Research contract Miguel Servet (ISCIII CD14/00032) to (G.N.-G.). Fellowship from the Netherlands Organization for Scientific Research &#8220;Graduate School of Translational Neuroscience Program&#8221; (022005019) to (C.H.).
Grants from the Prinses Beatrix Fonds (Project WAR08-12) and the Association Fran&#231;aise contre les Myopathies to (P.M.-M.); as well as by a Veni Fellowship of the Netherlands Organization for Scientific Research (916.10.148) a fellowship of the Brain Foundation of the Netherlands (FS2008(1)-28) and the Prinses Beatrix Fonds (Project WAR08-12) (to M.L.).
We thank Jozien Jaspers for her help in the B-cell sorting by flow cytometry.

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+detection+in+automated+DNA+sequence+analysis.">Fluorescence detection in automated DNA sequence analysis.</a> Nature. 321 (6071), 674-679 (1986).</li></ol>

The authors declare that they have no competing financial interests.

In this manuscript, all the steps for the generation of IgG antibodies from human PBMCs are presented in detail. This protocol includes some advantages over previously published techniques. One of the advantages is that the antibody produced keeps the heavy and light chains corresponding to the original pair in the B cell clone. The identification of IgG antibodies can be done in any type of human donor, and there is no need for exacerbation of the immune response due to vaccination5. The use of the fibroblast...

The goal of this article is to describe in detail a methodology to generate and characterize human IgG monoclonal antibodies obtained from human peripheral blood mononuclear cells (PBMCs).
The interest to study human antibodies has grown in many different fields of research. In particular, many research groups are interested in the pathology caused by auto-antibodies1-3. We have cloned and characterized pathogenic auto-antibodies1. The study of auto-antibodies can help to identify their targets and to develop therapeutic strategies, e.g., using competitor antibodies4. Moreover, the study of human antibodies can also be of interest in other fields of research, i.e., to evaluate the immune response after vaccination5, to characterize the antibody profile of individuals that were exposed and became resistant to specific pathogens6 or to study which antibodies are in the natural repertoire7,12.
Several techniques have been developed to generate recombinant human monoclonal antibodies8-12; most of these use phage display and B-cell immortalization. The use of phage display has been extensively applied for the discovery of new antibodies13. However it has a major disadvantage, namely that the heavy and light chain&#160;pairs of the human immunoglobulin become dissociated in the process. Production of hybridomas with human B cells or EBV transformation overcomes this drawback.
We use infection of thymic B cells with EBV in combination with polyclonal B cell stimulation via Toll-like receptor 9 (TLR-9)6,12.
In this paper, we describe in detail the technology that we use for the development of IgG human antibodies, with a complete overview of all the steps from PBMC isolation to the in vitro antibody generation. This protocol can be used for the analysis of any type of human IgG profile. In our laboratory, B cells producing IgG antibodies have been successfully separated from the rest of PBMCs after sorting. Fifty sorted B cells8 can then be plated in multi-well plates and immortalized by EBV and TLR-9 activation, for the clonal expansion of single B cells. As feeder cells, fibroblasts from human embryonic lung tissue have been used, cell line wi38, which facilitates the visualization of the immortalized B cells. From these B cells, the sequences of the heavy and light chains of the immunoglobulin can be obtained by PCR, and the antibodies&#39; genes cloned in immunoglobulin G expression vectors and produced in vitro. Using this technique, single antibodies with exactly the same antibody sequence found in the donor can be studied.

<table><tbody><tr><td>Histopaque-1077&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10771</td><td>solution containing polysucrose and sodium diatrizoate</td></tr><tr><td>FACSAria II cell sorter&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>96 U-bottom micro well plates&amp;nbsp;</td><td>Costar</td><td>3799</td><td></td></tr><tr><td>Advanced Roswell Park Memorial Institute (RPMI) 1640 medium&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>12633-020</td><td></td></tr><tr><td>30% v/v EBV-containing supernatant of the B95-8 cell line&amp;nbsp;&amp;nbsp;</td><td>ATCC</td><td>CRL-1612</td><td>3.4 x 108 copies/ml</td></tr><tr><td>CpG2006&amp;nbsp;</td><td>Invivogen</td><td>ODN 7909</td><td></td></tr><tr><td>Wi38 cells&amp;nbsp;</td><td>Sigma-Aldrich</td><td>90020107</td><td></td></tr><tr><td>Interleukin-2</td><td>Roche&amp;nbsp;</td><td>10799068001</td><td></td></tr><tr><td>ELISA plates</td><td>Greiner Bio-One, Microlon</td><td>655092</td><td></td></tr><tr><td>AffiniPure F(ab&#039;)2 Fragment Goat Anti-Human IgG, Fc&amp;gamma; Fragment Specific (unconjugated)</td><td>Jackson ImmunoResearch</td><td>109-006-008</td><td></td></tr><tr><td>4% non-fat dry milk (Blotting Grade Blocker)&amp;nbsp;</td><td>Biorad</td><td>170-6404</td><td></td></tr><tr><td>Human IgG&amp;nbsp;</td><td>Sigma</td><td>I 2511</td><td>HUMAN IgG purified Immunoglobulin, 5.6 mg/ml</td></tr><tr><td>Goat F(ab)&lt;sub&gt;2&lt;/sub&gt; antihuman IgG Fc&amp;gamma; (conjugated with peroxidase (PO))</td><td>Jackson ImmunoResearch</td><td>109-036-008</td><td></td></tr><tr><td>ELISA reader (Perkin Elmer 2030)&amp;nbsp;</td><td>Perkin Elmer&amp;nbsp;</td><td>2030-0050</td><td></td></tr><tr><td>Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc&lt;sub&gt;5&amp;micro;&amp;nbsp;&lt;/sub&gt;</td><td>Jackson ImmunoResearch</td><td>309-035-095</td><td></td></tr><tr><td>SuperScript III Cells Direct cDNA Synthesis System&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>18080-200</td><td></td></tr><tr><td>Applied Biosystems (ABI) GeneAm PCR System 2700</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>High Pure RNA Isolation Kit&amp;nbsp;</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>Reverse transcription system kit&amp;nbsp;</td><td>Promega</td><td>A3500</td><td></td></tr><tr><td>Recombinant Taq DNA Polymerase</td><td>TAKARA</td><td>R001A</td><td></td></tr><tr><td>Primers (2 &amp;mu;l)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Ultrapure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>100 bp ladder</td><td>Invitrogen</td><td>15628-019</td><td></td></tr><tr><td>Quantity One 4.5.2 (Gel Doc 2000)</td><td>Biorad</td><td>170-8100</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>QIAGEN</td><td>28106</td><td></td></tr><tr><td>BigDye Terminator v3.1 cycle sequencing kit&amp;nbsp;</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr><tr><td>0.1 ml reaction plate (MicroAMP Optical 96-well)</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>Genetic analyser ABI300&amp;nbsp;</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>DH5&amp;alpha; competent cells (E. coli)</td><td>Invitrogen</td><td>18263-012</td><td></td></tr><tr><td>pFUSEss-CHIg-hG1 (4,493 bp)</td><td>Invivogen</td><td>pfusess-hchg1</td><td></td></tr><tr><td>pFUSEss-CHIg-hG4 (4,484 bp)&amp;nbsp;</td><td>Invivogen</td><td>pfusess-hchg4</td><td></td></tr><tr><td>pFUSE2ss-CLIg-hk (3,875 bp)</td><td>Invivogen</td><td>pfuse2ss-hclk</td><td></td></tr><tr><td>pFUSE2ss-CLIg-hl2 (3,883 bp)&amp;nbsp;</td><td>Invivogen</td><td>pfuse2ss-hcll2</td><td></td></tr><tr><td>SOC medium</td><td>Invitrogen</td><td>15544-034</td><td></td></tr><tr><td>LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar)</td><td>Invivogen</td><td>fas-zn-s</td><td></td></tr><tr><td>Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB)</td><td>Invivogen</td><td>fas-zn-l</td><td></td></tr><tr><td>DNA Miniprep kit&amp;nbsp;</td><td>Omega Bio Technology</td><td>D6942-02</td><td></td></tr><tr><td>Nanodrop (ND1000 Spectrophotometer)</td><td>Nanodrop</td><td></td><td></td></tr><tr><td>LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar)</td><td>Invivogen</td><td>fas-bl-s</td><td></td></tr><tr><td>Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB)</td><td>Invivogen</td><td>fas-bl-l</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td>20,000 U/ml, in 10x NEBuffer EcoRI</td></tr><tr><td>NheI</td><td>New England Biolabs</td><td>R0131S</td><td>10,000 U/ml, in 10x NEBuffer 2.1</td></tr><tr><td>2-Log DNA ladder</td><td>New England Biolabs</td><td>N3200S</td><td>0.1-10.0 kb, 1,000 &amp;mu;g/ml</td></tr><tr><td>XmaI</td><td>New England Biolabs</td><td>R0180S</td><td>10,000 U/ml, in 10x CutSmart Buffer&amp;nbsp;</td></tr><tr><td>BsiWI</td><td>New England Biolabs</td><td>R0553S</td><td>10,000 U/ml, in 10x NEBuffer 3.1</td></tr><tr><td>AvrII</td><td>New England Biolabs</td><td>R0174S</td><td>5,000 U/ml, in 10x CutSmart Buffer&amp;nbsp;</td></tr><tr><td>FastAP Thermosensitive Alkaline Phosphatase</td><td>Thermo Scientific</td><td>EF0651</td><td>1 U/&amp;micro;l, in 10x FastAP Buffer</td></tr><tr><td>DH5&amp;alpha; competent cells&amp;nbsp;</td><td>Invitrogen</td><td>18263-012</td><td></td></tr><tr><td>PE Mouse Anti-Human IgG</td><td>BD Pharmingen</td><td>555787</td><td></td></tr><tr><td>anti-CD22, PerCP-Cy5.5, Clone: HIB22</td><td>Fisher scientific</td><td>BDB563942</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit&amp;nbsp;</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>BigDye Terminator v3.1</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr></tbody></table>

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.

The sorting gating after staining CD22 and IgG positive cells is shown in Figure 1. In this image the area of the double positive cells &#8211; B cells producing IgG antibodies &#8211; is selected to sort all these cells in a separate tube. In the analysis, approximately 1% of the total PBMCs correspond to this double positive population. The number of sorted cells obtained will depend on the number of cells obtained in section 1.
The different outcomes after 5 weeks of EBV im...

Watch this Scientific Journal Video about Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells at JoVE.com

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

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, ...

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Immunology and Infection

18.3K Views.  Maastricht University. 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.

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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.

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 ...

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 ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

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.

Wytwarzanie rekombinowanych przeciwciał monoklonalnych z kultur komórkowych ssaków

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

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

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.

Technika wytwarzania przeciwciał monoklonalnych z limfocytów B specyficznych dla antygenu

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.

Technologia Hybridoma

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