Detection of Platelet-Activating Antibodies Associated with Vaccine-Induced Thrombotic Thrombocytopenia by Flow Cytometry: An Italian Experience.

Rare cases of thrombocytopenia and thrombosis after anti-COVID-19 adenovirus-associated mRNA vaccines (VITT) due to platelet-activating anti-platelet-factor 4 (PF4)/polyanion antibodies have been reported. VITT laboratory diagnosis, similarly to heparin-induced thrombocytopenia (HIT) diagnosis, requires immunoassays for anti-PF4/polyanion antibodies identification, such as ELISA assays and platelet-activating functional tests, such as heparin-induced platelet activation test (HIPA), to confirm their pathogenicity. We compared the flow cytometry (FC) measurement of platelet p-selectin exposure to the gold standard functional test HIPA for diagnosis confirmation in 13 patients with a clinical VITT syndrome (6M/7F; median age 56 (33-78)) who resulted positive to anti-PF4/polyanion antibodies ELISA assays (12/13). FC and HIPA similarly identified three different patterns: (1) a typical non-heparin-dependent VITT pattern (seven and six patients by FC and HIPA, respectively); (2) low/no platelet activation in patients under IvIg therapy (five out of five and two out of four patients by FC and HIPA, respectively); (3) a HIT pattern. Antibodies investigated by FC became negative after 7, 17, and 24 days of therapy in three patients. FC measurement of P-selectin exposure was as sensitive as HIPA but simpler to detect anti-PF4/polyanion antibodies in VITT patients. FC could reliably discriminate VITT from HIT, thus helping for the choice of the anticoagulant.

Refinement and Neutralization Evaluation of the F(ab')2 Type of Antivenom against the Deadly Jellyfish Nemopilema nomurai Toxins.

Jellyfish stings threaten people's health and even life in coastal areas worldwide. Nemopilema nomurai is one of the most dangerous jellyfish in the East Asian Marginal Seas, which not only stings hundreds of thousands of people every year but also is assumed to be responsible for most deaths by jellyfish stings in China. However, there is no effective first-aid drug, such as antivenoms, for the treatment of severe stings by N. nomurai to date. In this study, we prepared a N. nomurai antiserum from rabbits using inactivated N. nomurai toxins (NnTXs) and isolated the IgG type of antivenom (IgG-AntiNnTXs) from the antiserum. Subsequently, IgG-AntiNnTXs were refined with multiple optimizations to remove Fc fragments. Finally, the F(ab')2 type of antivenom (F(ab')2-AntiNnTXs) was purified using Superdex 200 and protein A columns. The neutralization efficacy of both types of antivenom was analyzed in vitro and in vivo, and the results showed that both IgG and F(ab')2 types of antivenom have some neutralization effect on the metalloproteinase activity of NnTXs in vitro and could also decrease the mortality of mice in the first 4 h after injection. This study provides some useful information for the development of an effective antivenom for N. nomurai stings in the future.

A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.

Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis.

Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 µg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

Superantigen Recognition and Interactions: Functions, Mechanisms and Applications.

Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues.

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

Newly developed radioimmunoassay for Human Angiotensin-(1-12) measurements in plasma and urine.

The dodecapeptide angiotensin-(1-12) [Ang-(1-12)] functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) method for the measurement of Ang-(1-12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1-12) sequence. The RIA method was applied to quantify the Ang-(1-12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1-12) level was significantly higher (P < 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure <140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1-12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1-12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1-12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1-12)/Ang II-mediated hypertension and organ damage.

Improving oligo-conjugated antibody signal in multimodal single-cell analysis.

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.

Development of a novel capillary electrophoresis-based approach for detection of anti-drug antibody responses.

Peggy Sue is a capillary-based western/immunoassay platform that can separate and characterize proteins by size or charge. A quick and automated immunogenicity assay was developed on Peggy Sue based on charge separation and compared with a traditional bridging method using preclinical samples from non-human primate studies. The results generated with the Peggy Sue assay were comparable to those of the bridging assays. The Peggy Sue platform has several advantages, including time efficiency, low sample consumption, and easy automation. The platform is especially ideal for further characterization of anti-drug antibody (ADA) specificity against complex biologics such as bispecific or multi-specific biotherapeutics as it is easy to conduct domain specificity assessment of observed ADA responses. Our evaluation suggests that the Peggy Sue platform is a promising tool for preclinical ADA analysis.

Systemic delivery of a specific antibody targeting the pathological N-terminal truncated tau peptide reduces retinal degeneration in a mouse model of Alzheimer's Disease.

Retina and optic nerve are sites of extra-cerebral manifestations of Alzheimer's Disease (AD). Amyloid-ß (Aß) plaques and neurofibrillary tangles of hyperphosphorylated tau protein are detected in eyes from AD patients and transgenic animals in correlation with inflammation, reduction of synapses, visual deficits, loss of retinal cells and nerve fiber. However, neither the pathological relevance of other post-translational tau modifications-such as truncation with generation of toxic fragments-nor the potential neuroprotective action induced by their in vivo clearance have been investigated in the context of AD retinal degeneration. We have recently developed a monoclonal tau antibody (12A12mAb) which selectively targets the neurotoxic 20-22 kDa NH2-derived peptide generated from pathological truncation at the N-terminal domain of tau without cross-reacting with its full-length normal protein. Previous studies have shown that 12A12mAb, when intravenously (i.v.)-injected into 6-month-old Tg2576 animals, markedly improves their AD-like, behavioural and neuropathological syndrome. By taking advantage of this well-established tau-directed immunization regimen, we found that 12A12mAb administration also exerts a beneficial action on biochemical, morphological and metabolic parameters (i.e. APP/Aß processing, tau hyperphosphorylation, neuroinflammation, synaptic proteins, microtubule stability, mitochondria-based energy production, neuronal death) associated with ocular injury in the AD phenotype. These findings prospect translational implications in the AD field by: (1) showing for the first time that cleavage of tau takes part in several pathological changes occurring in vivo in affected retinas and vitreous bodies and that its deleterious effects are successfully antagonized by administration of the specific 12A12mAb; (2) shedding further insights on the tight connections between neurosensory retina and brain, in particular following tau-based immunotherapy. In our view, the parallel response we detected in this preclinical animal model, both in the eye and in the hippocampus, following i.v. 12A12mAb injection opens novel diagnostic and therapeutic avenues for the clinical management of cerebral and extracerebral AD signs in human beings.

A custom ÄKTA avant configuration enabling automated parallel protein purification over a range of process scales.

Biologics are making up an increasing proportion of the global drug discovery pipeline. Supporting the expansion of biologics drug discovery requires higher throughput techniques for the expression, purification and characterization of both therapeutic candidates and reagents. Here we describe the programming and development of a novel ÄKTA™ instrument configuration that enables automated parallel and multistep chromatography over a range of scales. The programming strategy is offered as open source and the custom plumbing configuration was developed with off the shelf components available from Cytiva. Combined with high flow resin technology we show how this strategy can reduce the duration of a standard antibody purification process by 4.5X, from 4.5 h down to 1 h per run. An automated loading strategy was also developed to enable true walk away application of up to 24 samples and around the clock processing capability. The techniques used here to accomplish parallel multistep chromatography can be duplicated or modified for specific applications and represent a straightforward and cost-effective means to eliminate protein purification bottlenecks.

Antibodies purification from human plasma using fractionation, chromatography and gel electrophoresis assisted by multivariate analysis of complimentary absorption and fluorescence spectra.

Employing simple precipitation (fractionation) using Cohn method and weak anion exchange chromatography with DEAE resin, antibodies such as Immunoglobulin G are purified from human plasma. Fractions are eluted from column in four different regions depending on washing NaCl concentrations. Absorbance and excitation-emission fluorescence spectral data are measured for separated chromatographic fractions and analyzed using Multivariate Curve Resolution- Alternating Least Squares (MCR-ALS) and Parallel Factor Analysis (PARAFAC) techniques. Resolved concentration and spectral profiles provided information about existing components in each fraction. Protein and non-protein components are distinguished considering their resolved pure spectra and information from the two applied spectroscopic techniques is complementary. A number of components displayed both fluorescence and absorbance signals. When concentration of component (protein or non-protein) in sample is low and no significant absorbance signal is observed, sensitive fluorescence is useful to recognize the component and for non-fluorescent components absorbance spectra are utilized. Electrophoresis is utilized for separation of proteins in each fraction and showed that one distinguished protein from fluorescence and/or absorbance data can be a group of proteins with similar pure spectra and retention volume. Results showed presence of two protein in the first region (IgM and IgA), a group of proteins in second region (IgM, α-globulin, and IgG), a pure protein in third region (IgG), and a group of ß-globulin proteins in fifth region. It is well and clearly shown that multivariate analysis of different data sets with complementary information is necessary for better interpretation of such technically simple and biochemically complicated systems.

Advances and applications of in-tube solid-phase microextraction for analysis of proteins.

In-tube solid-phase microextraction (IT-SPME) with capillary column as extraction device is a well-established green extraction technique with a lot of applications in the fields of biomedicine, food and environment. This article reviews the research contributions of IT-SPME for analysis of proteins. The paper first briefly describes the history of IT-SPME. Then, the development and principle of IT-SPME for analysis of proteins are introduced, in which capillary column configurations of IT-SPME and instruments for quantitative analysis of proteins are summarized. Subsequently, the synthesis strategy and recognition principle of different recognition units, including antibodies, aptamers, molecularly imprinted polymers, and boronate affinity materials, are discussed in detail. This part also introduces several rare recognition units, including lectins, restricted access materials, lysine modified with ß-cyclodextrin and cell membrane. The development trend and possible future direction of IT-SPME for analysis of proteins are mentioned.

An immunohistochemical study of lymphatic elements in the human brain.

Almost 150 papers about brain lymphatics have been published in the last 150 years. Recently, the information in these papers has been synthesized into a picture of central nervous system (CNS) "glymphatics," but the fine structure of lymphatic elements in the human brain based on imaging specific markers of lymphatic endothelium has not been described. We used LYVE1 and PDPN antibodies to visualize lymphatic marker-positive cells (LMPCs) in postmortem human brain samples, meninges, cavernous sinus (cavum trigeminale), and cranial nerves and bolstered our findings with a VEGFR3 antibody. LMPCs were present in the perivascular space, the walls of small and large arteries and veins, the media of large vessels along smooth muscle cell membranes, and the vascular adventitia. Lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater. There were many LMPCs in the perineurium and endoneurium of cranial nerves. Soluble waste may move from the brain parenchyma via perivascular and paravascular routes to the closest subarachnoid space and then travel along the dura mater and/or cranial nerves. Particulate waste products travel along the laminae of the dura mater toward the jugular fossa, lamina cribrosa, and perineurium of the cranial nerves to enter the cervical lymphatics. CD3-positive T cells appear to be in close proximity to LMPCs in perivascular/perineural spaces throughout the brain. Both immunostaining and qPCR confirmed the presence of adhesion molecules in the CNS known to be involved in T cell migration.

Nonionic detergent micelle aggregates: An economical alternative to protein A chromatography.

We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates led to high purity of the recovered Ab's (>95 %, by gel densitometry). Good overall Ab recovery yields were observed with Tween-20 (80-83 %), Brij-O20 (85-87 %) and Triton X-100 (87-90 %), while Pluronic F-127 was less efficient (42-53 %). Of additional importance is the finding that the process was performed by filtration rather than centrifugation, thereby allowing a continuous purification mode that led to the recovery of monomeric IgG, as determined by dynamic light scattering and preservation of Ab specificity as measured by ELISA. The amphiphilic chelator, bathophenanthroline (batho) was recycled almost quantitatively (95 %) by crystallization. Good IgG recovery yields of ∼80 % were also observed when Ab concentrations were increased from 1 mg/mL to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies, are discussed.

Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics.

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as ß-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.

Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.

Immunogenicity of Biologic and Biosimilar Therapies for Psoriasis and Impact of Novel Immunoassays for Immunogenicity Detection.

Anti-drug antibodies (ADAs) may develop against originator biologic and biosimilar therapies used for the treatment of psoriasis and may be the cause of initial therapeutic non-response or diminished therapeutic response over time. Comparing immunogenicity between therapeutic agents is challenging owing to the variation in assays used for detection, among other reasons. Using the results of a PubMed search for psoriasis clinical trials disclosing the rates of ADAs for originator biologic and biosimilar therapies approved for the treatment of psoriasis within the last 5 years, this review discusses the rates and potential clinical impact of ADA formation in patients with psoriasis managed with originator biologic and biosimilar therapies, along with novel methods of ADA testing. Anti-drug antibodies are detectable in all biologic and biosimilar therapies approved for the treatment of psoriasis in the last 5 years, and the effect of ADAs on clinical response varies by agent. Novel immunoassays used for the detection of ADAs may have increased sensitivity compared with traditional assays, although the increased rate of detection may not correlate with decreased clinical response and the decision to test for the presence of ADAs may vary from patient to patient. Though ADA formation seems ubiquitous with the use of biologic agents for the treatment of psoriasis, the increased rates of ADAs detected by novel immunoassays may not necessarily correlate with decreased treatment efficacy.

Impact of salt concentration in mobile phase on antibody retention in Protein A, Protein L and KappaSelect affinity chromatography.

Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions.

Studies of binding by sulfonylureas with glyoxal- and methylglyoxal-modified albumin by immunoextraction using affinity microcolumns.

Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20-50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.