Contrato de investigação Miguel Servet (ISCIII CD14 / 00032) a (GN-G.). Fellowship da Organização Holandesa para Pesquisa &quot;Graduate School of Translational Programa de Neurociência&quot; Scientific (022005019) para (CH). Concessões do Prinses Beatrix Fonds (Project WAR08-12) ea Association Française contre les Myopathies para (PM-M.); bem como por um Veni Fellowship da Organização Holandesa para Pesquisa Científica (916.10.148) uma bolsa da Fundação Cérebro dos Países Baixos (FS2008 (1) -28) eo Prinses Beatrix Fonds (Project WAR08-12) (a ML ). Agradecemos Jozien Jaspers por sua ajuda na triagem de células B por citometria de fluxo.

<ol>
	<li>Vrolix, K., 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=Clonal+heterogeneity+of+thymic+B+cells+from+early-onset+myasthenia+gravis+patients+with+antibodies+against+the+acetylcholine+receptor.">Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.</a> J Autoimmun. 52, 101-112 (2014).</li><li>Yamashita, M., Katakura, Y., Shirahata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+generation+of+human+monoclonal+antibody.">Recent advances in the generation of human monoclonal antibody.</a> Cytotechnology. 55 (2-3), 55-60 (2007).</li><li>Pereira, K. M., Dellavance, A., Andrade, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+identification+of+autoantibodies+specific+to+systemic+autoimmune+rheumatic+diseases+in+high+throughput+operation:+Proposal+of+reliable+and+feasible+strategies.">The challenge of identification of autoantibodies specific to systemic autoimmune rheumatic diseases in high throughput operation: Proposal of reliable and feasible strategies.</a> Clin Chim Acta. 437, 403-410 (2014).</li><li>Losen, 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=Treatment+of+myasthenia+gravis+by+preventing+acetylcholine+receptor+modulation.">Treatment of myasthenia gravis by preventing acetylcholine receptor modulation.</a> Ann N Y Acad Sci. 1132, 174-179 (2008).</li><li>Smith, K., 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=Rapid+generation+of+fully+human+monoclonal+antibodies+specific+to+a+vaccinating+antigen.">Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.</a> Nat Protoc. 4 (3), 372-384 (2009).</li><li>Fraussen, J., 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=A+novel+method+for+making+human+monoclonal+antibodies.">A novel method for making human monoclonal antibodies.</a> J Autoimmun. 35 (2), 130-134 (2010).</li><li>Traggiai, E., 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=An+efficient+method+to+make+human+monoclonal+antibodies+from+memory+B+cells:+potent+neutralization+of+SARS+coronavirus.">An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.</a> Nat Med. 10 (8), 871-875 (2004).</li><li>Fraussen, J., 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=Autoantigen+induced+clonal+expansion+in+immortalized+B+cells+from+the+peripheral+blood+of+multiple+sclerosis+patients.">Autoantigen induced clonal expansion in immortalized B cells from the peripheral blood of multiple sclerosis patients.</a> J Neuroimmunol. 261 (1-2), 98-107 (2013).</li><li>Yamashita, 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=Different+individual+immune+responses+elicited+by+in+vitro+immunization.">Different individual immune responses elicited by in vitro immunization.</a> Cytotechnology. 40 (1-3), 161-165 (2002).</li><li>Hui-Yuen, J., Koganti, S., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cell+immortalization+for+monoclonal+antibody+production.">Human B cell immortalization for monoclonal antibody production.</a> Methods Mol Biol. 1131, 183-189 (2014).</li><li>Tiller, T., 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=Efficient+generation+of+monoclonal+antibodies+from+single+human+B+cells+by+single+cell+RT-PCR+and+expression+vector+cloning.">Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.</a> J Immunol Methods. 329 (1-2), 112-124 (2008).</li><li>Lanzavecchia, A., Corti, D., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monoclonal+antibodies+by+immortalization+of+memory+B+cells.">Human monoclonal antibodies by immortalization of memory B cells.</a> Curr Opin Biotechnol. 18 (6), 523-528 (2007).</li><li>Traggiai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+B+cells:+analysis+of+B+cell+repertoire+and+production+of+human+monoclonal+antibodies.">Immortalization of human B cells: analysis of B cell repertoire and production of human monoclonal antibodies.</a> Methods Mol Biol. 901, 161-170 (2012).</li><li>Strober, W., 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=Monitoring+cell+growth.+Curr+Protoc+Immunol+/+edited+by.">Monitoring cell growth. Curr Protoc Immunol / edited by.</a> John E. Coligan ... [et al.]. Appendix 3, Appendix 3A (2001).</li><li>Ibrahim, S. F., van den Engh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+cell+sorting.">Flow cytometry and cell sorting.</a> Adv Biochem Eng Biotechnol. 106, 19-39 (2007).</li><li>Leith, J. T., Padfield, G., Faulkner, L. E., Quinn, P., Michelson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+feeder+cells+on+the+X-ray+sensitivity+of+human+colon+cancer+cells.">Effects of feeder cells on the X-ray sensitivity of human colon cancer cells.</a> Radiother Oncol. 21 (1), 53-59 (1991).</li><li>Hui-Yuen, J., McAllister, S., Koganti, S., Hill, E., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Epstein-Barr+virus+growth-transformed+lymphoblastoid+cell+lines.">Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines.</a> J Vis Exp. (57), 3321 (2011).</li><li>Smith, L. 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>

Os autores declaram que não têm interesses financeiros concorrentes.

Neste texto, todos os passos para a geração de anticorpos IgG a partir de PBMC humanas são apresentados em detalhe. Este protocolo inclui algumas vantagens em relação às técnicas anteriormente publicados. Uma das vantagens é que o anticorpo produzido mantém as cadeias pesada e leve correspondentes ao par original no clone de células B. A identificação de anticorpos IgG pode ser feito em qualquer tipo de dador humano, e não há necessidade de exacerbação da resposta imunitária devido à vacinação 5...

O objetivo deste artigo é descrever em detalhe uma metodologia para gerar e caracterizar anticorpos monoclonais IgG humanas obtidas a partir de células mononucleares do sangue periférico humano (PBMC). O interesse em estudar anticorpos humanos tem crescido em muitos campos diferentes de pesquisa. Em particular, muitos grupos de pesquisa estão interessados ​​na patologia causada por auto-anticorpos 1-3. Clonámos e caracterizado auto-anticorpos patogénicos 1. O estudo de auto-anticorpos podem ajudar a identificar as suas metas e desenvolver estratégias terapêuticas, por exemplo, utilizando anticorpos concorrente 4. Além disso, o estudo de anticorpos humanos podem também ser de interesse em outras áreas de pesquisa, ou seja, para avaliar a resposta imune após a vacinação 5, para caracterizar o perfil de anticorpos de indivíduos que foram expostos e se tornaram resistentes a patógenos específicos 6 ou para estudar quais Os anticorpos são emo repertório singular 7,12. Diversas técnicas têm sido desenvolvidas para gerar anticorpos monoclonais humanos recombinantes 8-12; A maioria destes usar exibição em fagos e imortalização de células B. O uso de exibição em fagos tem sido amplamente aplicada para a descoberta de novos anticorpos 13. No entanto, tem um grande inconveniente, isto é, que os pares de cadeia pesada e leve da imunoglobulina humana estão dissociados no processo. A produção de hibridomas com células B humanas ou transformação EBV supera esta desvantagem. Usamos a infecção das células B timo com EBV em associação com a estimulação das células B policlonais através do receptor Toll-like? 9 (TLR-9) 6,12. Neste artigo, descreve detalhadamente a tecnologia que nós usamos para o desenvolvimento de anticorpos humanos IgG, com uma visão completa de todos os passos do PBMC isolamento para a geração de anticorpos em vitro. Esteprotocolo pode ser utilizado para a análise de qualquer tipo de perfil de IgG humana. No nosso laboratório, as células B produtoras de anticorpos de IgG têm sido separados com sucesso a partir do resto de PBMC após separação. Cinquenta células B ordenadas 8 podem então ser plaqueadas em placas multi-poços e imortalizadas por EBV e TLR-9 activação, para a expansão clonal de células B individuais. Como células de alimentação, os fibroblastos a partir de tecido de pulmão embrionário humano têm sido utilizados, linha de células WI38, o que facilita a visualização das células B imortalizadas. A partir destas células B, as sequências das cadeias pesada e leve da imunoglobulina pode ser obtido por PCR, e os genes dos anticorpos de imunoglobulina G clonado em vectores de expressão e produzido in vitro. Usando esta técnica, os anticorpos individuais com exactamente a mesma sequência de anticorpo encontradas no dador pode ser estudada.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>solution containing polysucrose and sodium diatrizoate</td>
		</tr>
		<tr>
			<td>FACSAria II cell sorter&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 U-bottom micro well plates&#160;</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Roswell Park Memorial Institute (RPMI) 1640 medium&#160;</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&#160;&#160;</td>
			<td>ATCC</td>
			<td>CRL-1612</td>
			<td>3.4 x 108 copies/ml</td>
		</tr>
		<tr>
			<td>CpG2006&#160;</td>
			<td>Invivogen</td>
			<td>ODN 7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Wi38 cells&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>90020107</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin-2</td>
			<td>Roche&#160;</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&#39;)2 Fragment Goat Anti-Human IgG, Fc&#947; 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)&#160;</td>
			<td>Biorad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG&#160;</td>
			<td>Sigma</td>
			<td>I 2511</td>
			<td>HUMAN IgG purified Immunoglobulin, 5.6 mg/ml</td>
		</tr>
		<tr>
			<td>Goat F(ab)2 antihuman IgG Fc&#947; (conjugated with peroxidase (PO))</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-036-008</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA reader (Perkin Elmer 2030)&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td>2030-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5&#181;&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>309-035-095</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Cells Direct cDNA Synthesis System&#160;</td>
			<td>Invitrogen&#160;</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&#160;</td>
			<td>Roche</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcription system kit&#160;</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&#956;l)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Agarose&#160;</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&#160;</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&#160;</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells (E. coli)</td>
			<td>Invitrogen</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG1 (4493 bp)</td>
			<td>Invivogen</td>
			<td>pfusess-hchg1</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG4 (4484 bp)&#160;</td>
			<td>Invivogen</td>
			<td>pfusess-hchg4</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hk (3875 bp)</td>
			<td>Invivogen</td>
			<td>pfuse2ss-hclk</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hl2 (3883 bp)&#160;</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&#160;</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 &#956;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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>FastAP Thermosensitive Alkaline Phosphatase</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>1 U/&#181;L, in 10x FastAP Buffer</td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells&#160;</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&#160;</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.

O gate de triagem após coloração das células positivas de CD22 e IgG é mostrado na Figura 1 Nesta imagem da área das células duplamente positivas -. Células B produtoras de anticorpos IgG - é seleccionado para classificar todas essas células em um tubo separado. Na análise, cerca de 1% do total de PBMC correspondem a esta população positiva dupla. O número de células classificadas obtidos dependerá do número de células obtidas na secção 1. Os diferentes r...

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.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

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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JoVE Journal

Immunology and Infection

18.2K visualizações.  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.