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Related Concept Videos

Antibody Structure01:10

Antibody Structure

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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
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The Equilibrium Binding Constant and Binding Strength02:18

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The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
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Ligand Binding Sites

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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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Ligand Binding and Linkage

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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    Area of Science:

    • Immunology
    • Biotechnology

    Background:

    • Antibody discovery is crucial for therapeutic and diagnostic development.
    • Screening methods for antibody-antigen interactions require robust controls and flexibility.
    • Hybridoma technology and phage display are key platforms for antibody generation.

    Purpose of the Study:

    • To present a simple, powerful protocol for testing antibody binding to cell surface antigens.
    • To incorporate built-in negative controls for reliable screening.
    • To enable simultaneous screening of positive and negative cell lines using color-coded dyes.

    Main Methods:

    • Utilizing cell-based assays to detect antibody binding to target antigens.
    • Incorporating cell-permeant dyes for differential cell line identification.
    • Applying the protocol to screen hybridoma supernatants and bacterial periplasmic extracts from phage libraries.
    • A variant enzyme-linked immunosorbent assay (ELISA) using antigen-presenting beads is also described.

    Main Results:

    • The protocol demonstrates effective antibody screening with integrated negative controls.
    • Cell-permeant dyes allow for efficient, multiplexed screening within single wells.
    • The assay is versatile, applicable to both hybridoma and phage display library screening.

    Conclusions:

    • This protocol offers a simplified and powerful method for antibody-antigen interaction testing.
    • The built-in controls and color-coding enhance assay reliability and throughput.
    • The assay's adaptability makes it valuable for antibody discovery pipelines.