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 ...

The overall goal of this procedure is to generate antibody producing hybridoma cell lines for the biotechnological production of monoclonal antibodies. This method can be used to generate specific binders for the detection and visualization of biomolecules and for the development of effective detection systems, like ELISA, flow cytometry, or magnetic cell sorting. The main advantage of this technique is that it facilitates the generation of monoclonal antibody producing cell lines with a predefined specificity and a continuous availability.To prepare the myeloma cells, thaw a vial of murine SP2/0 cells in warm water. Then transfer the cells into 10 mL of culture medium and collect them by centrifugation. Re-suspend the pellet in 10 mL of fresh culture medium and seed the cells into a 25 square centimeter flask for incubation at 37 degrees celsius, and 5%CO2, preserving aliquots of the cells for longterm storage after the appropriate number of expansions.To prepare feeder cells, remove the fur from a 12 week old NMRI mouse and disinfect the abdomen with 70%ethanol. Next, inject 5 mL of ice cold RPMI medium into the abdominal cavity and use tweezers to massage the abdomen. Then withdraw the feeder cell suspension and dispense the cells into a new 50 mL tube.After transferring a second feeder cell harvest into the same 50 mL tube, as just demonstrated, bring the total volume in the tube to 50 mL with fresh full cell culture medium. Then transfer the suspension to a new 100 mL reservoir, and repeat the addition. Then mix the cell suspensions well and seed 100 mcL of cells per well into 10 96-well cell culture plates for overnight incubation at 37 degrees celsius and 5%CO2.For cell fusion, use the front end of a syringe plunger to press a freshly harvested spleen through a 40 micron strainer into a 50 mL tube. Suspend the cells in 20 mL of full cell culture medium, and then centrifuge. Next, use a 5 mL pipet to flush the myeloma cells with the culture supernatant and transfer the floating cells to a 50 mL conical tube for centrifugation, followed by four washes in 20 mL of balanced salt buffer.After the last wash, add three times the volume of splenocytes to one part volume of myeloma cells and obtain a cell pellet by centrifugation. Re-suspend the cell mixture in 1 mL of fresh balanced salt buffer and mix 200 mcL of cells with 200 mcL of PEG 8000. Transfer the cell mixture into an electroporation cuvette with a 2 mm diameter and pulse the cells at 600 to 650 V for 25 ms, then rest the cells at room temperature for 3 minutes, before seeding them drop-wise into an Erlenmeyer flask containing 100 mL of selection medium.After fusing the last 200 mcL of cells, incubate the pooled fusion products for three hours in a cell culture incubator. At the end of the incubation, supplement the cell culture with fresh hypoxanthine as a serine, and thymidine. Transfer the suspension to a reservoir and then add 100 mcL of the electroporated cell mixture to each well of feeder cells.Finally, return the plates to the cell culture incubator. After seven days, check the wells under a 10x magnification for the presence of mono-or polyclonal cells. The monoclonal cells will appear as one cluster of cells that originate from a single cell.The polyclonal cells will appear as multiple clusters of cells in one well. Then replace the supernatant with fresh selection medium, and return the cells to the incubator for another seven days. At the end of the second incubation, collect the supernatant for ELISA and add fresh full cell culture medium to the cells, for expansion of the hybridomas, into 24-well culture plates.To cryoconserve the positive mono-and polyclonal hybridomas for longterm storage, harvest the cells from the 24-well plate when they reach an at least 60%confluency. Centrifuge the collected cells and re-suspend the pellet in 500 mcL of full cell culture medium. Transfer the cells into cryotubes.Then mix the suspensions with 500 mcL of freeze medium and store the hybridomas at minus 80 degrees celsius for three days. For limiting dilution of the polyclonal hybridomas, after the first seven days of culture, perform an ELISA on the culture supernatants to identify the appropriate monoclonal hybridomas for mass culture. Next, transfer the monoclonal hybridomas that exhibited the appropriate ELISA signals to a cell culture flask.Add medium and then return the culture to the incubator. Cryopreserving three to five aliquots in total during mass culture is sufficient. After two to four weeks, filter the supernatant through a 0.45 micron cell strainer and mix the supernatant with binding buffer at a 3:1 ratio.Then wash a Protein A column with washing buffer and load the supernatant onto the column, collecting the flow-through. Elute the bound antibody with 0.1 molar citrate followed by the immediate neutralization of the pH with 500 mcL of TrisHCl. In this representative experiment, immune sera were titrated in comparison to serum from a naive mouse.Both sera from the immunized mice exhibited a significantly higher antigen-specific titer compared to the naive animal, with a dilution of 1:5000 sufficient for cell fusion. Here a standard elution profile of a monoclonal antibody with an IgG1 isotype, purified via Protein A mediated affinity chromatography as just demonstrated, is shown. Further characterization of the eluate by SDS-PAGE revealed clearly visible proteins at 50 and 25 kDa, weights consistent with that expected for heavy-and light-chain antibody proteins respectively, indicating a successful purification of the isolated antibody molecule.Once mastered, this technique can be completed within three and a half hours if performed properly. After watching this video, you should have a good understanding of how to generate monoclonal antibody producing cell lines.

generation-of-murine-monoclonal-antibodies-by-hybridoma-technology

Research

JoVE Journal

Immunology and Infection

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 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 marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

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

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 plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

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

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

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 transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

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, 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 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 glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing 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 Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

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 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 ...

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 vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

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