Survey of recent advances in analytical applications of immunoaffinity chromatography

Methods that use immunoaffinity chromatography (IAC) for sample preparation or detection are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds. This paper presents an overview of immunoaffinity chromatography and examines some recent developments of this technique in analytical applications. The emphasis is placed on HPLC-based IAC methods or those that combine IAC with other instrumental techniques; however, novel approaches that employ low-performance IAC columns for chemical quantitation are also considered. Particular applications that are examined include (1) the use of IAC in the direct detection of analytes, (2) the extraction of samples by IAC prior to on- or off-line detection by other methods, (3) the use of IAC in chromatographic-based immunoassays, and (4) the development of postcolumn reactors based on IAC for the detection of analytes as they elute from other types of chromatographic columns. The advantages and limitations for each approach are considered. In addition, a summary is provided of reports in the literature that have used IAC for these various formats.     

High-performance liquid chromatographic separation and detection methods for anabolic compounds

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.     

Liquid chromatography of active principles in Sophora flavescens root

Herbal medicines were one of the major resources for healthcare in earlier stages, and some traditional herbal medicines have been in use for more than 2000 years. Currently, they are attracting more and more attention of the modern pharmaceutical industry, as scientists has become aware that herbs have almost infinite resources for medicine development. This review provides an overview of the analytical approaches applied in the researches concentrated on various aspects of the matrine-type alkaloids in Sophora flavescens root. Emphasis will be laid on the analytical processes of high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), as well as gas chromatography (GC) methods. The sample extraction, separation and detection have been summarized. In addition, the applications of chromatographic determinations are introduced for the main matrine-type alkaloids in S. flavescens root, such as matrine, sophoridine, sophocarpine, lehmannine, sophoramine, oxymartine, oxysophocarpine, cytosine and aloperine. The advantages and limitations of HPLC, CE and GC methods in the analytical applications of the alkaloids are also discussed.     

Separation procedures capable of revealing DNA adducts

Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32P-postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.     

An antibody-based microarray assay for the simultaneous detection of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript>

Advances in microsystem technology have enabled protein and nucleic acid-based microarrays to be used in various applications, including the study of diseases, drug discovery, genetic screening, and clinical and food diagnostics. Analytical methods for the detection of mycotoxins, however, remain largely based on thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or enzyme-linked Immunosorbent assay (ELISA) . The aim of our work, therefore, was to transfer an immunological assay from microtitrr plates into microarray format, in order to develop a multiparametric, rapid, sensitive and inexpensive method for the detection of mycotoxins for use in food safety applications. Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests but also peptide, antigen, and antibody assays, using different formats of microarrays, have evolved within the last decade. Antibody-based microarrays provide a powerful tool that can be used to generate rapid and detailed expression profiles of a defined set of analytes in complex samples and are potentially useful for generating rapid immunological assays of food contaminants. In this paper, we report a feasibility study of the application of antibody microarrays for the simultaneous (or independent) detection of two common mycotoxins, Aflatoxin B₁ and Fumonisin B₁. We present the development of microarray detection of aflatoxin B1 and fumonisin B1 in standard solutions with detection limits of 3 ng/ml of AFB1 and 43 ng/ml for FB1, and have developed a competitive immunoassay in microarray format for simultaneous analyses. The quality of the microarray data is comparable to data generated by microplate-based immunoassay (ELISA), but further investigations are needed in order to characterise our method more fully. We hope that these preliminary results might suggest that further research is warranted in order to develop hapten microarrays for the immunochemical simultaneous analysis of mycotoxins, as well as for other small molecules (e.g. bacterial toxins or biological warfare agents).     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Immunoaffinity techniques coupled to mass spectrometry for the analysis of human peptide hormones: advances and applications

Introduction: The accurate and comprehensive determination of peptide hormones from biological fluids has represented a considerable challenge to analytical chemists for decades. Besides long-established bioanalytical ligand binding assays (or ELISA, RIA, etc.), more and more mass spectrometry-based methods have been developed recently for purposes commonly referred to as targeted proteomics. Eventually the combination of both, analyte extraction by immunoaffinity and subsequent detection by mass spectrometry, has shown to synergistically enhance the test methods’ performance characteristics.

Areas covered: The review provides an overview about the actual state of existing methods and applications concerning the analysis of endogenous peptide hormones. Here, special focus is on recent developments considering the extraction procedures with immobilized antibodies, the subsequent separation of target analytes, and their detection by mass spectrometry.

Expert commentary: Key aspects of procedures aiming at the detection and/or quantification of peptidic analytes in biological matrices have experienced considerable improvements in the last decade, particularly in terms of the assays’ sensitivity, the option of multiplexing target compounds, automatization, and high throughput operation. Despite these advances and progress as expected to be seen in the near future, immunoaffinity purification coupled to mass spectrometry is not yet a standard procedure in routine analysis compared to ELISA/RIA.     

Detection and isolation of human serum autoantibodies that recognize oxidatively modified autoantigens

The breakdown of human immune tolerance to self-proteins occurs by a number of mechanisms, including posttranslational modifications of host molecules by reactive oxygen, nitrogen, or chlorine species. This has led to great interest in detecting serum autoantibodies raised against small quantities of oxidatively modified host proteins in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis. Here, we provide protocols for the preparation and chemical characterization of oxidatively modified protein antigens and procedures for their use in immunoblotting and ELISAs that detect autoantibodies against these antigens in clinical samples. These gel electrophoresis- and plate reader-based immunochemical methods sometimes suffer from low analytical specificity and/or sensitivity when used for serum autoantibody detection. This is often because a single solid-phase protein (antigen) is exposed to a complex mixture of serum proteins that undergo nonspecific binding. Therefore more sensitive/specific techniques are required to detect autoantibodies specifically directed against oxidatively modified proteins. To address this, we describe novel affinity chromatography protocols by which purified autoantibodies are isolated from small volumes (<1 ml) of serum. We have also developed strategies to conjugate submilligram amounts of isolated immunoglobulins and other proteins to fluorophores. This set of methods will help facilitate the discovery of novel diagnostic autoantibodies in patients.     

A chemiluminescent immunosensor based on antibody immobilized carboxylic resin beads coupled with micro-bubble accelerated immunoreaction for fast flow-injection immunoassay

A novel immunoaffinity column used as an immunosensor for flow-injection chemiluminescent (CL) immunoassay was prepared by immobilizing antibody on carboxylic resin beads. The immunosensor could fast recognize and trap the immunocomplex of horseradish peroxidase (HRP)-labeled antibody and antigen, which was firstly formed with a micro-bubble accelerated pre-incubation process, to produce a sandwich immunocomplex. The HRP introduced in the immunoaffinity column could catalyze the CL reaction to produce enzyme-enhanced emission. With alpha-fetoprotein (AFP) as a mode, a flow-injection CL immunoassay was proposed. The whole assay for one sample, including the pre-incubation and the regeneration of immunoaffinity column, could be performed within 16min. The linear range was 1.0-80ng/ml with a correlation coefficient of 0.998 and a detection limit of 0.1ng/ml at a signal/noise ratio of 3. The intra- and inter-assay coefficients of variation at 20ng/ml AFP were 1.2% and 8.5%, respectively. The storage stability of the immunoaffinity column and the accuracy for sample detection were acceptable. This flexible, sensitive, low-cost, and rapid method is valuable for clinical immunoassay.     

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.     

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography

The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-microm x 25-mm column packed with 1-microm silica beads was chosen for use with a 500-microL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R2=0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.     

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Determination of cysteinyl leukotrienes in exhaled breath condensate: method combining immunoseparation with LC-ESI-MS/MS

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.     

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.     

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.     

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomics biomarker discovery

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.