Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Related Experiment Video

Updated: Oct 31, 2026

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study
11:10

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Published on: June 29, 2016

A Nechansky1, E Pursch, F Effenberger

  • 1Novartis Forschungsinstitut GmbH, Vienna, Austria.

Hybridoma
|December 5, 1997
PubMed

A panel of monoclonal antibodies (8H10/D11, 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, 8A4/G12/F9, and 8H10/F12) was raised in mice against the recombinant 20-kDa extracellular part of the alpha-chain of the human IgE high affinity receptors (ecFc epsilon RIalpha) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc epsilon RIalpha, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc epsilon RIalpha covalently immobilized on the surface of a sensor chip. The generated hybridomas can be divided into three groups. Hybridoma supernatants 8A4/G12/F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc epsilon RIalpha in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/kappa type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc epsilon RIalpha in ELISA with intensities comparable to each other. Isotype analysis of antibodies secreted by these hybridomas showed that the antibodies 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, and 8H10/D11 were IgG1/kappa. The hybridoma supernatants were purified via protein A chromatography. In a SPR experiment, ecFc epsilon RIalpha, displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expected for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they do not inhibit the binding of human IgE to ecFc epsilon RIalpha. All purified monoclonal antibodies gave positive signals in Western blotting.

More Related Videos

Detection of True IgE-expressing Mouse B Lineage Cells09:40

Detection of True IgE-expressing Mouse B Lineage Cells

Published on: December 1, 2014

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood13:14

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Published on: February 6, 2018

Related Concept Videos

Antibody Structure01:10

Antibody Structure

Overview
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
Antibodies consist of four polypeptide chains: two identical heavy...
Antibody Structure01:10

Antibody Structure

Overview
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
Antibodies consist of four polypeptide chains: two identical heavy...
Antibody Structure and Classes01:25

Antibody Structure and Classes

Antibodies, also known as immunoglobulins, are produced by B cells in response to foreign substances, such as bacteria and viruses. These proteins are critical for recognizing and neutralizing these substances, protecting the body from potential harm.
The basic structure of an antibody consists of four protein chains: two identical heavy chains and two identical light chains. These chains are held together by disulfide bonds and other non-covalent interactions, forming a Y-shaped structure.
Affinity and Avidity01:41

Affinity and Avidity

Overview
Hybridoma Technology01:31

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol...
Antibody Actions01:26

Antibody Actions

Antibodies, or immunoglobulins, are critical players in the immune system's arsenal against invading pathogens. Produced by B cells and plasma cells, their primary role is to detect and bind to specific antigens, molecules found on the surface of pathogens like bacteria or viruses. Beyond antigen recognition, antibodies perform several vital functions that contribute to immune defense.
Neutralization
Antibodies can bind to pathogens, preventing them from infecting host cells. This process...