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Updated: Aug 7, 2026
09:42
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
Generation of sequence-specific, high affinity anti-DNA antibodies
M L Cerutti1, J M Centeno, F A Goldbaum
1Instituto de Investigaciones Bioquimicas, Fundación Campomar and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, (1405) Buenos Aires, Argentina.
The Journal of Biological Chemistry
|March 30, 2001
Summary
Researchers developed highly specific monoclonal antibodies targeting a human papillomavirus (HPV-16) DNA sequence. These antibodies exhibit high affinity and specificity, crucial for understanding DNA-protein interactions and developing diagnostic tools.
Area of Science:
- Immunology
- Molecular Biology
- Virology
Background:
- The E2 transcriptional regulator of human papillomavirus (HPV-16) plays a key role in viral replication.
- Developing specific antibodies against DNA sequences is challenging due to DNA's nature as a poor immunogen.
- Protein-DNA complexes offer a stable platform for generating anti-DNA antibodies.
Purpose of the Study:
- To generate highly specific monoclonal antibodies against a predetermined DNA sequence.
- To characterize the binding affinity and specificity of these antibodies for the HPV-16 E2 binding site.
- To investigate the potential of DNA-protein complexes in eliciting specific anti-DNA immune responses.
Main Methods:
- Utilized a stable protein-DNA complex of HPV-16 E2 and its binding site for immunization.
- Produced and purified univalent antibody fragments from monoclonal antibodies.
- Employed spectroscopic methods to analyze antibody-DNA binding kinetics and affinity (dissociation constants).
- Tested antibody specificity against various double- and single-stranded synthetic DNA sequences.
Main Results:
- Obtained two monoclonal antibodies with high specificity for the target HPV-16 E2 DNA sequence.
- Antibody fragments demonstrated low and subnanomolar dissociation constants, matching natural binding domain affinity.
- Antibodies showed significant discrimination (125- to 20,000-fold) against non-specific DNA sequences.
- Antibodies exhibited fine specificity tuning, binding less tightly to a related DNA sequence differing by one base pair.
Conclusions:
- Established a method for generating highly specific anti-DNA antibodies using protein-DNA complexes.
- The developed antibodies provide a valuable tool for studying HPV-16 E2 DNA interactions.
- These findings offer insights into the mechanisms of antibody recognition of DNA as an antigen.
- The high affinity and specificity suggest potential applications in diagnostics and molecular biology research.
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01:10Antibody 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...
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.
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Antibodies consist of four polypeptide chains: two identical heavy...
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01:31Hybridoma Technology
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01:28Diversity of Antigen Receptors
Antigen receptors are essential components of the immune system crucial in defending the body against foreign invaders. These receptors are present on the surface of B and T cells, enabling them to recognize antigens and mount an appropriate immune response.
Before encountering any antigen, lymphocytes express these receptors. On B cells, the antigen receptor is a membrane-bound antibody molecule called BCR; on T cells, it is a T cell receptor or TCR. B and T cell receptors are composed of two...
Before encountering any antigen, lymphocytes express these receptors. On B cells, the antigen receptor is a membrane-bound antibody molecule called BCR; on T cells, it is a T cell receptor or TCR. B and T cell receptors are composed of two...
01:25Antibody 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.
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.