Name: chemical conjugation of a purified dec-205-directed antibody with full-length protein for targeting mouse dendritic cells in vitro and in vivo
Uploaded: 2021-02-05T14:00:00
Description: Watch this Scientific Journal Video about Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo at JoVE.com

作者感谢S. Prettin的专家技术援助。这项工作得到了亥姆霍兹德国研究中心协会（HGF）的资助，该资助是亥姆霍兹联盟"癌症免疫疗法"（HCC_WP2b）的一部分。

所有描述的动物实验都得到了当地政府机构的批准（Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit;文件编号33.12-42502-04-10/0108），并根据国家和机构指南进行。
1. 从杂交瘤细胞系NLDC-145生产αDEC-205
<ol><li>对于抗体生产，在37°C的水浴中解冻冷冻保存的NLDC-145细胞产生αDEC-205。在37°C和5%CO下扩增细胞2.两个 75 厘米2 需要瓶子才能进行抗体生产。细胞?...

<ol>
	<li>Amon, L., Hatscher, L., Heger, L., Dudziak, D., Lehmann, C. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+complete+repertoire+of+conventional+dendritic+cell+functions+for+cancer+immunotherapy.">Harnessing the complete repertoire of conventional dendritic cell functions for cancer immunotherapy.</a> Pharmaceutics. 12 (7), 12070663 (2020).</li><li>Banchereau, 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=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Wculek, S. 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=Dendritic+cells+in+cancer+immunology+and+immunotherapy.">Dendritic cells in cancer immunology and immunotherapy.</a> Nature Reviews: Immunology. 20 (1), 7-24 (2020).</li><li>Kastenmuller, W., Kastenmuller, K., Kurts, C., Seder, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell-targeted+vaccines--hope+or+hype.">Dendritic cell-targeted vaccines--hope or hype.</a> Nature Reviews: Immunology. 14 (10), 705-711 (2014).</li><li>Bonifaz, L. C., 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=In+vivo+targeting+of+antigens+to+maturing+dendritic+cells+via+the+DEC-205+receptor+improves+T+cell+vaccination.">In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.</a> Journal of Experimental Medicine. 199 (6), 815-824 (2004).</li><li>Boscardin, S. B., 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=Antigen+targeting+to+dendritic+cells+elicits+long-lived+T+cell+help+for+antibody+responses.">Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses.</a> Journal of Experimental Medicine. 203 (3), 599-606 (2006).</li><li>Hossain, M. K., Wall, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Dendritic+Cell+Receptors+as+Targets+for+Enhancing+Anti-Cancer+Immune+Responses.">Use of Dendritic Cell Receptors as Targets for Enhancing Anti-Cancer Immune Responses.</a> Cancers. 11 (3), 11030418 (2019).</li><li>Johnson, T. S., 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=Inhibition+of+melanoma+growth+by+targeting+of+antigen+to+dendritic+cells+via+an+anti-DEC-205+single-chain+fragment+variable+molecule.">Inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-DEC-205 single-chain fragment variable molecule.</a> Clinical Cancer Research. 14 (24), 8169-8177 (2008).</li><li>Trumpfheller, C., 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=The+microbial+mimic+poly+IC+induces+durable+and+protective+CD4++T+cell+immunity+together+with+a+dendritic+cell+targeted+vaccine.">The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2574-2579 (2008).</li><li>Idoyaga, 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=Comparable+T+helper+1+(Th1)+and+CD8+T-cell+immunity+by+targeting+HIV+gag+p24+to+CD8+dendritic+cells+within+antibodies+to+Langerin,+DEC205,+and+Clec9A.">Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (6), 2384-2389 (2011).</li><li>Sancho, D., 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=Tumor+therapy+in+mice+via+antigen+targeting+to+a+novel,+DC-restricted+C-type+lectin.">Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin.</a> Journal of Clinical Investigation. 118 (6), 2098-2110 (2008).</li><li>Volckmar, 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=Targeted+antigen+delivery+to+dendritic+cells+elicits+robust+antiviral+T+cell-mediated+immunity+in+the+liver.">Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.</a> Scientific Reports. 7, 43985 (2017).</li><li>Kraal, G., Breel, M., Janse, M., Bruin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans'+cells,+veiled+cells,+and+interdigitating+cells+in+the+mouse+recognized+by+a+monoclonal+antibody.">Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.</a> Journal of Experimental Medicine. 163 (4), 981-997 (1986).</li><li>Volckmar, 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=The+STING+activator+c-di-AMP+exerts+superior+adjuvant+properties+than+the+formulation+poly(I:C)/CpG+after+subcutaneous+vaccination+with+soluble+protein+antigen+or+DEC-205-mediated+antigen+targeting+to+dendritic+cells.">The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.</a> Vaccine. 37 (35), 4963-4974 (2019).</li></ol>

内吞作用受体特异性抗体和蛋白质抗原的化学偶联为临床前小鼠模型 中的体内 DC靶向提供了一种有效且重要的灵活方法。通过我们的方案，我们为模型抗原OVA与DEC-205特异性IgG抗体的成功偶联提供了一种有效的方法。
在我们的方案中，αDEC-205是从杂交瘤细胞系中纯化的，过去，我们已经使用如上所述的蛋白G琼脂糖纯化了抗体。值得注意的是，在后来的研究中，我们还?...

树突状细胞（DC）是免疫系统的核心参与者。它们是一组专门用于抗原呈递的多样化细胞，其主要功能是桥接先天性和适应性免疫1，2。重要的是，DC不仅在有效和特异性的病原体导向反应中起重要作用，而且还参与抗肿瘤免疫1，3的许多方面。
由于它们在宿主免疫中的排他性作用，DC成为疫苗接种靶细胞4的焦点。一种方法是在体内将抗原靶向DC以诱导抗原特异性免疫反应，并且在过去几年中，大量研究致力于确定合适的受体和靶向策略1，4。一个例子是C型凝集素受体DEC-205，其可以通过DEC-205特异性抗体靶向诱导内吞作用。重要的是，DEC-205靶向与合适的佐剂组合已被证明可以有效地诱导长寿命和保护性的CD4+和CD8+ T细胞，以及抗体反应，也针对肿瘤抗原3，5，6，7，8，9。
有许多研究表明，靶向DC的偶联抗原优于游离的未偶联抗原3，5，10，11，12。这使得抗原与相应的DC靶向部分的共轭成为DC靶向方法的核心步骤。在通过抗体或抗体片段靶向DC的情况下，抗原可以是化学或遗传连接的，并且任何一种策略都提供了自己的（dis）优势1。一方面，在基因工程抗体 - 抗原构建体中，抗原剂量以及位置的控制在批次1之间提供卓越的可比性。然而，与此同时，化学偶联需要较少的准备工作，并提供更大的灵活性，特别是在尝试在实验和临床前模型中测试和比较不同的抗原和/或疫苗接种策略时。
在这里，我们提出了一种方案，用于将卵清蛋白（OVA）作为模型蛋白抗原与适合小鼠体内DC靶向的DEC-205特异性IgG2a抗体（αDEC-205）进行有效和可靠的化学偶联。首先，从NLDC-145杂交瘤细胞13中纯化αDEC-205。对于化学偶联，使用含有NHS（N-羟基琥珀酰亚胺基）和马来酰亚胺基的异双功能交联剂4-[N-马来酰亚胺甲基]环己烷-1-羧酸酯（磺基-SMCC），允许含胺和巯基分子的共价共轭。具体而言，抗体的伯胺最初与磺基-SMCC反应，所得的马来酰亚胺活化的αDEC-205随后与通过TCEP-HCl（三（2-羧乙基）膦盐酸盐）还原的含巯基OVA蛋白反应。最终产物是化学偶联的αDEC-205 / OVA（图1）。除了化学偶联本身之外，我们的方案还描述了从偶联物中去除过量的OVA，以及通过蛋白质印迹分析和特定的酶联免疫吸附测定来验证成功偶联。我们过去曾成功采用这种方法将OVA和其他蛋白质或免疫原性肽化学偶联至αDEC-205。我们证明了在体外与CD11c+细胞的有效结合以及体内细胞和体液免疫的有效诱导 。
当然，这种方法存在缺点，例如批次间可比性以及最终偶联物中抗原的精确剂量。然而，与基因工程构建体相比，化学偶联在选择抗体和蛋白质抗原方面提供了实验灵活性。因此，我们认为这种方法在评估临床前小鼠模型中不同抗原在DC靶向中的效率方面特别有价值，重要的是在特定的抗肿瘤免疫反应的背景下也是如此。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>antibody buffer 2 %</td>
			<td></td>
			<td></td>
			<td>2 % (w/v) Slim-Fast Chocolate powder in TBS-T</td>
		</tr>
		<tr>
			<td>antibody buffer 5 %</td>
			<td></td>
			<td></td>
			<td>5 % milk powder (w/v) in TBS-T</td>
		</tr>
		<tr>
			<td>blocking buffer (ELISA)</td>
			<td></td>
			<td></td>
			<td>10 % FBS in PBS</td>
		</tr>
		<tr>
			<td>blocking buffer 4 %</td>
			<td></td>
			<td></td>
			<td>4 % (w/v) Slim-Fast Chocolate powder in TBS-T</td>
		</tr>
		<tr>
			<td>blocking buffer 10 %</td>
			<td></td>
			<td></td>
			<td>10 % milk powder (w/v) in TBS-T</td>
		</tr>
		<tr>
			<td>cell culture flask T25</td>
			<td>Greiner Bio-One</td>
			<td>690175</td>
			<td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 )</td>
		</tr>
		<tr>
			<td>cell culture flask T75</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>we use standard CELLSTAR&#160; filter cap cell culture flasks; alternatively use suspension culture flask (658195)&#160;</td>
		</tr>
		<tr>
			<td>cell culture flask T175</td>
			<td>Greiner Bio-One</td>
			<td>661175</td>
			<td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195)</td>
		</tr>
		<tr>
			<td>centrifugal concentrator MWCO 10 kDa</td>
			<td>Sartorius</td>
			<td>VS2001</td>
			<td>Vivaspin 20 centrifugal concentrator</td>
		</tr>
		<tr>
			<td>centrifugal protein concentrator MWCO 100 kDa, 5 - 20 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>88532</td>
			<td>Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available&#160;</td>
		</tr>
		<tr>
			<td>centrifuge bottles</td>
			<td>Nalgene</td>
			<td>525-2314</td>
			<td>PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany</td>
		</tr>
		<tr>
			<td>coating buffer (ELISA)</td>
			<td></td>
			<td></td>
			<td>0.1 M sodium bicarbonate (NaHCO3) in H2O (pH 9.6)</td>
		</tr>
		<tr>
			<td>desalting columns MWCO 7 kDa</td>
			<td>Thermo Fisher Scientific</td>
			<td>89891</td>
			<td>Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL</td>
		</tr>
		<tr>
			<td>detection reagent ELISA (HRPO substrate)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>T8665-100ML</td>
			<td>3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB) liquid substrate system</td>
		</tr>
		<tr>
			<td>detection reagent western blot (HRPO substrate)</td>
			<td>Roche/Merck</td>
			<td>12 015 200 01</td>
			<td>Lumi-Light Western Blotting Substrate (Roche)</td>
		</tr>
		<tr>
			<td>dialysis tubing MWCO 12 - 14 kDa</td>
			<td>SERVA Electrophoresis</td>
			<td>44110</td>
			<td>Visking dialysis tubing, 16 mm diameter</td>
		</tr>
		<tr>
			<td>ELISA 96-well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>442404</td>
			<td>MaxiSorp Nunc-Immuno Plate</td>
		</tr>
		<tr>
			<td>fetal calf serum</td>
			<td>PAN-BIOtech</td>
			<td>P40-47500</td>
			<td>FBS Good forte</td>
		</tr>
		<tr>
			<td>ISF-1 medium</td>
			<td>Biochrom/bioswisstec</td>
			<td>F 9061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td>Carl Roth</td>
			<td>T145.2</td>
			<td>powdered milk, blotting grade, low in fat; alternatively we have also used conventional skimmed milk powder from the supermarket</td>
		</tr>
		<tr>
			<td>NLDC-145 hybridoma</td>
			<td>ATCC</td>
			<td>HB-290</td>
			<td>if not already at hand, the hybridoma cells can be acquired from ATCC</td>
		</tr>
		<tr>
			<td>non-reducing SDS sample buffer 4 x&#160;</td>
			<td></td>
			<td></td>
			<td>for 12 ml: 4 ml of 10 % SDS, 600 &#181;l 0.5 M Tris-HCl (ph 6.8), 3.3 ml sterile H2O, 4 ml glycerine, 100 &#181;l of 5 % Bromphenol Blue</td>
		</tr>
		<tr>
			<td>ovalbumin</td>
			<td>Hyglos (via BioVendor)</td>
			<td>321000</td>
			<td>EndoGrade OVA ultrapure with &#60;0.1 EU/mg</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used</td>
		</tr>
		<tr>
			<td>PETG polyethylene terephthalate glycol cell culture roller bottles</td>
			<td>Nunc In Vitro</td>
			<td>734-2394</td>
			<td>standard PDL-coated, vented (1.2X), 1050 cm&#178;, 100 - 500 ml volume; obtained from VWR, Germany&#160;&#160;</td>
		</tr>
		<tr>
			<td>pH indicator strips</td>
			<td>Merck</td>
			<td>109535</td>
			<td>pH indicator strips 0-14</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rat-IgG(H+L)-HRPO (western blot)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>112-035-062</td>
			<td>obtained from Dianova, Germany; used at 1:5000 for western blot</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rat-IgG+IgM-HRPO antibody&#160; (ELISA)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>112-035-068</td>
			<td>obtained from Dianova, Germany; used at 1:2000 for ELISA</td>
		</tr>
		<tr>
			<td>polyclonal goat &#945;rabbit-IgG-HRPO (western blot)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-035-045&#160;</td>
			<td>obtained from Dianova, Germany; used at 1:2000 for western blot</td>
		</tr>
		<tr>
			<td>polyclonal rabbit &#945;OVA (ELISA)</td>
			<td>Abcam</td>
			<td>ab181688</td>
			<td>used at 3 ng/&#181;l</td>
		</tr>
		<tr>
			<td>polyclonal rabbit &#945;OVA antibody (western blot)</td>
			<td>OriGene</td>
			<td>R1101</td>
			<td>used at 1:3,000 for western blot</td>
		</tr>
		<tr>
			<td>Protein G Sepharose column</td>
			<td>Merck/Millipore</td>
			<td>P3296</td>
			<td>5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01)</td>
		</tr>
		<tr>
			<td>protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>26616</td>
			<td>PageRuler Prestained Protein ladder 10 - 180 kDa</td>
		</tr>
		<tr>
			<td>PVDF (polyvinylidene difluoride) membrane</td>
			<td>Merck/Millipore</td>
			<td>IPVH00010</td>
			<td>immobilon-P PVDF (polyvinylidene difluoride) membrane</td>
		</tr>
		<tr>
			<td>rubber plug</td>
			<td>Omnilab</td>
			<td>5230217</td>
			<td>DEUTSCH &#38; NEUMANN rubber stoppers (lower &#934; 17 mm; upper &#934; 22 mm)</td>
		</tr>
		<tr>
			<td>silicone tube</td>
			<td>Omnilab</td>
			<td>5430925</td>
			<td>DEUTSCH &#38; NEUMANN (inside &#934; 1 mm; outer &#934; 3 mm)</td>
		</tr>
		<tr>
			<td>Slim-Fast</td>
			<td></td>
			<td></td>
			<td>we have used regular Slim-Fast Chocolate freely available at the pharmacy as in this western blot approach it yielded better results than milk powder</td>
		</tr>
		<tr>
			<td>stopping solution (ELISA)</td>
			<td></td>
			<td></td>
			<td>1M H2SO4</td>
		</tr>
		<tr>
			<td>sulfo-SMCC</td>
			<td>Thermo Fisher Scientific</td>
			<td>22322</td>
			<td>Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg)</td>
		</tr>
		<tr>
			<td>syringe filter unit 0.22 &#181;m&#160;</td>
			<td>Merck/Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV Syringe Filter Unit, 0.22 &#181;m, PVDF, 33 mm, gamma sterilized&#160;</td>
		</tr>
		<tr>
			<td>syringe 10 ml</td>
			<td>Omnilab</td>
			<td></td>
			<td>Disposable syringes Injekt&#174; Solo B.Braun</td>
		</tr>
		<tr>
			<td>Sterican&#174; cannulas</td>
			<td>B. Braun</td>
			<td></td>
			<td>Sterican&#174; G 20 x 1 1/2&#34;&#34;; 0.90 x 40 mm; yellow</td>
		</tr>
		<tr>
			<td>TBS-T</td>
			<td></td>
			<td></td>
			<td>Tris-buffered saline containing 0.1 % (v/v) Tween 20</td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>A35349</td>
			<td></td>
		</tr>
		<tr>
			<td>tubing connector</td>
			<td>Omnilab</td>
			<td></td>
			<td>Kleinfeld miniature tubing connectors for silicone tube</td>
		</tr>
	</tbody>
</table>

chemical conjugation of a purified dec-205-directed antibody with full-length protein for targeting mouse dendritic cells in vitro and in vivo

靶向抗原递送到 体内 交叉呈递树突状细胞（DC）可有效诱导T效应细胞反应，并在疫苗设计中显示出一种有价值的方法。抗原通过内吞作用受体（如 DEC-205）特异性抗体递送到 DC，这些受体可诱导摄取、加工和 MHC I 类和 II 类表现。
将所需抗原高效可靠地偶联到合适的抗体是DC靶向的关键步骤，除其他因素外，还取决于抗原的格式。全长蛋白质与纯化抗体的化学偶联是一种可能的策略。过去，我们已经成功地建立了模型抗原卵清蛋白（OVA）和DEC-205特异性IgG2a抗体（αDEC-205）的交联，用于小鼠 体内 DC靶向研究。该协议的第一步是通过亲和色谱法从NLDC（非淋巴树突状细胞）-145杂交瘤的上清液中纯化抗体。纯化的抗体通过磺基-SMCC（磺基琥珀酰亚胺基4-[N-马来亚胺基]环己烷-1-羧酸盐）激活以进行化学偶联，同时通过与TCEP-HCl（三（2-羧乙基）盐酸膦）孵育暴露于OVA蛋白的巩基。除去过量的TCEP-HCl和磺基-SMCC，并将抗原与活化的抗体混合过夜偶联。所得的αDEC-205/OVA偶联物被浓缩并从未结合的OVA中释放出来。通过蛋白质印迹分析和酶联免疫吸附测定（ELISA）验证OVA与αDEC-205的成功偶联。
我们已经成功地使用化学交联的αDEC-205 / OVA在肝脏中诱导细胞毒性T细胞反应，并比较了不同的佐剂在 体内 靶向DEC-205+ DC后诱导体液和细胞免疫的潜力。除此之外，这种化学偶联的抗体/抗原偶联物为有效诱导疫苗对肿瘤抗原的反应提供了有价值的工具，并且已被证明在预防和治疗各种类型肿瘤方面优于经典免疫方法。

使用该协议将αDEC-205与OVA蛋白的化学偶联通常允许有效地生成αDEC-205 / OVA用于体内DC靶向方法。有不同的策略来验证技术本身并测试所产生共轭物的功能。蛋白质印迹分析和ELISA用于验证成功的偶联，同时检测可能留下的游离OVA（图2）。体外结合研究（图3）和体内免疫（图4）证实了偶...

Watch this Scientific Journal Video about Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo at JoVE.com

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

我们描述了一种将模型抗原卵清蛋白化学偶联到内吞作用受体特异性抗体的方案，用于体内树突状细胞靶向。该协议包括抗体的纯化，抗原的化学偶联，以及偶联物的纯化和有效偶联的验证。

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

该方案的所有实验条件都经过仔细优化，可用于促进DEC-205抗体与OVA抗原的成功和有效的化学偶联。该技术的主要优点是它允许灵活选择蛋白质抗原和抗体，并且不依赖于这些组分的遗传融合。我们还使用该协议来生成丙型肝炎病毒蛋白的偶联物和用于体内DC靶向的抗DEC-205。这对于抗肿瘤疫苗接种方法同样有价值。为了开始抗DEC-205生产，将一毫升体积的1至500万个解冻的冷冻保存的NLDC 145细胞重悬于9毫升37摄氏度的ISF-1培养基中，并补充1%青霉素和链霉素，并将细胞接种到25平方厘米的细胞培养瓶中，在37摄氏度和5%二氧化碳下孵育24至48小时。当细胞达到70%汇合度时，将整个细胞悬浮液转移到15毫升的锥形管中并通过离心收集细胞。将沉淀重悬于12毫升新鲜完整的37度ISF-1培养基中，并将细胞重新接种在75平方厘米的烧瓶中。培养48至72小时后，将70%汇合培养物分成两个新的75平方厘米烧瓶，并在每个烧瓶中加入6毫升新鲜完整的ISF-1培养基。当培养物达到70%汇合度时，使用10毫升移液管用细胞悬浮液冲洗细胞培养瓶底部和培养表面，以收集所有细胞并将每个膨胀的NLDC 145细胞悬浮液的10毫升转移到单独的PETG辊瓶中。然后将140毫升完整的ISF-1培养基加入每个辊瓶培养物中，并以37摄氏度，5%二氧化碳和每分钟25轮的速度培养辊瓶，持续三天。对于来自NLDC 145上清液的抗体纯化，将硅管的末端放入充满上清液的烧杯中，并允许800毫升上清液滴通过蛋白质G琼脂糖柱。对于洗脱，将100微升1.5摩尔Tris-HCL加入20个1.5毫升管中，并从色谱柱上取下橡胶塞。将一毫升0.1摩尔甘氨酸转移到柱的上腔室，并将含有淋洗液的抗体收集到制备的1.5毫升管之一中，并为每个管重复。当收集完所有抗体并汇集含抗体洗脱物后，用适当的透析管闭合20厘米长透析管的底部，并小心地将抗体洗脱液入管中。用第二个夹子关闭透析管的顶部，并将透析管的上夹固定在浮动支架上。将磁性搅拌棒加入PBS烧杯中，然后将烧杯放在磁力搅拌器上。在四摄氏度下透析过夜后，打开一个夹具，将整个透析液装入分子量截止为10千达尔顿的离心浓缩器，然后在693倍G和4摄氏度下离心浓缩器30分钟。在离心结束时，将10毫升PBS加载离心浓缩器进行第二次离心，直到获得最终的1至1.5毫升体积的抗体溶液。要将OVA与抗DEC-205抗体偶联，将200微升PBS加入补充500微克OVA蛋白，240微升新鲜制备的TCEP-HCL溶液和560微升无菌超纯水中至1.5毫升管中，在室温下孵育1.5小时。同时，将2.5毫克抗DEC-205在900微升PBS和100微升新鲜制备的磺胺-SMCC中加入第二个1.5毫升管中，在37摄氏度下以每分钟550转的速度在加热块中孵育30分钟。在孵育结束时，将脱盐柱与松开的盖子和扭曲的底部放入单独的15毫升锥形管中，并离心柱以除去液体。离心后，将色谱柱置于新管中，取下盖子，并将抗体磺基-SMCC和OVA TCEP-HCL溶液缓慢上样到每个溶液一个色谱柱的紧凑树脂床的中心。离心后，丢弃色谱柱，并立即用温和移液混合抗体和OVA溶液。将抗 DEC-205/OVA 溶液加载到离心蛋白质浓缩器中，并在浓缩器中加入 PBS 至 15 毫升体积。在2， 000倍G和室温下离心浓缩器五分钟，并在浓缩器中加入额外的10毫升PBS。在相同的离心机条件下离心浓度至少八分钟后，从上腔室轻轻收集浓缩样品。为了通过蛋白质印迹分析验证偶联的成功，将蛋白质标准品上每个样品的等分试样上样上样到SDS凝胶上，并根据标准SDS-PAGE分析方案运行凝胶。凝胶印迹后，根据标准方案用辣根过氧化物酶偶联抗体染色膜，以检测抗DEC-205或OVA。然后可以分析膜中每个样品中相应蛋白质的存在。为了验证ELISA偶联的成功，在每孔中涂覆100微升抗OVA抗体的适当96孔ELISA板，并以1至2的比例和封闭缓冲液连续稀释抗DEC-205 / OVA，以获得从每毫升6微克到每毫升93.8纳克的稀释液。将每个稀释液中的100微升加入抗体包被板的适当孔中，在室温下孵育一小时。在孵育结束时，向板中加入100微升针对抗DEC-205 / OVA偶联物的辣根过氧化物酶偶联抗体，在室温下孵育一小时，然后向每个孔中加入50微升辣根过氧化物酶底物，以便通过分光光度法分析颜色反应。平行蛋白质印迹分析可用于检测共轭OVA和共轭抗DEC-205。OVA染色将允许检测过量的游离OVA（如果存在），并且抗DEC-205染色通过增加裸抗DEC-205和偶联物之间的分子量来验证偶联的成功。ELISA还可用于评估偶联的成功，检测信号与分析的蛋白质量之间存在正相关，表明抗DEC-205 / OVA偶联剂的成功生成。流式细胞术和免疫荧光分析清楚地表明，抗 DEC-205 核心可有效结合骨髓来源的 CD11c 阳性细胞。与单独接种OVA和佐剂相比，抗 DEC-205/OVA 联合佐剂接种疫苗可诱导小鼠受试者 OVA 特异性 IgG 滴度增加。此外，抗 DEC-205/OVA 可有效诱导超特异性 CD4 和 CD8 阳性 T 细胞反应，抗 DEC-205/OVA 诱导的 CD8 阳性 T 细胞反应显著超过单独 OVA 诱导的反应。为了使OVA抗原成功与抗DEC-205抗体偶联，重要的是要按照证明制备材料并在一致的条件下执行偶联步骤。在抗原和抗体成功偶联后，许多后续方法是可能的，包括结合分析和在体内诱导病原体或肿瘤相关免疫。

Research

JoVE Journal

Cancer Research

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

体外成像和荧光标记的GnRH类似物的药物靶向性的定量分析

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

科研

癌症研究

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Counting Proteins in Single Cells with Addressable Droplet Microarrays

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

GPC3-targeting 双特异性抗体, GPC3-S-Fab, 具有强大的细胞毒性

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...