have compared these two methods and it is apparent that certain elements of these assays, such as the clones of monoclonal antibodies used for detection and reporting, are pivotal in obtaining similar results from both assays. By careful consideration of these variables, it should be possible to utilize multiplex bead array assays in lieu of ELISAs for studies requiring high throughput analysis of numerous analytes.     

Rapid antibody biosensor assays for environmental analysis

Traditionally, biosensor development has focused on molecules with a defined metabolic role that can be exploited by enzyme-based systems. Antibodies have the ability to move beyond this range of analytes, and are particularly useful in detecting small, hapten molecules. Electrochemically based biosensor developments have been less fruitful in this regard, as enzyme labelling is required, and such assays require the separation from bound and unbound species. These separations and the removal of background signals result in the increased complexity of the assay format, making it unsuitable for rapid sensor analysis. We have developed an electrochemical sensor based on antibodies that does not require the separation of bound and unbound molecules in a competition immunoassay format. This removes the need for several washing and separation steps as is normally employed in this type of assay. This allows single-step immunoassays to be performed using this system, and also allows for the real-time monitoring of antibody-antigen interactions. We have shown that such assays are possible in both batch and flow-injection formats and we are currently developing an assay for the pesticide atrazine. Tentative results show that analysis with this system is possible in the p.p.m. to p.p.b. range.     

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.     

紫杉醇免疫检测方法的研究进展

紫杉醇是一种有效的抗肿瘤药物,广泛应用于治疗卵巢癌、乳腺癌和肺癌等癌症。紫杉醇在紫杉树皮中的含量极低（仅为0．01％）,而且紫杉醇是一种对蛋白质有着高亲和力的小分子,在体液中约有98％的分子与蛋白质结合,因此需要一种高灵敏度、高通量的检测方法对紫杉醇进行鉴定。在分析紫杉醇检测方法的基础之上,综述了紫杉醇免疫学检测方法的研究进展,包括紫杉醇半抗原的分子修饰、蛋白偶联物的构建和鉴定以及免疫学检测方法在植物组织和病人血浆中紫杉醇定性和定量中的应用。     

Immunoassay for thyroxine (T4) in serum using capillary electrophoresis and micromachined devices

As part of our ongoing effort to develop electrophoretic assay technology for clinical diagnostics, we describe a competitive immunoassay for the determination of serum thyroxine (T4) based on electrophoresis and laser induced fluorescence (LIF). Measurements of total T4 are useful for the clinical evaluation of thyroid function. A fluorescein thyroxine conjugate was utilized in conjunction with a polyclonal antibody preparation as assay reagents. Capillary electrophoresis (CE) conditions tolerant of the direct injection of serum without extraction or other sample preparation steps were developed and used for quantitation of total T4 in serum. We have been exploring the use of micromachined devices with arrays of channels for high assay throughput. Our assay protocol was carried in a microchip format. The results illustrate that gains in speed can be additionally achieved, with the electrophoretic separation of free from bound labelled T4 being performed in about 15 s for serum samples.     

Fast analysis using monolithic columns coupled with high-flow on-line extraction and electrospray mass spectrometric detection for the direct and simultaneous quantitation of multiple components in plasma

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors' laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.     

A magnetite suspension-based washing method for immunoassays using Escherichia coli cells with autodisplayed Z-domains

Escherichia coli cells with autodisplayed Z-domains have been used for immunoassays of specific target analytes. In this study, a magnetite suspension was used for the washing step in immunoassays of E. coli cells with autodisplayed Z-domains. This approach enhanced the washing conditions for these immunoassays by determining (1) the optimal concentration of the magnetite suspension, (2) the capacity of the magnetite suspension-based washing method to recover E. coli cells, and (3) the level at which the activity of autodisplayed Z-domains is maintained. In immunoassays of C-reactive protein (CRP), the immunoassay incorporating the magnetite suspension-based washing method showed a sensitivity and limit of detection considerably higher than those of the conventional centrifugation-based washing method. The results indicated that immunoassays incorporating the magnetite suspension-based washing method are effective for medical diagnoses based on CRP assay.     

Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS

Recent technological advances have made available reverse phase chromatographic media with a 1.7 microm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC-MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC-MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.     

Fluorescence immunoassay system based on the use of a pH-sensitive phase-separating polymer

Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linear water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This polymer precipitates out of water below a critical pH 5.8 at 37 degrees C and redissolves when the pH of solution is above 6.2. The characteristic of this polymer makes it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. The above approach was applied to determination of alpha-fetoprotein with the competitive immunoassay format. Compared with traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100-120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carried out through a sandwich or a competitive method. This general technique may also be used for a wide variety of separation processes in addition to immunoassay, in which a specific component is to be isolated for analysis, recovery, or disposal.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.     

Continuous monitoring of analyte concentrations.

We have investigated the application of a modified, heterogeneous, competitive enzyme immunoassay for the continuous measurement of small analytes in a medium stream. The analytical system contains two antibodies that are immobilized on spatially separated areas, one binding the analyte (Ab1) and the other binding the enzyme (Ab2). An analyte-enzyme conjugate serves as signal generator. The analyte-enzyme conjugate functions as a heterobifunctional shuttle that can bind to either antibody. A semipermeable membrane retains the enzyme shuttle in the internal volume of the sensor but permits the passage of small analytes from the medium stream. The amount of enzyme bound to Ab1 is inversely proportional and the amount of enzyme bound to Ab2 is directly proportional to the analyte concentration. We have demonstrated that this analytical system (1) can provide a larger total signal; (2) has a sensitivity comparable with conventional competitive immunoassays; (3) does not require the separation of bound from free antigens; and (4) is therefore suitable for the continuous measurement of analytes in a medium stream. With a model system, an increase from 0 ng ml-1 to 20 ng ml-1 of the steroid hormone progesterone and the subsequent fall to 0 ng ml-1 could be monitored.     

Cytokine profiling by multiplex immunoassay as an effective approach to assess immunomodulatory activity of bacterial product CANTASTIM

The bacterial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specific protection against bacterial infection, enhances macrophage effector functions and modulates production of cytokines. Most likely, it interacts with components of the innate immune response. Cytokine production can be used to assess the bioactivity of this product but these biomolecules operate in vivo in a complex regulatory network with reciprocal influences so there is a need for profiling an array of cytokines rather than an individual analysis. Current technology development of multiplex immunoassay for simultaneous measurement of multiple analytes in a single assay has greatly improved the throughput and cost effectiveness of cytokine profiling and proved to be an effective approach to evaluate the immunomodulatory activity of the bacterial product CS.     

Fluorescence polarization competition immunoassay for tyrosine kinases

To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used (32)PO(4) transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the (32)PO(4) transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3, 4-d]pyrimidine in these two FP assays was similar to that obtained in the (32)PO(4) transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.