Name: determining the phagocytic activity of clinical antibody samples
Uploaded: 2011-11-30T13:00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

लेखकों 3R01AI080289 02S1 एनआईएच, और NIH / NIAID 2P30AI060354 07 के तहत एड्स रिसर्च स्कॉलर फैलोशिप के लिए एक हार्वर्ड विश्वविद्यालय केंद्र से धन स्वीकार करते करने की कामना करते हैं.

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Immunopathol. 28, 303-304 (2006).</li><li>Yamaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoform-dependent+conformational+alteration+of+the+Fc+region+of+human+immunoglobulin+G1+as+revealed+by+NMR+spectroscopy.">Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.</a> Biochim. Biophys. Acta. 1760, 693-700 (2006).</li><li>Anthony, R. M., Ravetch, J. V. <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+role+for+the+IgG+Fc+glycan:+the+anti-inflammatory+activity+of+sialylated+IgG.">A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG.</a> Fcs. J. Clin. Immunol. 30, S9-S14 (2010).</li><li>Nimmerjahn, F., Anthony, R. M., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agalactosylated+IgG+antibodies+depend+on+cellular+Fc+receptors+for+in+vivo+activity.">Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8433-8437 (2007).</li><li>Marchette, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+immune+status+on+dengue+2+virus+replication+in+cultured+leukocytes+from+infants+and+children.">Effect of immune status on dengue 2 virus replication in cultured leukocytes from infants and children.</a> Infect. Immun. 24, 47-50 (1979).</li><li>Fust, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+and+enhancing+antibodies+measured+in+complement-restored+serum+samples+from+HIV-1-infected+individuals+correlate+with+immunosuppression+and+disease.">Neutralizing and enhancing antibodies measured in complement-restored serum samples from HIV-1-infected individuals correlate with immunosuppression and disease.</a> AIDS. 8, 603-609 (1994).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

परख यहाँ वर्णित एक monocytic सेल लाइन लंबे समय के लिए phagocytic 10 प्रक्रियाओं और प्लेट आधारित स्वचालित प्रवाह cytometry अध्ययन के लिए उपयोग का उपयोग करके चिकित्सीय एंटीबॉडी नमूनों की phagocytic गतिविधि का उच्च throughput विश्लेषण की अनुमति देता है. इस परख ठीक विशिष्ट प्रतिजन प्रतिरक्षी सबसेट विशेषताएँ करने की क्षमता में दूसरों से अधिक फायदेमंद है, phagocytosis में मतभेद रोग विशिष्ट एंटीबॉडी द्वारा संचालित विभिन्न विषयों से अध्ययन के लिए न केवल अनुमति देता है, लेकिन यह भी एक ही विषय के भीतर एकाधिक प्रतिजन specificities के अध्ययन है. क्योंकि जन्मजात effector कोशिकाओं, monocytes और अन्य प्रतिजन कोशिकाओं पेश सहित एंटीबॉडी भर्ती जोरदार रोग 11-13 परिणाम प्रभावों, और जैविक चर Asparagine297 एफसी डोमेन 14-16 के एंटीबॉडी ज्यामिति, आईजीजी उपवर्ग, और ग्लाइकोसिलेशन निर्भर करता है, इस पद्धति के लिए प्रदान करता है कार्रवाई का एक महत्वपूर्ण प्रतिरक्षी तंत्र का मूल्यांकन. इसके अलावा, एंटीबॉडी दवा क्योंकि7,17,18 संक्रमण के दौरान पैदा phagocytosis कुछ रोगजनकों द्वारा शोषण किया जाता है, इस परख एंटीबॉडी निर्भर वृद्धि की प्रक्रिया के मूल्यांकन में वादा रखती है, और एक संभावित सुरक्षा, के रूप में अच्छी तरह के रूप में संभावित हानिकारक के रूप में phagocytosis की दोहरी प्रकृति पर प्रकाश डाला गया एंटीबॉडी गतिविधि. वर्णित परख रोगी आबादी के नैदानिक ​​नमूनों से एंटीबॉडी का विश्लेषण का उपयोग किया जा सकता है है, लेकिन यह भी vaccinees की बजाय शुद्ध आईजीजी सीरम का उपयोग करने के लिए अनुकूलित किया जा सकता है. इसके अतिरिक्त, phagocytosis के जवाब में cytokine स्राव संस्कृति सतह पर तैरनेवाला से विश्लेषण किया जा सकता है, और विशिष्ट FCR के प्रभाव FCR अवरुद्ध एंटीबॉडी का उपयोग, phagocytic शक्ति में न केवल मतभेदों को निर्धारित किया जा अनुमति के द्वारा निर्धारित किया जा सकता है है, लेकिन बहाव घटनाओं अंतर्दृष्टि के साथ, संकेत तंत्र में, और हल करने और इस प्रतिरक्षा तंत्र की समझ को परिष्कृत करने में मदद कर सकते हैं.

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> ईज़ी - लिंक माइक्रो Sulfo - एन एच एस-LC-Biotinylation किट </td><td> थर्मो Scientfic </td><td> 21935 </td><td></td></tr><tr><td> Amicon अल्ट्रा 15 Ultracel-50 झिल्ली के साथ केन्द्रापसारक फ़िल्टर यूनिट </td><td> Millipore </td><td> UFC905024 </td><td> फ़िल्टर इकाई आकार प्रतिजन आकार के अनुसार भिन्न हो सकते हैं </td></tr><tr><td> FluoSpheres NeutrAvidin microspheres, 1.0 सुक्ष्ममापी, पीले हरे रंग का फ्लोरोसेंट (505/515) लेबल </td><td> Invitrogen </td><td> F-8776 </td><td> निर्माता विभिन्न रंगों में इस उत्पाद का उत्पादन </td></tr><tr><td> तरबूज जेल आईजीजी शोधन किट </td><td> थर्मो </td><td> 45212 </td><td> केयर एसडीएस - पेज द्वारा अब नमूने की शुद्धता की पुष्टि करने के लिए लिया जाना चाहिए. </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

Determining the Phagocytic Activity of Clinical Antibody Samples

हम उच्च throughput प्रवाह cytometric परख वर्तमान नैदानिक ​​नमूनों से प्रतिजन विशिष्ट एंटीबॉडी की phagocytic गतिविधि का निर्धारण, फ्लोरोसेंट प्रतिजन में लिपटे माला और एक monocytic सेल लाइन में एकाधिक एफसी व्यक्त उपयोग रिसेप्टर्स - प्रदान रिसेप्टर उपयोग और phagocytic गतिविधि determinations में एक मानकीकृत और ब्याज की किसी भी प्रतिजन के लिए प्रतिलिपि प्रस्तुत करने योग्य फैशन.

क्लीनिकल एंटीबॉडी नमूने के Phagocytic गतिविधि निर्धारण

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Research

JoVE Journal

Immunology and Infection

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लार लार ग्रंथि, Ixodes Scapularis Ticks से और Hemolymph संग्रह

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

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फेज-प्रदर्शित सिंथेटिक एंटीबॉडी पुस्तकालयों से स्केलेबल उच्च Throughput चुनाव

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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पर्यावरण के नमूनों से नई Faustoviruses के अलगाव का उपयोग के लिए एक तेजी रणनीति<em&gt; Vermamoeba vermiformis</em

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