Name: generation of murine monoclonal antibodies by hybridoma technology
Uploaded: 2017-01-02T12:30:00
Description: Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

The authors acknowledge the German Federal Ministry of Education and Research (BMBF, Grant No: 03IPT7030X, 03IPT703A, and 03IP703) for funding our projects, "Artificial immune reactions," "Camelid antibodies," and "Antibody technologies." We thank Prof. Burkhard Micheel for proofreading the manuscript and for the helpful comments.

Balb/c or NMRI mice (Mus musculus) from our breeding colony at the University of Potsdam (Potsdam, Germany) were used for the production of monoclonal antibodies. The animal work was conducted according to relevant national and international guidelines. The study was approved by the Brandenburg Ministry of Environment, Health, and Consumer Protection (reference number V3-2347-A16-4-2012).
1. Preparation of Immunoconjugates
<ol>
	<li>For coupling the antigen to a carrier, use standar...

<ol>
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The generation of monoclonal antibodies by hybridoma technology requires an intense and detailed epitope analysis, especially with regard to the final application, when the antibody should recognize the target. This is often underestimated by users and leads to antibodies with weak performances. The fusion process is always random, which means that the outcome of specific hybridomas is highly dependent upon the cell ratio and the vitality at this point. After limited dilution, the cells are very unstable and require stri...

The hybridoma technology presented&#160;in this protocol was first described by K&#246;hler and Milstein1 in 1975 and, except for some technical improvements, the main procedure has not changed dramatically during the last 40 years2. The aim of this protocol is to explain a more appropriate immunization strategy, a standard method for the generation of monoclonal antibodies, and an example for a validation method (ELISA).
Antibodies are incredible tools and contribute to a wide range of technological approaches, such as flow cytometry, magnetic cell sorting, or immunofluorescence, as well as to diagnostic and therapeutic options for disease monitoring and treatment3. The commercial availability of monoclonal antibodies for desired targets is demonstrated by the presence of over 24 different web databases with nearly countless quantities of antibodies or antibody-related products4. In 2015, antibody molecules were part of an international discussion5-7 due to serious problems in proper validation and characterization of commercially available antibodies.
It can be difficult and expensive to find specific antibodies for a targeted antigen, and often, they do not have the affinity or specificity needed. Although generating an antibody is still time-consuming and requires skilled personnel to develop and validate the antibody, producing an antibody individually might better than buying one.
Due to the fact that antibody production is time-consuming and requires experience, alternative methods for the production of binding molecules were developed to overcome these problems. The most commonly used alternative method is the recombinant production of single-chain antibodies via phage display. The genes for the variable binding region are extracted from cells and combined with the coating protein of a phage. The single chain is then expressed on the surface of a bacteriophage and screened in several panning steps8. The production of single-chain antibodies is a bit faster, but it also requires a skilled experimentalist. The disadvantages of some recombinant single-chain antibodies are poor stability and a lack of suitability for in vitro diagnostics. In most diagnostic tests, an Fc-receptor for detection is necessary, which needs to be added to a recombinant single-chain antibody afterwards. Again, this is time-consuming and even more complex than the hybridoma technique. In in vitro diagnostics, full-length monoclonal mouse and rabbit antibodies have been demonstrated to be the best choice.
One of the major steps in generating monoclonal antibodies must be done before the work in the lab starts: the design of the immunoconjugate. Questions that need to be addressed are: What is the physical composition of the target in the final application, and which matrices are present? Which concentration will the target have in the application? What is the final application, and what are the requirements that the antibody must fulfill?
Always take into account that if a linear peptide fragment is used, it also has to be linear in the final epitope in the target of choice; otherwise, the antibody will not bind. Of course, independent of the screening method, antibodies could be selected to recognize different antigenic formats in different applications, but this must be validated very precisely. These are the reasons why antibody development and validation are such ambitious processes.
The choice of the antigenic format for immunization is fundamental for antibody development and determines the success or failure of this process. Once the mice express a relevant antibody titer, the spleen cells are isolated and fused with myeloma cells. The most common myeloma cell lines for murine monoclonal antibody development are X63-Ag 8.6539 and Sp2/0-Ag 1410 from a Balb/c mouse strain. The cells descend from a malignant B cell lymphoma and were selected because they do not secrete any of their own heavy or light chains. The cells can be adapted to a ratio between 1:10 and 10:1 (splenocytes versus myeloma cells). In this protocol, the cells were adapted to a ratio of 3:1 and fused by polyethylene glycol (PEG) and electrofusion, according to Stoicheva and Hui11.
The fusion of B cells and myeloma cells is a random process. Therefore, hybrids of two B lymphocytes or two myeloma cells could be generated, but those hybrids would not be able to survive for a long time in culture. The cells undergo a hypoxanthine, aminopterin, and thymidine (HAT) selection, by which only fused hybridoma cells can survive due to the possibility of using the de novo pathway of pyrimidine synthesis. For the generation of monoclonal antibodies, it is necessary to obtain a cell line originating from one mother cell. The monoclonality is ensured by limiting the dilution techniques and the microscopic analysis of cell growth. The hybridoma culture supernatants are screened for specific antibody production, mostly by ELISA or flow cytometry, and the best binders are selected. After mass culture and purification, the antibody molecule can finally be characterized and validated for the desired application.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">glutaraldehyde</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">G5882</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC)</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">39391-10ML</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sulfo-GMBS</td>
			<td style="width:81px;">Perbio Science Germany</td>
			<td style="width:136px;">22324</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">ovalbumin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A5503</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">bovine serum albumin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A2153</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">keyhole limpet hemocyanin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">H8283</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Falcon tubes 15 mL</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P91015</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">reaction vials, 1.5 mL</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">CNT2.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">hollow needle</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">C724.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">glass wool</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">6574.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Sephadex G25 coarse</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">GE-17-0034-02</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Freund&#180;s adjuvant, complete</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">F5881-10ML</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">ELISA plates, 96 well</td>
			<td style="width:81px;">Greiner bio-one</td>
			<td style="width:136px;">655101</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">neonatal calf serum</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">S1025</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">TipOne Tips 1000 &#181;L</td>
			<td style="width:81px;">Starlab</td>
			<td style="width:136px;">S1111-2021</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Pipette tips 200 &#181;L</td>
			<td style="width:81px;">Greiner bio-one</td>
			<td style="width:136px;">739291</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">HRP-conjugated goat-anti-mouse IgG antibody</td>
			<td style="width:81px;">Dianova</td>
			<td style="width:136px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">tetramethylbenzidine</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">6350.2</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Natriumdihydrogenphosphat</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">K300.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">peroxide/urea</td>
			<td style="width:81px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">sulphuric acid</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">4623.3</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">RPMI 1640</td>
			<td style="width:81px;">Life technologies GmbH</td>
			<td style="width:136px;">31870074</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">L-glutamine</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">HN08.2</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">beta-mercaptoethanol</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">fetal calf serum</td>
			<td style="width:81px;">Invitrogen</td>
			<td style="width:136px;">10270106</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-flask 25 cm2</td>
			<td style="width:81px;">Peske GmbH</td>
			<td style="width:136px;">86-V025</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-flask 75 cm2</td>
			<td style="width:81px;">Peske GmbH</td>
			<td style="width:136px;">86-V075</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">ethanol, 96%</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">P075.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">cell strainer</td>
			<td style="width:81px;">VWR international</td>
			<td style="width:136px;">734-0002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Falcon tubes 50 mL</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P91050</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">PEG 8000</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">1546605</td>
			<td></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:247px;">electroporation cuvette, 2mm</td>
			<td style="width:81px;">Biodeal Handelsvertretung Edelmann e.K.</td>
			<td style="width:136px;">EKL2,25</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">hypoxanthine</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">H9636-25G</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">azaserine</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A4142</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">thymidine</td>
			<td style="width:81px;">USB Europa GmbH</td>
			<td style="width:136px;">22305 1 GM</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-plates 96 well</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P92696</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-plates 24 well</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P92424</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">cryotubes, 1mL</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">V7384-1CS</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">dimethylsulfoxid</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">4720.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">protein A sepharose</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">P3391-1G</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">SDS sample loading buffer, Roti-Load 1</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">K929.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">unstained protein ladder</td>
			<td style="width:81px;">BioRad Laboratories</td>
			<td style="width:136px;">161-0363</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">comassie brilliant blue R-250</td>
			<td style="width:81px;">BioRad Laboratories</td>
			<td style="width:136px;">161-0406</td>
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generation of murine monoclonal antibodies by hybridoma technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Figure 1 shows an example of the antigen-specific serum titer for one immunization performed in the lab. In this figure, the immune sera A and B were titrated from 1:50 to 1:5,000,000 in comparison to a serum of a na&#239;ve mouse. The antigen was coated on the solid phase, and the specific antibodies were detected with a POD-conjugated goat anti-mouse Ig antibody. Both sera of the immunized mice showed a significantly higher antigen-specific titer compared to the na&#239...

Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Research

JoVE Journal

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