Antibodies

Research

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13 pages, 4269 KiB

Open AccessArticle

Evaluation of Molecular Simulations and Deep Learning Prediction of Antibodies’ Recognition of TRBC1 and TRBC2

by Xincheng Zeng, Tianqun Wang, Yue Kang, Ganggang Bai and Buyong Ma

Antibodies 2023, 12(3), 58; https://doi.org/10.3390/antib12030058 - 17 Sep 2023

Cited by 3 | Viewed by 2810

Abstract

T cell receptor β-chain constant (TRBC) is a promising class of cancer targets consisting of two highly homologous proteins, TRBC1 and TRBC2. Developing targeted antibody therapeutics against TRBC1 or TRBC2 is expected to eradicate the malignant T cells and preserve half of the [...] Read more.

T cell receptor β-chain constant (TRBC) is a promising class of cancer targets consisting of two highly homologous proteins, TRBC1 and TRBC2. Developing targeted antibody therapeutics against TRBC1 or TRBC2 is expected to eradicate the malignant T cells and preserve half of the normal T cells. Recently, several antibody engineering strategies have been used to modulate the TRBC1 and TRBC2 specificity of antibodies. Here, we used molecular simulation and artificial intelligence methods to quantify the affinity difference in antibodies with various mutations for TRBC1 and TRBC2. The affinity of the existing mutants was verified by FEP calculations aided by the AI. We also performed long-time molecular dynamics simulations to reveal the dynamical antigen recognition mechanisms of the TRBC antibodies. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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20 pages, 3558 KiB

Open AccessArticle

Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

by Judith Cruz Amaya, Bruce Walcheck, Julie Smith-Gagen, Vincent C. Lombardi and Dorothy Hudig

Antibodies 2023, 12(3), 44; https://doi.org/10.3390/antib12030044 - 27 Jun 2023

Viewed by 2575

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity [...] Read more.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals’ variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC₅₀s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC₅₀s for CD107a externalization vs. ⁵¹Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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16 pages, 2690 KiB

Open AccessArticle

Polyclonal Antibodies Derived from Transchromosomic Bovines Vaccinated with the Recombinant F1-V Vaccine Increase Bacterial Opsonization In Vitro and Protect Mice from Pneumonic Plague

by Sergei S. Biryukov, Hua Wu, Jennifer L. Dankmeyer, Nathaniel O. Rill, Christopher P. Klimko, Kristi A. Egland, Jennifer L. Shoe, Melissa Hunter, David P. Fetterer, Ju Qiu, Michael L. Davies, Christoph L. Bausch, Eddie J. Sullivan, Thomas Luke and Christopher K. Cote

Antibodies 2023, 12(2), 33; https://doi.org/10.3390/antib12020033 - 8 May 2023

Cited by 1 | Viewed by 2919

Abstract

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized [...] Read more.

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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9 pages, 644 KiB

Open AccessCommunication

Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C₆Mab-13) Using 1× Alanine Scanning

by Tomohiro Tanaka, Mayuki Tawara, Hiroyuki Suzuki, Mika K. Kaneko and Yukinari Kato

Antibodies 2023, 12(2), 32; https://doi.org/10.3390/antib12020032 - 28 Apr 2023

Cited by 1 | Viewed by 2377

Abstract

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 [...] Read more.

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C₆Mab-13 (rat IgG₁, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C₆Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1–20 amino acid region of mCCR6. In the ELISA results, C₆Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C₆Mab-13. In our SPR analysis, the dissociation constants (K_D) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C₆Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C₆Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C₆Mab-13 could be useful for further functional analysis of mCCR6 in future studies. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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25 pages, 5519 KiB

Open AccessArticle

Defensin Interactions in Relation to Monoclonal and Disease-Related Proteinase 3 Antibodies Binding at the Catalytic Site

by Morten Zoega, Nicole Hartwig Trier, Rikke Guldhammer Nejrup, Anna Chailyan, Tina Friis, Peter Højrup and Gunnar Houen

Antibodies 2023, 12(1), 23; https://doi.org/10.3390/antib12010023 - 13 Mar 2023

Viewed by 2418

Abstract

Proteinase 3 (PR3) is a neutrophil granulocyte enzyme and an autoantigen found in several forms of vasculitis. Due to the diagnostic and clinical importance of antibodies (Abs) to PR3, it is important to characterize the protein and the nature of its epitopes. Here, [...] Read more.

Proteinase 3 (PR3) is a neutrophil granulocyte enzyme and an autoantigen found in several forms of vasculitis. Due to the diagnostic and clinical importance of antibodies (Abs) to PR3, it is important to characterize the protein and the nature of its epitopes. Here, we have characterized PR3 monoclonal antibodies (MAbs) and disease-associated Abs and their dependency on the PR3 structure and modifications, especially interactions with α-defensins. Three MAbs (HYB 172-01, 172-04, 172-05), which bind to PR3 in its native and denatured forms and provide the disulphide bridges, were intact. α-1-antitrypsin (AT) binds to purified human neutrophil granulocyte PR3 and inhibits its proteolytic activity, towards a small synthetic peptide substrate and a large protein substrate (casein). AT also inhibited the binding of the three MAbs to PR3, indicating that they bind in a region affected by AT binding. However, the MAbs did not inhibit PR3 proteolytic activity with a small substrate, showing that they bound at the active site without restricting access to the substrate cleft. Patient-derived Abs showed essentially the same characteristics as the MAbs, with important implications for vasculitis diagnostics and pathophysiology. Current findings illustrate that PR3 epitopes depend on the three-dimensional structure of the PR3/defensin complex, and that the epitopes depend to a smaller or larger degree on PR3/defensin associations. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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9 pages, 960 KiB

Open AccessCommunication

Determination of the Binding Epitope of an Anti-Mouse CCR9 Monoclonal Antibody (C₉Mab-24) Using the 1× Alanine and 2× Alanine-Substitution Method

by Hiyori Kobayashi, Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Mika K. Kaneko and Yukinari Kato

Antibodies 2023, 12(1), 11; https://doi.org/10.3390/antib12010011 - 31 Jan 2023

Cited by 1 | Viewed by 2686

Abstract

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical [...] Read more.

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C₉Mab-24 (rat IgG_2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1–19). C₉Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C₉Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C₉Mab-24 did not react with four peptides (M13A–F14A, F14A–D15A, D16A–F17A, and F17A–S18A), indicating that _13-MFDDFS_-18 is involved in C₉Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target–antibody interaction. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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13 pages, 6949 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

by Maile Velasquez, Chris O’Sullivan, Robert Brockett, Amanda Mikels-Vigdal, Igor Mikaelian, Victoria Smith and Andrew E. Greenstein

Antibodies 2023, 12(1), 9; https://doi.org/10.3390/antib12010009 - 18 Jan 2023

Cited by 6 | Viewed by 3604

Abstract

Matrix metalloproteinase 9 (MMP9), a protease implicated in multiple diseases, is secreted as an inactive zymogen and requires proteolytic removal of the pro-domain for activation. The relative levels and functionality of the pro- and active-MMP9 isoforms in tissues are not characterized. We generated [...] Read more.

Matrix metalloproteinase 9 (MMP9), a protease implicated in multiple diseases, is secreted as an inactive zymogen and requires proteolytic removal of the pro-domain for activation. The relative levels and functionality of the pro- and active-MMP9 isoforms in tissues are not characterized. We generated a specific antibody that distinguishes an active form of MMP9, F107-MMP9, from the inactive pro-MMP9 isoform. Using multiple in vitro assays and specimen types, we show that F107-MMP9 expression is localized and disease-specific compared with its more abundant parental pro-form. It is detected around sites of active tissue remodeling, including fistulae of inflammatory bowel and dermal fissures in hidradenitis suppurativa, and is expressed by myeloid cells, including macrophages and neutrophils. Together, our findings provide insights into the distribution and potential role of MMP9 in inflammatory diseases. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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10 pages, 1067 KiB

Open AccessArticle

Integrating Single Domain Antibodies into Field-Deployable Rapid Assays

by George P. Anderson, Lisa C. Shriver-Lake, Jinny L. Liu and Ellen R. Goldman

Antibodies 2022, 11(4), 64; https://doi.org/10.3390/antib11040064 - 17 Oct 2022

Cited by 3 | Viewed by 2451

Abstract

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to [...] Read more.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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Review

Jump to: Research

14 pages, 2437 KiB

Open AccessEditor’s ChoiceReview

Recent Progress in Antibody Epitope Prediction

by Xincheng Zeng, Ganggang Bai, Chuance Sun and Buyong Ma

Antibodies 2023, 12(3), 52; https://doi.org/10.3390/antib12030052 - 8 Aug 2023

Cited by 10 | Viewed by 7088

Abstract

Recent progress in epitope prediction has shown promising results in the development of vaccines and therapeutics against various diseases. However, the overall accuracy and success rate need to be improved greatly to gain practical application significance, especially conformational epitope prediction. In this review, [...] Read more.

Recent progress in epitope prediction has shown promising results in the development of vaccines and therapeutics against various diseases. However, the overall accuracy and success rate need to be improved greatly to gain practical application significance, especially conformational epitope prediction. In this review, we examined the general features of antibody–antigen recognition, highlighting the conformation selection mechanism in flexible antibody–antigen binding. We recently highlighted the success and warning signs of antibody epitope predictions, including linear and conformation epitope predictions. While deep learning-based models gradually outperform traditional feature-based machine learning, sequence and structure features still provide insight into antibody–antigen recognition problems. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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21 pages, 2940 KiB

Open AccessEditor’s ChoiceReview

Proteolysis-Targeting Chimera (PROTAC) Delivery into the Brain across the Blood-Brain Barrier

by Toshihiko Tashima

Antibodies 2023, 12(3), 43; https://doi.org/10.3390/antib12030043 - 26 Jun 2023

Cited by 8 | Viewed by 6749

Abstract

Drug development for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease has challenging difficulties due to the pharmacokinetic impermeability based on the blood-brain barrier (BBB) as well as the blurriness of pharmacodynamic targets based on their unclarified pathogenesis and complicated [...] Read more.

Drug development for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease has challenging difficulties due to the pharmacokinetic impermeability based on the blood-brain barrier (BBB) as well as the blurriness of pharmacodynamic targets based on their unclarified pathogenesis and complicated progression mechanisms. Thus, in order to produce innovative central nervous system (CNS) agents for patients suffering from CNS diseases, effective, selective delivery of CNS agents into the brain across the BBB should be developed. Currently, proteolysis-targeting chimeras (PROTACs) attract rising attention as a new modality to degrade arbitrary intracellular proteins by the ubiquitin-proteasome system. The internalizations of peptide-based PROTACs by cell-penetrating peptides and that of small molecule-based PROTACs through passive diffusion lack cell selectivity. Therefore, these approaches may bring off-target side effects due to wrong distribution. Furthermore, efflux transporters such as multiple drug resistance 1 (MDR1) expressed at the BBB might interrupt the entry of small molecule-based PROTACs into the brain. Nonetheless, intelligent delivery using machinery systems to absorb the nutrition into the brain for homeostasis, such as carrier-mediated transport (CMT) or receptor-mediated transcytosis (RMT), can be established. PROTACs with N-containing groups that are recognized by the proton-coupled organic cation antiporter might cross the BBB through CMT. PROTAC-antibody conjugates (PACs) might cross the BBB through RMT. Subsequently, such small molecule-based PROTACs released in the brain interstitial fluid would be transported into cells such as neurons through passive diffusion and then demonstrate arbitrary protein degradation. In this review, I introduce the potential and advantages of PROTAC delivery into the brain across the BBB through CMT or RMT using PACs in a non-invasive way. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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16 pages, 1239 KiB

Open AccessEditor’s ChoiceReview

CCR6 as a Potential Target for Therapeutic Antibodies for the Treatment of Inflammatory Diseases

by Sara Gómez-Melero and Javier Caballero-Villarraso

Antibodies 2023, 12(2), 30; https://doi.org/10.3390/antib12020030 - 20 Apr 2023

Cited by 11 | Viewed by 6442

Abstract

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor (GPCR) involved in a wide range of biological processes. When CCR6 binds to its sole ligand CCL20, a signaling network is produced. This pathway is implicated in mechanisms related to many diseases, [...] Read more.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor (GPCR) involved in a wide range of biological processes. When CCR6 binds to its sole ligand CCL20, a signaling network is produced. This pathway is implicated in mechanisms related to many diseases, such as cancer, psoriasis, multiple sclerosis, HIV infection or rheumatoid arthritis. The CCR6/CCL20 axis plays a fundamental role in immune homeostasis and activation. Th17 cells express the CCR6 receptor and inflammatory cytokines, including IL-17, IL-21 and IL-22, which are involved in the spread of inflammatory response. The CCL20/CCR6 mechanism plays a crucial role in the recruitment of these pro-inflammatory cells to local tissues. To date, there are no drugs against CCR6 approved, and the development of small molecules against CCR6 is complicated due to the difficulty in screenings. This review highlights the potential as a therapeutic target of the CCR6 receptor in numerous diseases and the importance of the development of antibodies against CCR6 that could be a promising alternative to small molecules in the treatment of CCR6/CCL20 axis-related pathologies. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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24 pages, 1399 KiB

Open AccessEditor’s ChoiceReview

Intravenous Immunoglobulins as Immunomodulators in Autoimmune Diseases and Reproductive Medicine

by Tsvetelina Velikova, Metodija Sekulovski, Simona Bogdanova, Georgi Vasilev, Monika Peshevska-Sekulovska, Dimitrina Miteva and Tsvetoslav Georgiev

Antibodies 2023, 12(1), 20; https://doi.org/10.3390/antib12010020 - 2 Mar 2023

Cited by 16 | Viewed by 5933

Abstract

Intravenous administration of immunoglobulins has been routinely used for more than 60 years in clinical practice, developed initially as replacement therapy in immunodeficiency disorders. Today, the use of intravenous immunoglobulins (IVIGs) is embedded in the modern algorithms for the management of a few [...] Read more.

Intravenous administration of immunoglobulins has been routinely used for more than 60 years in clinical practice, developed initially as replacement therapy in immunodeficiency disorders. Today, the use of intravenous immunoglobulins (IVIGs) is embedded in the modern algorithms for the management of a few diseases, while in most cases, their application is off-label and thus different from their registered therapeutic indications according to the summary of product characteristics. In this review, we present the state-of-the-art use of IVIGs in various autoimmune conditions and immune-mediated disorders associated with reproductive failure, as approved therapy, based on indications or off-label. IVIGs are often an alternative to other treatments, and the administration of IVIGs continues to expand as data accumulate. Additionally, new insights into the pathophysiology of immune-mediated disorders have been gained. Therefore, the need for immunomodulation has increased, where IVIG therapy represents an option for stimulating, inhibiting and regulating various immune processes. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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19 pages, 6552 KiB

Open AccessEditor’s ChoiceReview

Non-Affinity Purification of Antibodies

by Tsutomu Arakawa, Yui Tomioka, Masataka Nakagawa, Chiaki Sakuma, Yasunori Kurosawa, Daisuke Ejima, Kouhei Tsumoto and Teruo Akuta

Antibodies 2023, 12(1), 15; https://doi.org/10.3390/antib12010015 - 13 Feb 2023

Cited by 6 | Viewed by 5798

Abstract

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an [...] Read more.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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