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>
	<li>K&#246;hler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 7 (256), 495-497 (1975).</li><li>Hanack, K., Messerschmidt, K., Listek, M., B&#246;ldicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+and+Selection.">Antibodies and Selection.</a> Protein Targeting Compounds. , 11-22 (2015).</li><li>Walsh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopharmaceutical+benchmarks.">Biopharmaceutical benchmarks.</a> Nat Biotechnol. 32 (10), 992-1000 (2014).</li><li>Pauly, D., Hanack, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+avoid+pitfalls+in+antibody+use.">How to avoid pitfalls in antibody use.</a> F1000 Res. 7 (4), 691-698 (2015).</li><li>Bradbury, A., Pl&#252;ckthun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility:+Standardize+antibodies+used+in+research.">Reproducibility: Standardize antibodies used in research.</a> Nature. 5 (518), 27-29 (2015).</li><li>Polakiewicz, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies:+The+solution+is+validation.">Antibodies: The solution is validation.</a> Nature. 26 (518), 483 (2015).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+anarchy:+A+call+to+order.">Antibody anarchy: A call to order.</a> Nature. 26 (527), 545-551 (2015).</li><li>Smith, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fusion+phage:+novel+expression+vectors+that+display+cloned+antigens+on+the+virion+surface.">Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.</a> Science. 228 (4705), 1315-1317 (1985).</li><li>Kearney, J. F., Radbruch, A., Liesegang, B., Rajewsky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mouse+myeloma+cell+line+that+has+lost+immunoglobulin+expression+but+permits+the+construction+of+antibody-secreting+hybrid+cell+lines.">A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.</a> J Immunol. 123 (4), 1548-1550 (1979).</li><li>Shulman, M., Wilde, C. D., K&#246;hler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+better+cell+line+for+making+hybridomas+secreting+specific+antibodies.">A better cell line for making hybridomas secreting specific antibodies.</a> Nature. 16 (276), 269-270 (1978).</li><li>Stoicheva, N. G., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+induced+fusion+of+mammalian+cells+in+the+presence+of+polyethylene+glycol.">Electrically induced fusion of mammalian cells in the presence of polyethylene glycol.</a> J Membr Biol. 141 (2), 177-182 (1994).</li><li>Osterhaus, A. D., Uytdehaag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+hybridoma+technology.">Current developments in hybridoma technology.</a> Tijdschr Diergeneeskd. 15 (110), 835-839 (1985).</li><li>Migneault, I., Dartiguenave, C., Bertrand, M. J., Waldron, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaraldehyde:+behavior+in+aqueous+solution,+reaction+with+proteins,+and+application+to+enzyme+crosslinking.">Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.</a> Biotechniques. 37 (5), 798-802 (2004).</li><li>Vitetta, E. S., Baur, S., Uhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Surface+Immunoglobulin+II.+Isolation+and+characterization+of+immunoglobulin+from+mouse+splenic+lymphocytes.">Cell Surface Immunoglobulin II. Isolation and characterization of immunoglobulin from mouse splenic lymphocytes.</a> Journal of experimental medicine. 134, 242-264 (1971).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Browne, S. M., Al-Rubeai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+methods+for+high-producing+mammalian+cell+lines.">Selection methods for high-producing mammalian cell lines.</a> Trends Biotechnol. 25 (9), 425-432 (2007).</li><li>Holmes, P., Al-Rubeai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+cell+line+development+by+a+high+throughput+affinity+capture+surface+display+technique+to+select+for+high+secretors.">Improved cell line development by a high throughput affinity capture surface display technique to select for high secretors.</a> J Immunol Methods. 19 (230), 141-147 (1999).</li><li>Weaver, J. C., McGrath, P., Adams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+microdrop+technology+for+rapid+isolation+of+rare+and+high+producer+cells.">Gel microdrop technology for rapid isolation of rare and high producer cells.</a> Nat Med. 3 (5), 583-585 (1997).</li><li>Messerschmidt, K., Hempel, S., Holzl&#246;hner, P., Ulrich, R. G., Wagner, D., Heilmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+antibody+production+by+intrarectal+immunization+of+mice+using+recombinant+major+capsid+protein+of+hamster+polyomavirus.">IgA antibody production by intrarectal immunization of mice using recombinant major capsid protein of hamster polyomavirus.</a> Eur J Microbiol Immunol. 2 (3), 231-238 (2012).</li><li>Listek, M., Micheel, B., Hanack, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomoleküle+freisetzende+zelle+und+deren+selektion+mittels+eines+oberflächenproteins.">Biomoleküle freisetzende zelle und deren selektion mittels eines oberflächenproteins.</a> neweramabs GmbH. , (2014).</li></ol>

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>
			<td></td>
		</tr>
	</tbody>
</table>

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

Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...