DOCSLIB.ORG

Antibody Generation: Challenges and Solutions

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Antibody Generation: Challenges and Solutions Antibody Generation: challenges and solutions Glen Marszalowicz, PHD May 10, 2015 9AM GenScript – the most cited biology CRO Gene Services Peptide Services CRISPR Services Protein Services Antibody Services Discovery Biology Catalog Products Services Make Research Easy 2 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 3 Antibody applications Research • Track biomarker location, expression -Enzyme linked immunosorbent assay(ELISA) -Fluorescent activated cell sorting (FACS) -Western Blot -Immunohistochemistry (IHC), Immunocytochemistry (ICC) -Neutralize receptors/ligands in lieu of knock-out systems Industry • Traditional assays, but also - Therapeutic Antibody Drugs - Antibody Drug Conjugates (ADCs) - Recombinant antibodies (rAbs) result in many formats Make Research Easy 4 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 5 How are antibodies generated Antibodies are created in response to foreign (non-self) antigen via the adaptive immune system • B-cells recognize foreign antigens and, with further activation by Helper T cells, release antibodies to that antigen. B-cell recognizes Cognate helper T-cell Transformation to plasma cell exogenous antigen activates through cytokine signaling and antibody secretion Make Research Easy 6 Creating an immune response Primary immunization (prime) • Inject antigen, with carrier protein or adjuvant if required Measure response via ELISA Provided additional exposures (boost) with antigen in same or in various format (stable cell expressing, alternative carrier protein, etc.) Measure response via ELISA If satisfied with serum titer, proceed to antibody recovery Make Research Easy 7 How to recover antibodies Polyclonal antibodies: • Collect serum • (optional) purify by protein A/G or antigen affinity Monoclonal antibodies • Create hybridomas • Reach clonality through limiting dilutions • Select for positive clones • Expand, produce, purify, and preserve Make Research Easy 8 Creating hybridomas Lymphocyte fusion with myelomas via electrofusion. Selection of only lymphocyte/myeloma fusion products through HAT medium Make Research Easy 9 Achieving monoclonality Repetitive limiting dilutions statistically insures a single cell/well Selection of positive clones via ELISA Make Research Easy 10 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 11 Challenges with Antibody Generation Antibody generation requires many steps, therefore many chances for difficulty including: Antigen Production • Difficult to produce antigens Immune response • Self-tolerance Final product • Low affinity antibodies Make Research Easy 12 Challenge: Difficult Antigens Possible antigen production difficulties • Low yield- not enough antigen produced • Low stability- antigen does not survive long enough for durable exposure • Low solubility- achieving concentrations necessary for immunization are difficult • Complexity- ex; GPCRs, ion channels Additional antigen issues • Toxicity- exposure harms or kills host Make Research Easy 13 Solution to difficult antigens: DNA Immunization Monoclonal Antibody Development Fusion & Screening Immune Response Immunization Cloning Target cDNA In vivo expression overcomes antigen generation issues. Make Research Easy 14 Case Study: GLP1R mAb by DNA immunization FACS ELISA Glucagon-like peptide receptor-1 (GLP1R): • 3 Disulfide bonds • N-terminal glycosylation Western Blot ICC Make Research Easy 15 Challenge: self-tolerance (to high homology antigens) Make Research Easy 16 Solution to self-tolerance Modify immunization such that signaling mimics foreign antigen exposure • GenScript proprietary ImmunoPlus™ technology Overcomes self tolerance through antigen modification Make Research Easy 17 Challenge: low affinity antibodies Despite high serum and hybridoma supernatant titers during initial screening, ultimate affinity is unknown until sufficient antibodies quantities can be generated Only low affinity antibodies may be recovered at which time the opportunity to generate high affinity antibodies in vivo is lost. Make Research Easy 18 Solution to low affinity antibodies Germinal Center Associated Nuclear Protein (GANP) transgenic mice B-cells experience somatic hypermutation (SHM) which create a wider variety of variable domains. Antibodies have greater chances of subtle changes in the complemetarity determining region (CDR), increasing the opportunity to recover high affinity antibodies. Make Research Easy 19 Conclusions Antibody generation has may steps for potential difficulties, but also many opportunities for optimization Novel techniques such as DNA immunization, immunomodulation, and GANP mice offer elegant solutions to previously insurmountable problems Immunization approach must be tailored to fit the characteristics of the target Ultimately, collaborating or outsourcing to experience hands can expedite the process Make Research Easy 20 Antibody Generation: challenges and solutions 1 Antibody applications 2 Traditional antibody generation 3 3 Common challenges and solutions 4 Related antibody generation services Make Research Easy 21 GenScript’s antibody generation solutions •antibodies in 45 days with just your target Polyclonal sequence starting at $649. •high quality, purified monoclonal antibodies Monoclonal guaranteed to work in your application Phospho- •monoclonal or polyclonal with <10% non- specific phospho peptide cross-reactivity guaranteed •Ideal for studying PK/PD of therapeutic Anti-idiotype antibodies DNA Immunization Applicable to ImmunoPlus™ any antibody GANP® Mice project! Make Research Easy 22 Q&A Thank you for attending! Type your questions into the “Questions” field on your Go to Webinar toolbar Please complete the survey you receive by email Check out upcoming and archived webinars at www.genscript.com/webinars.html Email me at [email protected] Make Research Easy 23 .
Load more

Recommended publications
  • Perspectives on in Vitro Diagnostic Devices, Regulated by the Office Of
    1 2 “This transcript appears as received from the commercial transcribing service after inclusion of minor corrections to typographical and factual errors recommended by the Technical Point of Contact.” 1 PERSPECTIVES ON IN VITRO DIAGNOSTIC DEVICES, 2 REGULATED BY THE OFFICE OF BLOOD RESEARCH AND REVIEW 3 4 FOOD AND DRUG ADMINISTRATION 5 WHITE OAK CAMPUS 6 BUILDING 31 7 10903 NEW HAMPSHIRE AVENUE 8 SILVER SPRING, MARYLAND 20903 9 10 TUESDAY, JULY 16, 2019 11 8:30 A.M. 12 13 APPEARANCES: 14 MODERATOR: TERESITA C. MERCADO, MS 15 16 WELCOME: 17 JULIA LATHROP, PHD, DETTD/OBRR 18 19 INTRODUCTION: 20 WENDY PAUL, MD, DBCD/OBRR 21 22 PRESENTERS: 23 KIMBERLY BIGLER, MLS(ASCP)CMSBB, DBCD/OBRR 1 ANNETTE RAGOSTA, MT(ASCP)SBB, DBCD/OBRR 2 ZHUGONG "JASON" LIU, PHD, DRB/DBCD 3 4 CONFERENCE 5 TUESDAY, JULY 16, 2019 6 8:30 A.M. 7 DR. LATHROP: Okay, everybody. Thank you for coming to the second day of 8 our workshop which is going to be focused on considerations for IVDs regulated by the Division 9 of Blood Components and Devices in OBRR. 10 Again, the slides will be available on the website in about two weeks, so take a 11 look for them there. And moderating today’s session is Teresita Mercado from the Division. 12 MS. MERCADO: Good morning. Welcome to session three of the IVD 13 workshop. Our first speaker will be Dr. Wendy Paul. She is the deputy director of DBCD who 14 will give us an introduction to devices in DBCD. 15 DR.
    [Show full text]
  • Monoclonal Antibodies As Tools to Combat Fungal Infections
    Journal of Fungi Review Monoclonal Antibodies as Tools to Combat Fungal Infections Sebastian Ulrich and Frank Ebel * Institute for Infectious Diseases and Zoonoses, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, D-80539 Munich, Germany; [email protected] * Correspondence: [email protected] Received: 26 November 2019; Accepted: 31 January 2020; Published: 4 February 2020 Abstract: Antibodies represent an important element in the adaptive immune response and a major tool to eliminate microbial pathogens. For many bacterial and viral infections, efficient vaccines exist, but not for fungal pathogens. For a long time, antibodies have been assumed to be of minor importance for a successful clearance of fungal infections; however this perception has been challenged by a large number of studies over the last three decades. In this review, we focus on the potential therapeutic and prophylactic use of monoclonal antibodies. Since systemic mycoses normally occur in severely immunocompromised patients, a passive immunization using monoclonal antibodies is a promising approach to directly attack the fungal pathogen and/or to activate and strengthen the residual antifungal immune response in these patients. Keywords: monoclonal antibodies; invasive fungal infections; therapy; prophylaxis; opsonization 1. Introduction Fungal pathogens represent a major threat for immunocompromised individuals [1]. Mortality rates associated with deep mycoses are generally high, reflecting shortcomings in diagnostics as well as limited and often insufficient treatment options. Apart from the development of novel antifungal agents, it is a promising approach to activate antimicrobial mechanisms employed by the immune system to eliminate microbial intruders. Antibodies represent a major tool to mark and combat microbes. Moreover, monoclonal antibodies (mAbs) are highly specific reagents that opened new avenues for the treatment of cancer and other diseases.
    [Show full text]
  • Generation of High Affinity Anti-Peptide Polyclonal Antibodies
    molecules Article Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein Aliah Zannierah Mohsin 1 , Rashidah Sukor 1,2,* , Jinap Selamat 1,2 , Anis Shobirin Meor Hussin 2, Intan Hakimah Ismail 3, Nuzul Noorahya Jambari 1,2 and Farina Mustaffa-Kamal 4 1 Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang 43400, Malaysia; [email protected] (A.Z.M.); [email protected] (J.S.); [email protected] (N.N.J.) 2 Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang 43400, Malaysia; [email protected] 3 Faculty of Medicine, Universiti Putra Malaysia, Serdang 43400, Malaysia; [email protected] 4 Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Malaysia; [email protected] * Correspondence: [email protected]; Tel.: +603-9769-8254 Academic Editor: Paolo Iadarola Received: 18 April 2020; Accepted: 5 May 2020; Published: 5 June 2020 Abstract: The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography.
    [Show full text]
  • Monoclonal Antibodies Vs Polyclonal Antibodies
    ARP American Research Products Inc http://blog.arp1.com Monoclonal Antibodies vs Polyclonal Antibodies Author : Dan Souw Monoclonal Antibodies vs Polyclonal Antibodies: what are the differences and how they will affect your experiments Monoclonal antibodies vs Polyclonal antibodies: The different production processes Both polyclonal and monoclonal antibodies share a huge number of applications, such as IHC and WB, in common. However, the very nature of how they are produced can often make one more suitable for a given application over another. So what are the difference in their production? And how does this affect their various features? The Polyclonal Antibody Production Process In nature, the primary immune response to a foreign antigen involves the activation of large numbers of B-cells to protect the body. Each B-cell targets a single antigen, and produces antibodies against a specific epitope of that antigen. The result is the production large numbers of antibodies, each with different specificities and epitope affinities, known as ‘polyclonal antibodies’. Polyclonal antibodies can be raised in many different species, such as goats, sheep, or rabbits. To do this, the animals are typically immunized with a specific antigen to elicit an immune response. This is usually followed by further immunizations to boost the levels of circulating antibodies – the titer. Following this, the serum containing the desired antibodies is collected. This is then typically enriched using affinity purification. This process – multiple immunizations followed by affinity purification – ultimately lends itself to the production of high titer, high-affinity polyclonal antibodies against the antigen of interest. The Monoclonal Antibody Production Process Monoclonal antibodies are created with a single B-cell clone from one animal, generating 1 / 4 ARP American Research Products Inc http://blog.arp1.com antibodies to a single specific epitope.
    [Show full text]
  • Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9
    applied sciences Communication Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9 Eun-Jung Kim 1, Gyu-Min Im 2, Chang-Soo Lee 3, Yun-Gon Kim 4, Byoung Joon Ko 5, Hee-Jin Jeong 6,* and Byung-Gee Kim 1,2,* 1 Bio-MAX/N-Bio, Seoul National University, Seoul 08826, Korea; [email protected] 2 Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 08826, Korea; [email protected] 3 Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, Korea; [email protected] 4 Department of Chemical Engineering, Soongsil University, Seoul 06978, Korea; [email protected] 5 School of Biopharmaceutical and Medical Sciences, Sungshin Women’s University, Seoul 02844, Korea; [email protected] 6 Department of Biological and Chemical Engineering, Hongik University, Sejong 30016, Korea * Correspondence: [email protected] (H.-J.J.); [email protected] (B.-G.K.) Abstract: The calcium-binding protein S100A9 regulates inflammatory processes and the immune response. It is overexpressed in a variety of inflammatory and oncologic conditions. In this study, we produced a recombinant human S100A9 (hS100A9) antigen with high yield and purity and used it to generate a hybridoma cell culture-based monoclonal anti-hS100A9 antibody. We selected five anti-hS100A9 antibodies from cell supernatants that showed high antigen binding efficiency and identified the nucleotide sequences of three antibodies: two with high effective concentration values and one with the lowest value. The antigen and antibody development procedures described herein Citation: Kim, E.-J.; Im, G.-M.; Lee, are useful for producing large amounts of monoclonal antibodies against hS100A9 and other antigens C.-S.; Kim, Y.-G.; Ko, B.J.; Jeong, H.-J.; of interest.
    [Show full text]
  • 1. Vaccinations What Is Vaccination?
    Chapter 18: Applications of Immunology 1. Vaccinations 2. Monoclonal vs Polyclonal Ab 3. Diagnostic Immunology 1. Vaccinations What is Vaccination? A method of inducing artificial immunity by exposing the individual to some portion or form of the pathogen (aka “immunization”): • triggers an adaptive immune response resulting in the production of memory T and B cells specific for antigens from the pathogen • a secondary exposure will result in a potent and immediate immune response to the specific pathogen due to the memory cells **the vaccination itself should NOT cause an infection** 1 Different Types of Vaccines 1) Attenuated whole agent vaccines: • live but “weakened” pathogen • genetically modified • mutant or less virulent strains of the pathogen 2) Inactivated whole agent vaccines: • pathogen that has been killed in some way • usually by chemical treatment, also by heat **whole agents are generally more effective due to containing multiple antigens, but also carry more risk of infection** 3) Toxoid vaccines: • chemically inactivated protein exotoxins • inactivated toxins are referred to as toxoids 4) Subunit vaccines: • a specific protein or protein fragment from pathogen • purified from pathogen directly OR • produced as a recombinant vaccine in other organism 5) Conjugated vaccines: • small or non-protein antigens attached to a “carrier” • necessary to enhance immune response **these “molecular vaccines” tend to be less effective however they are safer than whole agents** Haptens & Conjugated Antigens • some molecules (haptens) are too small to induce an IR • cannot be processed and presented on MHC class II • others (e.g. polysaccharides) are not very immunogenic • in either case, conjugation to a protein carrier is nec.
    [Show full text]
  • Antibody Production
    Canadian Council on Animal Care guidelines on: antibody production This document, the CCAC guidelines on: antibody production, has been developed by the ad hoc subcommittee on immunological procedures of the Canadian Council on Animal Care (CCAC) Guidelines Committee: Dr Albert Clark, Queen's University (Chair) Dr Dean Befus, University of Alberta (Canadian Society for Immunology representative) Dr Pamela O'Hashi, University of Toronto Dr Fred Hart, Aventis Pasteur Dr Michael Schunk, Aventis Pasteur Dr Andrew Fletch, McMaster University Dr Gilly Griffin, Canadian Council on Animal Care In addition, the CCAC is grateful to the many individuals, organizations and associations that pro- vided comments on earlier drafts of this guidelines' document. In particular thanks are extended to: the Canadian Society of Immunology; the Canadian Association for Laboratory Animal Science and the Canadian Association for Laboratory Animal Medicine; Dr Terry Pearson, University of Victoria; Dr Mavanur Suresh, University of Alberta; Drs Ernest Olfert and Barry Ziola, University of Saskatchewan; and Dr Patricia Shewen, University of Guelph. © Canadian Council on Animal Care, 2002 ISBN: 0-919087-37-X Canadian Council on Animal Care 315-350 Albert Street Ottawa ON CANADA K1R 1B1 http://www.ccac.ca CCAC guidelines on: antibody production, 2002 TABLE OF CONTENTS A. PREFACE . .1 E. REFERENCES . .20 B. INTRODUCTION . .2 1. General . .20 2. Polyclonal Antibody Production . .21 C. POLYCLONAL ANTIBODY PRODUCTION 3 3. Monoclonal Antibody Production . .25 3.1 Other useful references . .27 1. Animal Selection and Care . .3 3.2 Additional useful information . .27 2. Immunization Protocol . .6 3. Standard Operating Procedures . .6 F. GLOSSARY . .27 4.
    [Show full text]
  • The Immune System
    CAMPBELL BIOLOGY IN FOCUS URRY • CAIN • WASSERMAN • MINORSKY • REECE 35 The Immune System Lecture Presentations by Kathleen Fitzpatrick and Nicole Tunbridge, Simon Fraser University © 2016 Pearson Education, Inc. SECOND EDITION Overview: Recognition and Response . Pathogens, agents that cause disease, infect a wide range of animals, including humans . The immune system enables an animal to avoid or limit many infections . All animals have innate immunity, a defense that is active immediately upon infection . Vertebrates also have adaptive immunity © 2016 Pearson Education, Inc. Figure 35.1 © 2016 Pearson Education, Inc. Innate immunity includes barrier defenses . A small set of receptor proteins bind molecules or structures common to viruses, bacteria, or other microbes . Binding an innate immune receptor activates internal defensive responses to a broad range of pathogens © 2016 Pearson Education, Inc. Adaptive immunity, or acquired immunity, develops after exposure to agents such as microbes, toxins, or other foreign substances . It involves a very specific response to pathogens © 2016 Pearson Education, Inc. Figure 35.2 Pathogens (such as bacteria, fungi, and viruses) INNATE IMMUNITY Barrier defenses: (all animals) Skin • Recognition of traits shared Mucous membranes by broad ranges of Secretions pathogens, using a small set of receptors Internal defenses: Phagocytic cells • Rapid response Natural killer cells Antimicrobial proteins Inflammatory response ADAPTIVE IMMUNITY Humoral response: (vertebrates only) Antibodies defend against • Recognition of traits infection in body fluids. specific to particular pathogens, using a vast Cell-mediated response: array of receptors Cytotoxic cells defend against infection in body cells. • Slower response © 2016 Pearson Education, Inc. Concept 35.1: In innate immunity, recognition and response rely on traits common to groups of pathogens .
    [Show full text]
  • Assessment of the Immune Response to Posthodiplostomum Minimum Infection in Bluegills
    Eastern Illinois University The Keep Masters Theses Student Theses & Publications Summer 2020 Assessment of the Immune Response to Posthodiplostomum minimum Infection in Bluegills Olamide S. Olayinka Eastern Illinois University Follow this and additional works at: https://thekeep.eiu.edu/theses Part of the Immunity Commons, Immunology of Infectious Disease Commons, and the Parasitology Commons Recommended Citation Olayinka, Olamide S., "Assessment of the Immune Response to Posthodiplostomum minimum Infection in Bluegills" (2020). Masters Theses. 4823. https://thekeep.eiu.edu/theses/4823 This Dissertation/Thesis is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. ASSESSMENT OF THE IMMUNE RESPONSE TO POSTHODIPLOSTOMUM MINIMUM INFECTION IN BLUEGILLS by Olamide S. Olayinka D.V.M., University of Ibadan, 2015 (Under the Direction of Dr Jeffrey R. Laursen) A Thesis Submitted for the Requirements for the Degree of Master of Science Department of Biological Sciences Eastern Illinois University August 2020 1 ABSTRACT Bluegills (Lepomis macrochrius) are common intermediate hosts for white grub (Posthodiplostomum minimum). They tolerate heavy infections with minimal effect on condition and continue to accumulate metacercariae as they age. This suggests that any immune response to this parasite might not be effective. This study was conducted to better understand the immune mechanisms underlying P. minimum infection in bluegills. Infected organs (liver, kidney, and heart) were examined histologically, and serum from infected fish was tested for antibodies to white grub. Juvenile flukes were recovered from isolated metacercarial cysts.
    [Show full text]
  • Different Types of Glomerulopathic Light Chains Interact with Mesangial Cells Using a Common Receptor but Exhibit Different Intracellular Trafficking Patterns
    Laboratory Investigation (2004) 84, 440–451 & 2004 USCAP, Inc All rights reserved 0023-6837/04 $25.00 www.laboratoryinvestigation.org Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns Jiamin Teng1, William J. Russell1, Xin Gu1, James Cardelli2, M Lamar Jones1 and Guillermo A. Herrera1,3,4 1Department of Pathology; 2Department of Microbiology; 3Department of Cell Biology and Anatomy and 4Department of Medicine, Louisiana State University Health Sciences Center, Shreveport, LA, USA Patients with plasma cell dyscrasias may have circulating light chains (LCs), some of which are nephrotoxic. Nephrotoxic LCs can affect the various renal compartments. Some of these LCs may produce predominantly proximal tubular damage, while others are associated with distal nephron obstruction (the so-called ‘myeloma kidney’). Both these are considered tubulopathic (T) LCs. A receptor has been found in proximal tubular cells (cubilin/megalin complex), which mediates the absorption of LCs and is involved in the pathogenesis of tubulopathies that occurs in these patients. Another group of nephrotoxic LCs is associated with glomerular damage and these are considered as glomerulopathic (G). These patients with G-LCs may develop AL- amyloidosis (AL-Am) or LC deposition disease (LCDD). Recent evidence indicates that the physicochemical characteristics (amino-acid composition and conformation of the variable region) of a given nephrotoxic LC may be the most significant factor in determining the type and location of renal damage within the nephron. Other factors may also be involved, including yet uncharacterized host factors that may include genetic polymorphism, among others. Interestingly, the amount of LC production by the clone of plasma cells does not correlate directly with the severity of the renal alterations.
    [Show full text]
  • Antigens & Antibodies II Polyclonal Antibodies Vs Monoclonal Antibodies
    A Brief Review of Antibody Structure A Brief Review of Antibody Structure Primary structure Tertiary structure The basic antibody is a dimer of dimer (2 heavy chain-light chain pairs) composed of repeats of a single structural unit known as the “immunoglobulin Secondary structure domain” Quaternary structure Antigens & Antibodies II Polyclonal antibodies vs Monoclonal antibodies Definitions A comparison of antigen recognition by B and T cells Polyclonal antibodies: antibody preparations from Factors that influence immunogenicity immunized animals. Consist of complex mixtures of different antibodies produced by many different B cell Quantitating the strength of antibody-antigen interactions clones Equilibrium constants equilibrium dialysis Monoclonal Antibody: homogeneous antibody preparations impact of multivalency produced in the laboratory. Consist of a single type of antigen binding site, produced by a single B cell clone Cross-reactivity of antibodies (later we’ll talk about how these are made). Measuring antibody-antigen binding Affinity between two macromolecules can measured using a biosensor Technique: Surface Plasmon Resonance Instrument: Biocore -Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here) - Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to calculate equilibrium constants (Ka, Kd). -Rate of dissociation and association (koff, kon) can also be calculated. 1 Avidity (strength of binding) is influenced by both Affinity refers to strength of binding of single Affinity (Ka of single binding site) x Valence of epitope to single antigen binding site. interaction (number of interacting binding sites) But antibodies have 2 or more identical binding sites.
    [Show full text]
  • Passive Immunotherapy of Viral Infections: 'Super-Antibodies'
    REVIEWS Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray Laura M. Walker1 and Dennis R. Burton2–4 Abstract | Antibodies have been used for more than 100 years in the therapy of infectious diseases, but a new generation of highly potent and/or broadly cross-reactive human monoclonal antibodies (sometimes referred to as ‘super-antibodies’) offers new opportunities for intervention. The isolation of these antibodies, most of which are rarely induced in human infections, has primarily been achieved by large-scale screening for suitable donors and new single B cell approaches to human monoclonal antibody generation. Engineering the antibodies to improve half-life and effector functions has further augmented their in vivo activity in some cases. Super-antibodies offer promise for the prophylaxis and therapy of infections with a range of viruses, including those that are highly antigenically variable and those that are newly emerging or that have pandemic potential. The next few years will be decisive in the realization of the promise of super-antibodies. The use of antibodies to ameliorate the adverse clini­ then the generation of fully human antibodies by vari­ cal effects of microbial infection can be traced back to ous techniques described below. Such antibodies have the late 19th century and the work of von Behring and been largely applied in the fields of oncology and auto­ Kitasato on the serum therapy of diphtheria and tetanus immunity. Only a single antiviral mAb, the RSV­specific (reviewed in REFS 1,2). In these settings, the antibodies antibody palivizumab, is in widespread clinical use. act to neutralize bacterial toxins.
    [Show full text]