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 standard coupling protocols via amino-, carboxy, or sulfo-groups, such as by glutaraldehyde, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), or N-[y-maleimidobutyryloxy]succinimide ester (sulfo-GMBS).</li>
	<li>For coupling (e.g., with glutaraldehyde)13, use the target protein or peptide and the carrier (ovalbumin, bovine serum albumin, or keyhole limpet hemocyanin) in a 1:1 ratio. Dilute the target protein or peptide in phosphate-buffered saline (PBS) and the carrier protein in 50 mM NaHCO3 buffer. Adapt the volume and the concentration to the amount necessary for immunizations.
	<ol>
		<li>Mix both solutions and add 1.25% glutaraldehyde. Mix and incubate the sample for 4 h at room temperature (RT). To select the hybridoma cells, make the same immunoconjugate, but with another carrier, in order to avoid the selection of unspecific carrier binders.</li>
	</ol>
	</li>
	<li>Dialyze the coupling sample by performing a fast dialysis with commercial coarse resin (e.g., Sephadex G25).
	<ol>
		<li>Perforate the bottom of a 1.5-mL reaction vial with a hollow needle and place it in a 15-mL tube. Put glass wool in the bottom for sealing and cover it with 300 mg of coarse resin (to the calibration mark of 0.5 mL).</li>
		<li>Carefully, put 1 mL PBS dropwise into a 1.5-mL reaction tube and make sure that the glass wool is still sealing the perforation. Let the coarse resin swell and centrifuge the column at 500 x g for 2 min. One such column can be used to dialyze 200 &#181;L of the coupling sample. For higher volumes, make more columns.</li>
		<li>Change the tube and drop the coupling solution onto the swelled coarse resin. Centrifuge again at 500 x g for 2 min. Remove the eluent from the tube and use it for immunization.</li>
	</ol>
	</li>
</ol>
2. Immunization of Animals
<ol>
	<li>Choose two or three 6- to 12-week-old Balb/c mice for immunization.</li>
	<li>For one animal, prepare 150 - 200 &#181;g of the immunoconjugate in 200 &#181;L of PBS and mix it with 100 &#181;L of complete Freund&#180;s adjuvant (CFA). Mix the solution 3 times with a short and thin needle (0.6 mm diameter) on a 1-mL syringe. Inject the solution intraperitoneally into the mouse.</li>
	<li>Give a booster injection 6 weeks later with the same amount of the immunoconjugate, but without CFA. Take blood 7 days later (out of the retroorbital sinus) and prepare the serum by incubating the blood for 3 - 4 h at room temperature (RT).
	<ol>
		<li>Centrifuge the mixture at 3,000 x g for 5 min. Take the serum supernatant and transfer it into a new tube. Store it at -20 &#176;C. Discard the pellet.</li>
	</ol>
	</li>
	<li>Analyze the sera via ELISA, as described in Step 3. For fusion, choose the mouse with the highest serum titer.</li>
	<li>In preparation for cell fusion, boost the mouse with 150 - 200 &#181;g of the immunoconjugate in 300 &#181;L of PBS without adjuvant only 4 days before the fusion.</li>
</ol>
3. ELISA
<ol>
	<li>Prepare an antigen solution with a concentration of 5 &#181;g/mL in PBS and transfer 50 &#181;L into each ELISA well of a 96-well plate.</li>
	<li>Incubate the plate for 2 h at 37 &#176;C or overnight at 4 &#176;C in a humid chamber.</li>
	<li>Wash the plate 3 times with tap water. Block the wells with 80 &#181;L of PBS/5% neonatal calf serum (NCS) per well for 1 h at RT in a humid chamber. Prepare the serum dilutions in a 1:5 series starting with 1:50. For analysis of the cell culture, use the undiluted culture supernatant.</li>
	<li>Wash the plate 3 times with tap water. Pipette 50 &#181;L of the sample per well and incubate for 1 h at RT in a humid chamber. Prepare the secondary antibody solution in a 1:5,000 dilution of a goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP).</li>
	<li>Wash the plate 3 times with tap water and add 50 &#181;L of the secondary antibody solution per well. Incubate for 45 min at RT in a humid chamber.</li>
	<li>Prepare a fresh substrate solution. Make stock solutions of 0.1 M NaH2PO4 and 0.1% hydrogen peroxide. Take 5 parts NaH2PO4, 4 parts hydrogen peroxide, and 1 part tetramethylbenzidine for the fresh substrate solution.
	<ol>
		<li>Wash the plate 10 times with tap water. Pipette 50 &#181;L of fresh substrate solution into the wells and incubate for 5 - 10 min in the dark.</li>
	</ol>
	</li>
	<li>Stop the reaction with 50 &#181;L 1 M H2SO4 per well. Using a plate reader, measure the optical density at 450 nm with a reference wavelength at 630 nm.</li>
</ol>
4. Preparation of Myeloma Cell Cultures
<ol>
	<li>From now on, perform all work under sterile conditions.</li>
	<li>Prepare a fresh culture medium for cell cultivation. Supplement 500 mL of RPMI1640 with 10% fetal calf serum (50 mL), 2 mM L-glutamine (5 mL), and 50 &#181;M mercaptoethanol (5 mL). Substitute the glutamine every 7 days.</li>
	<li>Thaw a vial of murine SP2/0 cells in warm water. Transfer the cell solution into 10 mL of fresh culture medium and centrifuge for 10 min at 200 x g. Discard the supernatant and resuspend the pellet in 1 mL of fresh culture medium.</li>
	<li>Transfer the cells in a 25-cm2 flask and incubate them at 37 &#176;C and 5% CO2 in an incubator. Expand the cells to two 75-m2 flasks before fusion. Conserve aliquots for long-term storage, as described in Step 7.</li>
</ol>
5. Preparation of Feeder Cells
<ol>
	<li>Sacrifice a 12-week-old NMRI mouse (according to national and institutional guidelines; e.g., by CO2 inhalation). Remove the fur and clean the abdomen with 70% ethanol.</li>
	<li>Inject 5 mL of ice cold RPMI medium into the abdomen, massage the abdomen with a tweezer, and aspirate the feeder cell suspension. Place the cells in an empty 50-mL tube. Perform this step once again. 7 to 10 mL of the feeder cell suspension should be retrieved.</li>
	<li>Prepare a 100-mL feeder cell suspension by adding 90 mL of full cell culture medium (RPMI 1640, 10% fetal calf serum, 2 mM glutamine, and 50 &#181;M mercaptoethanol). Mix it and transfer 100 &#181;L/well into 10 x 96-well cell culture plates. Incubate the plates overnight at 37 &#176;C and 5% CO2 in an incubator.</li>
</ol>
6. Cell Fusion
<ol>
	<li>Sacrifice the immunized mouse by cervical dislocation or CO2 inhalation and excise the spleen14. Take additional blood from the heart and prepare the serum, as described in step 2.3, as a positive control in the ELISA.</li>
	<li>Prepare a spleen cell suspension by pressing the spleen through a cell strainer. Fill up to 20 mL with full cell culture medium. Harvest the cells in 50-mL tubes by centrifugation (200 x g, 10 min, and 4 &#176;C). Discard the supernatant.</li>
	<li>Rinse the cell culture flask and harvest the myeloma cells by centrifugation (200 x g, 10 min, and 4 &#176;C) and discard the supernatant.</li>
	<li>Resuspend the cell pellets in 20 mL of balanced salt buffer (BSS) (125 mM NaCl, 5 mM KCl, 4 mM CaCl2, 2.5 mM MgCl2, and 5 mM Tris-HCl; pH 7.4) and centrifuge again. Repeat the washing steps three times.</li>
	<li>For adjusting the splenocyte:myeloma cell ratio, count the cells after the second washing step by first resuspending the pellet in 1 mL of BSS buffer. Make a 1:10 dilution and transfer 10 &#181;L to a cell counting chamber. Count the cells and determine the cell concentration in 1 mL.</li>
	<li>Adjust the cell numbers in the BSS buffer to three-parts splenocytes and one-part myeloma cells in a 1-mL final volume. Take 200 &#181;L of the cell suspension and mix it with 200 &#181;L of PEG8000 (1 g in 4 mL of BSS buffer). Transfer the 400 &#181;L into an electroporation cuvette with a diameter of 2 mm and set the pulse (600 - 650 V and 25 ms).
	<ol>
		<li>After the pulse, incubate the cells at RT for 3 min in the cuvette. Remove the cells and put them dropwise into an Erlenmeyer flask with 100 mL of HAT Medium (RPMI 1640, 10% FCS, 2 mM glutamine, 50 &#181;M mercaptoethanol, 100 &#181;M hypoxanthine, 5.8 &#181;M azaserine, and 16 &#181;M thymidine).</li>
		<li>Repeat the fusion 4 times until the 1-mL batch is spent. Incubate the Erlenmeyer flask for 3 h at 37 &#176;C and 5% CO2 in an incubator.</li>
	</ol>
	</li>
	<li>Supplement the cell suspension again with hypoxanthine, azaserine, and thymidine before plating the cells on the feeder layer. Add 100 &#181;L of the cell suspension per well to the feeder layer plates and incubate the plates at 37 &#176;C and 5% CO2 (in an incubator). The final concentration within the well should be 100 &#181;M hypoxanthine, 5.8 &#181;M azaserine, and 16 &#181;M thymidine.</li>
	<li>Perform a microscopic analysis (magnification: 10X objective lens, 10X eye-piece) for clonal growth in each well of the plate after 7 days in order to determine if the wells are mono- or polyclonal. Monoclonal cells are one cluster of cells that originate from one single cell. Polyclonal cells are multiple clusters of cells in one well. Change the complete media after 7 days and incubate again for 7 days in HAT medium.</li>
	<li>Change the HAT medium and cultivate the cells in full cell culture medium. Use the supernatant to perform an ELISA, as described in Step 3. Positive hybridoma cells must be expanded in 24-well plates according to the tests for the production of specific antibodies. If the cells are polyclonal, a limited dilution has to be performed (described in step 8).</li>
</ol>
7. Cryoconservation of Hybridomas
<ol>
	<li>Cryoconserve positive mono- and polyclonal hybridomas for long-term storage. Harvest the cells from a 24-well plate with a confluence of &#62; 60% by centrifugation (200 x g, 10 min, and 4 &#176;C).</li>
	<li>Resuspend the pellet in 500 &#181;L of full cell culture media and transfer it into a cryotube. Add 500 &#181;L of freeze medium (RPMI 1640, 20% FCS, and 15% dimethylsulfoxide), mix it, and put it in in a Styrofoam box or a special freezing box. Store at -80 &#176;C for 3 days. After 3 days, the cryotube can be transferred to liquid nitrogen for long-term storage.</li>
</ol>
8. Limiting Dilution of Polyclonal Hybridomas
<ol>
	<li>Make a feeder layer culture, as described in Step 5. Harvest positive polyclonal cells, as described in Step 7.1. Count the cells, as described in Step 6.5, and adjust the cell number to 10 cells/mL, with a final volume of 10 mL for a 96-well plate. Add 100 &#181;L/well onto the feeder layer.</li>
	<li>Incubate for 7 days at 37 &#176;C and analyze the clonal growth microscopically (magnification: 10X objective lens, 10X eye-piece). Make an ELISA of the culture supernatant, as described in Step 3, and identify the appropriate monoclonal hybridomas for mass culture.</li>
</ol>
9. Mass Culture and Purification of Monoclonal Antibodies
<ol>
	<li>Transfer the monoclonal hybridomas with the appropriate ELISA signals to 25 cm2 or 75 cm2 flasks and collect up to 500 mL of the culture supernatant. Test the culture weekly for specific antibody production and conserve 3 - 5 aliquots per hybridoma for long-term storage, as described in step 7.</li>
	<li>Centrifuge the culture supernatant at 4,900 x g and 4 &#176;C for 15 min and filter it through a 0.45-&#181;m filter. Mix the supernatant with binding buffer (4 M NaCl and 2 M glycine NaOH, pH 8.9) in a 3:1 ratio.</li>
	<li>Prepare a protein A column by washing it with washing buffer (dilute the binding buffer 1:3 in dH2O). Pass the supernatant from Step 9.2 over the column and collect the flow-through. Elute the bound antibody with 0.1 M citrate, pH 3.5 and neutralize the pH immediately with 500 &#181;L of Tris-HCl, pH 9.0.</li>
	<li>Characterize the eluted antibody by SDS-PAGE, Western Blot, and ELISA, and validate it for the final application.
	<ol>
		<li>For the SDS PAGE, take 3 - 5 &#181;g of purified antibody solution per lane (a 20-&#181;L sample), add 5 &#181;L of 4x SDS sample loading buffer (8% SDS, 20% 2-mercaptoethanol, 40% glycerin, and 0.015% bromophenol, reduced), and heat the probes at 95 &#176;C for 5 min. As a control, use the same sample with a purified antibody of the same isotype but irrelevant specificity.</li>
		<li>Load the samples on a 12.5% SDS gel, place 5 &#181;L of an unstained protein ladder in an additional lane, and separate them by electrophoresis. Stain the gel with Coomassie Brilliant Blue and document the results. A basic SDS protocol was established by Laemmli in 197015.</li>
	</ol>
	</li>
</ol>

<ol><li>Köhler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Continous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 7 (256), 495-497 (1975).</li>
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<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+anarchy%3A+A+call+to+order%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Filamentous+fusion+phage%3A+novel+expression+vectors+that+display+cloned+antigens+on+the+virion+surface%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((A+new+mouse+myeloma+cell+line+that+has+lost+immunoglobulin+expression+but+permits+the+construction+of+antibody-secreting+hybrid+cell+lines%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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., Köhler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+better+cell+line+for+making+hybridomas+secreting+specific+antibodies%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Electrically+induced+fusion+of+mammalian+cells+in+the+presence+of+polyethylene+glycol%5BTitle%5D)+AND+%22J+Membr+Biol%22%5BJournal%5D)">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/pubmed?term=((Current+developments+in+hybridoma+technology%5BTitle%5D)+AND+%22Tijdschr+Diergeneeskd%22%5BJournal%5D)">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/pubmed?term=((Glutaraldehyde%3A+behavior+in+aqueous+solution%2C+reaction+with+proteins%2C+and+application+to+enzyme+crosslinking%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.</a> Biotechniques. 37 (5), 798-802 (2004).</li>
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</ol>

The authors Pamela Holzl&#246;hner and Katja Hanack are the co-founders of new/era/mabs GmbH, a company that offers the customized generation of monoclonal antibodies.

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><tbody><tr><td>glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&lt;/em&gt;-ethylcarbodiimide (EDC)</td><td>Sigma Aldrich</td><td>39391-10ML</td><td></td></tr><tr><td>Sulfo-GMBS</td><td>Perbio Science Germany</td><td>22324</td><td></td></tr><tr><td>ovalbumin</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>keyhole limpet hemocyanin</td><td>Sigma Aldrich</td><td>H8283</td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Biochrom GmbH</td><td>P91015</td><td></td></tr><tr><td>reaction vials, 1.5 mL</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>CNT2.1</td><td></td></tr><tr><td>hollow needle</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>C724.1</td><td></td></tr><tr><td>glass wool</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6574.1</td><td></td></tr><tr><td>Sephadex G25 coarse</td><td>Sigma Aldrich</td><td>GE-17-0034-02</td><td></td></tr><tr><td>Freund&amp;acute;s adjuvant, complete</td><td>Sigma Aldrich</td><td>F5881-10ML</td><td></td></tr><tr><td>ELISA plates, 96 well</td><td>Greiner bio-one</td><td>655101</td><td></td></tr><tr><td>neonatal calf serum</td><td>Biochrom GmbH</td><td>S1025</td><td></td></tr><tr><td>TipOne Tips 1,000 &amp;micro;L</td><td>Starlab</td><td>S1111-2021</td><td></td></tr><tr><td>Pipette tips 200 &amp;micro;L</td><td>Greiner bio-one</td><td>739291</td><td></td></tr><tr><td>HRP-conjugated goat-anti-mouse IgG antibody</td><td>Dianova</td><td>115-035-003</td><td></td></tr><tr><td>tetramethylbenzidine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6350.2</td><td></td></tr><tr><td>Natriumdihydrogenphosphat</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K300.2</td><td></td></tr><tr><td>peroxide/urea</td><td></td><td></td><td></td></tr><tr><td>sulphuric acid</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4623.3</td><td></td></tr><tr><td>RPMI 1640</td><td>Life technologies GmbH</td><td>31870074</td><td></td></tr><tr><td>L-glutamine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>HN08.2</td><td></td></tr><tr><td>beta-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270106</td><td></td></tr><tr><td>TC-flask 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V025</td><td></td></tr><tr><td>TC-flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V075</td><td></td></tr><tr><td>ethanol, 96%</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>P075.1</td><td></td></tr><tr><td>cell strainer</td><td>VWR international</td><td>734-0002</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Biochrom GmbH</td><td>P91050</td><td></td></tr><tr><td>PEG 8000</td><td>Sigma Aldrich</td><td>1546605</td><td></td></tr><tr><td>electroporation cuvette, 2 mm</td><td>Biodeal Handelsvertretung Edelmann e.K.</td><td>EKL2,25</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma Aldrich</td><td>H9636-25G</td><td></td></tr><tr><td>azaserine</td><td>Sigma Aldrich</td><td>A4142</td><td></td></tr><tr><td>thymidine</td><td>USB Europa GmbH</td><td>22305 1 GM</td><td></td></tr><tr><td>TC-plates 96 well</td><td>Biochrom GmbH</td><td>P92696</td><td></td></tr><tr><td>TC-plates 24 well</td><td>Biochrom GmbH</td><td>P92424</td><td></td></tr><tr><td>cryotubes, 1 mL</td><td>Sigma Aldrich</td><td>V7384-1CS</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4720.1</td><td></td></tr><tr><td>protein A sepharose</td><td>Sigma Aldrich</td><td>P3391-1G</td><td></td></tr><tr><td>SDS sample loading buffer, Roti-Load 1</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K929.1</td><td></td></tr><tr><td>unstained protein ladder</td><td>BioRad Laboratories</td><td>161-0363</td><td></td></tr><tr><td>comassie brilliant blue R-250</td><td>BioRad Laboratories</td><td>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 ...

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Immunology and Infection

41.6K Views.  University of Potsdam. 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. 

Video: Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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 histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

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

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

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 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, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

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

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 antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

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 Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

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

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

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Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Source: Nogales-Gadea, G. et al., <a href="https://app.jove.com/v/52830">Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Peripheral Blood ...

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Antibody-Based Technologies

Production and Purification

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Source: Xia, P., et al. <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Source: Devilder, M., et al. <a href="https://app.jove.com/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges K&#246;hler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques &#8212; electroporation, polyethylene glycol (PEG) mediated fusion, and the use of fusogenic viruses &#8212; generally produce hybridomas at a very low frequency. Fusion products thus have a high ratio of self-fused B-cells and myeloma cells, or unfused cells, with a low number of hybrid cells.
The hypoxanthine-aminopterin-thymidine (HAT) medium is used to select hybridomas by selectively allowing their growth. The aminopterin blocks the default nucleotide synthesis in cells. However, cells can utilize hypoxanthine and thymidine from the medium to synthesize nucleotides via the salvage pathway.
Myeloma cells deficient in the HGPRT enzyme cannot synthesize nucleotides via this pathway and thus, do not grow in HAT medium. Although B-cells produce functional HGPRT enzymes, they cannot divide indefinitely. Thus, only the hybrid cells with functional HGPT from the B-cells and immortality from the myeloma line can grow on the HAT selection medium. Such a culture of hybrid cells is called a hybridoma, which can be screened for monoclonal antibody production.
Hybridoma Screening
Hybridoma cultures from the HAT selection are plated onto a 96-well plate; each well containing only one cell. Using Enzyme-linked Immunosorbent Assay (ELISA), each cell is screened for the production of antibodies specific to the epitope of interest. Cells that test positive are then selected and grown in a larger culture vessel to establish hybridoma cell lines. These cell lines are a permanent source of unlimited monoclonal antibodies. Thus hybridoma technology is a convenient and cost-effective method for mass production of monoclonal antibodies.

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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