Immunoaffinity Chromatography for Protein Purification and Analysis.

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low-performance supports such as agarose for off-line immunoextraction and supports used in high-performance IAC for on-line immunoextraction. The use of IAC for protein analysis as a flow-based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Off-line immunoextraction by traditional immunoaffinity chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.

Approaches for the detection and analysis of antidrug antibodies to biopharmaceuticals: A review.

Antibody-based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side-effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography-mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis.

Studies of binding by 2-imidazolines to human serum albumin and alpha1-acid glycoprotein by high-performance affinity chromatography.

2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25 mm × 2.1 mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62 × 102 to 1.07 × 104 M-1 at pH 7.4 and 37 °C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80 × 102 to 1.85 × 104 M-1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.

Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction.

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α1-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~105-106 M-1 for the given drugs with AGP at 37°C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Recent Advances in Supramolecular Affinity Separations: Affinity Chromatography and Related Methods.

Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.

Affinity chromatography: A review of trends and developments over the past 50 years.

The field of affinity chromatography, which employs a biologically-related agent as the stationary phase, has seen significant growth since the modern era of this method began in 1968. This review examines the major developments and trends that have occurred in this technique over the past five decades. The basic principles and history of this area are first discussed. This is followed by an overview of the various supports, immobilization strategies, and types of binding agents that have been used in this field. The general types of applications and fields of use that have appeared for affinity chromatography are also considered. A survey of the literature is used to identify major trends in these topics and important areas of use for affinity chromatography in the separation, analysis, or characterization of chemicals and biochemicals.

Clinical and pharmaceutical applications of affinity ligands in capillary electrophoresis: A review.

Affinity capillary electrophoresis (ACE) is a separation technique that combines a biologically-related binding agent with the separating power and efficiency of capillary electrophoresis. This review will examine several classes of binding agents that have been used in ACE and applications that have been described for the resulting methods in clinical or pharmaceutical analysis. Binding agents that will be considered are antibodies, aptamers, lectins, serum proteins, carbohydrates, and enzymes. This review will also describe the various formats in which each type of binding agent has been used in CE, including both homogeneous and heterogeneous methods. Specific areas of applications that will be considered are CE-based immunoassays, glycoprotein/glycan separations, chiral separations, and biointeraction studies. The general principles and formats of ACE for each of these applications will be examined, along with the potential advantages or limitations of these methods.

Testosterone meets albumin - the molecular mechanism of sex hormone transport by serum albumins.

Serum albumin is the most abundant protein in mammalian blood plasma and is responsible for the transport of metals, drugs, and various metabolites, including hormones. We report the first albumin structure in complex with testosterone, the primary male sex hormone. Testosterone is bound in two sites, neither of which overlaps with the previously suggested Sudlow site I. We determined the binding constant of testosterone to equine and human albumins by two different methods: tryptophan fluorescence quenching and ultrafast affinity extraction. The binding studies and similarities between residues comprising the binding sites on serum albumins suggest that testosterone binds to the same sites on both proteins. Our comparative analysis of albumin complexes with hormones, drugs, and other biologically relevant compounds strongly suggests interference between a number of compounds present in blood and testosterone transport by serum albumin. We discuss a possible link between our findings and some phenomena observed in human patients, such as low testosterone levels in diabetic patients.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents.

The last few decades have witnessed the development of many high-performance separation methods that use biologically related binding agents. The combination of HPLC with these binding agents results in a technique known as high performance affinity chromatography (HPAC). This review will discuss the general principles of HPAC and related techniques, with an emphasis on their use for the analysis of biological compounds and pharmaceutical agents. Various types of binding agents for these methods will be considered, including antibodies, immunoglobulin-binding proteins, aptamers, enzymes, lectins, transport proteins, lipids, and carbohydrates. Formats that will be discussed for these methods range from the direct detection of an analyte to indirect detection based on chromatographic immunoassays, as well as schemes based on analyte extraction or depletion, post-column detection, and multi-column systems. The use of biological agents in HPLC for chiral separations will also be considered, along with the use of HPAC as a tool to screen or study biological interactions. Various examples will be presented to illustrate these approaches and their applications in fields such as biochemistry, clinical chemistry, and pharmaceutical research.