Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Pamela Holzl&#246;hner; Katja Hanack

doi:10.3791/54832

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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09:42 min

January 2, 2017

DOI: 10.3791/54832-v

Pamela Holzlöhner¹, Katja Hanack¹

¹Department of Biochemistry and Biology,University of Potsdam

Overview

This article presents an optimized protocol for generating monoclonal antibodies using hybridoma technology. The method involves immunizing mice, fusing spleen cells with myeloma cells, and selecting for antibody-producing hybridomas.

Key Study Components

Area of Science

Immunology
Biotechnology
Cell Biology

Background

Hybridoma technology is a key method for producing monoclonal antibodies.
Monoclonal antibodies are essential for various detection systems.
The technique allows for specific binders to be generated for biomolecule visualization.
Continuous availability of specific antibodies is a significant advantage.

Purpose of Study

To develop a reliable protocol for monoclonal antibody production.
To facilitate the generation of hybridoma cell lines with predefined specificity.
To enhance the efficiency of antibody production for research applications.

Methods Used

Mice were immunized with an immunoconjugate.
Spleen cells were fused with immortal myeloma cells using PEG and electric impulse.
Hybridoma cells were selected using HAT and antigen-specific ELISA screening.
Cell culture techniques were employed for the maintenance of myeloma cells.

Main Results

Successful generation of antibody-producing hybridoma cell lines.
Demonstrated specificity of generated monoclonal antibodies.
Established a protocol that can be replicated for various applications.
Highlighted the advantages of using hybridoma technology in research.

Conclusions

The optimized protocol provides a robust method for monoclonal antibody generation.
Hybridoma technology is effective for producing antibodies with specific targets.
This method supports advancements in detection systems and biotechnological applications.

Frequently Asked Questions

What is hybridoma technology?

Hybridoma technology is a method used to produce monoclonal antibodies by fusing B cells with myeloma cells.

Why are monoclonal antibodies important?

Monoclonal antibodies are crucial for specific detection and visualization of biomolecules in various research and clinical applications.

How are hybridoma cells selected?

Hybridoma cells are selected using HAT medium and antigen-specific ELISA screening to ensure they produce the desired antibodies.

What are the advantages of using this protocol?

The protocol allows for the generation of monoclonal antibodies with predefined specificity and ensures continuous availability of these antibodies.

What applications can benefit from monoclonal antibodies?

Monoclonal antibodies can be used in ELISA, flow cytometry, and magnetic cell sorting, among other applications.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

The overall goal of this procedure is to generate antibody producing hybridoma cell lines for the biotechnological production of monoclonal antibodies. This method can be used to generate specific binders for the detection and visualization of biomolecules and for the development of effective detection systems, like ELISA, flow cytometry, or magnetic cell sorting. The main advantage of this technique is that it facilitates the generation of monoclonal antibody producing cell lines with a predefined specificity and a continuous availability.

To prepare the myeloma cells, thaw a vial of murine SP2/0 cells in warm water. Then transfer the cells into 10 mL of culture medium and collect them by centrifugation. Re-suspend the pellet in 10 mL of fresh culture medium and seed the cells into a 25 square centimeter flask for incubation at 37 degrees celsius, and 5%CO2, preserving aliquots of the cells for longterm storage after the appropriate number of expansions.

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