저자는 NIH 3R01AI080289 - 02S1, 그리고 NIH / NIAID 2P30AI060354 - 07에 따라 에이즈 연구 학술 반지 원정대 하버드 대학교 센터에서 자금을 인정하고 싶습니다.

1. 문화 Phagocytic 셀 <ol><li> 문화 THP - 1 RPMI 1640에서 10 세포는 T의 flasks에 고정 중지 10 % 태아 소 혈청과 보충. 세포 밀도가 FcγR 표현 및 분석 성능을 일관된 수준을 유지하기 위해서는 0.5x10 6 / ML 아래에 유지되어야합니다. </li></ol> 2. Biotinylated 항원을 준비 <ol><li> 제조 업체의 지시에 따라 관심의 대상 항원을 biotinylate에 필요한 sulfo - 보건국 LC 비오틴 시약 키트의 양을 계산합니다. </li><li> 물에 biotinylation 시약을 용해하고 즉시 기본 아민을 포함하지 않는 버퍼에 항원으로 계산된 금액을 추가합니다. 반응이 때때로 혼합, 상온에서 1 시간 동안 진행하실 수 있습니다. </li><li> targe을 유지하기 위해 적절한 분자량의 컷오프의 Amicon 원심 농도 단위로 버퍼 교환에 의한 초과, unconjugated 비오틴을 제거t의 항원. 농도 단위의 상단 챔버에 샘플을 추가하고 최대 15 ML 채우기 라인에 전체 볼륨을 가지고 PBS를 추가합니다. 약 1.5 MLS에 볼륨을 가지고 4000 XG에 원심 분리기. 이 과정을 반복하면 두 번 구슬로 항원의 최대한의 코팅을 보장하기 위해 무료 비오틴의 99 %를 제거합니다. </li></ol> 3. 항원 포화 비즈 준비 <ol><li> 두 번 1 1mm 형광 neutravidin 비즈 100 μl를 씻으 ML 0.1 % PBS - BSA 고속 microcentrifuge 아래로 회전 후. Resuspend 10 튜브에 100 μl PBS - BSA, 그리고 나누어지는의 구슬을 빨기도 했죠. </li><li> 구슬을 포화 항원 코팅 조건을 확인하려면, 두 배 단계에 biotinylated 항원의 농도 변화로 씻은 비드 정지 10 UL을 결합. 회전에 microcentrifuge 튜브 4 ° C에서 밤새 품어. </li></ol> 참고 : 구슬의 채도가 실험적으로 결정되어야합니다. 이 수행할 수 있습니다구슬은 이후 컨트롤 단클론 항체와 opsonized 때 최대한의 phagocytosis를 얻을 비드 코팅 조건을 식별하여. <ol start="3"><li> 구슬이 pelleted 때까지 고속 1 ML PBS - BSA와 원심 분리기로 세척하여 언바운드 항원을 제거합니다. PBS - BSA를 제거하고 반복합니다. </li><li> PBS - BSA 1 ML의 최종 볼륨 Resuspend 항원 코팅 비즈. 비즈는 4 ° C 이전에 사용시 일주일에 저장할 수 있습니다. </li></ol> 4. 항체 샘플을 준비 <ol><li> 임상 항체 샘플 플라즈마는 제조 업체의 지침에 따라 멜론 젤 IgG 정화 키트를 사용하여 정화 수 있습니다. 정화 IgG는 4 ° C 사용을위한 준비까지 저장할 수 있습니다. </li></ol> 참고 : 인간의 샘플을 처리에 대한 적절한 예방 조치는 항상 촬영되어 있어야합니다. <ol start="2"><li> A280의 흡광도에 의한 항체를 정화의 농도를 결정하고, PBS 1 MG / ML에 샘플을 희석. </li><li> 멋부리다 준비1 MG / PBS에서 ML에 희석하여 필자와 부정적인 단클론 항체 제어. </li></ol> 5. 실험을 도금 <ol><li> vortexing에 의해 위에 준비한 씻어, 항원 포화 비드 솔루션을 Resuspend, 그리고 둥근 바닥 96 잘 플레이트의 각 우물에 10 μl를 전송합니다. 관리는 각 잘에 추가됩니다 지속적으로 형광 구슬의 같은 번호를 보장하기 위해 비드 중단을 선동로 이동합니다. </li><li> 각 항체에 대한 투여 - 반응 곡선을 만들고, 각 잘 관심 항체의 농도 변화에 추가합니다. 최적의 농도는 현재 항체의 titer에 따라 샘플을 사이에 다를 것이지만 0.01-100 μg / ML 최종 농도의 범위는 좋은 혜택을 제공하고 관심의 농도 범위의 신분을하실 수 있습니다. 확인 항체는 20 μl 이하 큰 볼륨에 추가되었습니다. </li><li> 37에서 2 시간 동안 품어 구슬과 항체 샘플 ° C 항체가 구슬을 opsonize 수 있도록. &lt;/ 리&gt; <li> 2.5 × 10 5 세포 / ML에서 THP - 1 세포 현탁액을 준비, 5 X의 총 각 잘이 정지 200 μl를 추가 10 4 THP - 1 각 우물에 세포. </li><li> 37 밤새 품어 ° C 5 % 고정 인큐베이터에서 CO 2, 중력을 통해 펠렛 수 있도록 세포와 구슬. </li></ol> 참고 : 노이즈에 신호가 최적의 비드를 결정하여 향상시킬 수 있습니다 항체 : THP - 1 주어진 항원과 항체 소스에 대한 세포 비율. 6. 흐름 Cytometric 분석 <ol><li> 각각 잘 세포 펠렛을 방해하지 않도록주의되는 뜨는 100 μl를 제거합니다. 시토킨 분비의 결정이나 다른 분석이 필요한 경우에는 뜨는을 저장할 수 있습니다. </li><li> 고정 들면, 세포를 resuspend과 섞어 각 잘하고 pipet에 4 % paraformaldehyde 100 μl를 추가합니다. </li><li> 높은 처리량의 흐름 cytometric 분석 HTS 플레이트 리더를 갖춘 BD LSR II를 사용하여 수행할 수 있습니다.</li><li> 프로그램 소프트웨어를 각각 잘 세 번 (100 μl 믹스 볼륨)를 혼합하고 각 시료 30 μl, 또는 적어도 2,000 셀 이벤트를 분석합니다. </li><li> 데이터 수집은 FlowJo 또는 이와 동등한 소프트웨어의 분석하실 수 있습니다. 유용한 통계는 비드 +의 퍼센트, 또는 형광 세포, 현재 phagocytic 세포의 수를 측정을 제공뿐만 아니라 phagocytosed 구슬의 수를 측정을 제공하는 phagocytic 세포의 형광 강도를 포함합니다. 이러한 값을 곱한 것은 통합된 의미 형광 강도, 또는 iMFI를 생성합니다. </li><li> 각 phagocytic 세포에 의해 phagocytosed 구슬의 평균 개수는 1 비드의 평균 형광하여 iMFI를 나누어 계산하실 수 있습니다. </li></ol> 7. 대표 결과 영향 및 영향을받지 과목에서 항체 샘플 명확한 차별이 있어야합니다. 그림 1A는 HIV nega에서 항체 샘플의 FACS histograms를 제공합니다tive (블랙 추적)와 HIV 양성 (회색 추적) 주제하고 항원에 특정한 항체의 존재에 의해 주도 증가 phagocytosis를 보여줍니다. 분석의 최적 감도 biotinylated 항원과 비즈의 채도에 따라 좌우됩니다. 그림 1B는 항원의 다양한 양의 코팅 구슬이 (구속력 항체, 삼각형 등) 3 제어 단클론 항체와 opsonized 때 관찰 phagocytosis을 제시,이 항원에 대한 포화 농도로 2μg 항원 / μl 구슬을 설정합니다. 선량 - 반응 곡선은 그림 2에 제시하고, 0.05-5 μg / ML의 항체 농도 범위 phagocytosis를 유도에 따라 항체 샘플 차등 용량을 보여줍니다. 이 차동 phagocytosis는 titer 또는 IgG의 하위 클래스와 글리코 실화 상태로 FC 도메인 속성의 차이에 의해 구동 수 있습니다. THP - 1 세포가 나는 경우maged은 형광 현미경으로 비드 phagocytosis의 명백한 증거가있다. 그림 3은 항체의 부재에 phagocytic 이해의 부족을 보여주는, 항체 opsonized (녹색) 및 비 opsonized (적색) 구슬과 부화 후에도 THP - 1 세포의 이미지 두 63x를 제공합니다. 시간 경과 현미경을 수행할 때, 형광 구슬의 항체 특정 phagocytic 이해는 (영화 1, 20x 배율) 훨씬 더 강렬합니다. 이전 작품은 세포와 관련된 구슬의 내면화를 확인하고, 기본 monocytes와 실험이 높은 처리량 분석 (데이터가 표시되지 않음)에서 phagocytic 점수 (iMFI 값)과 잘 합의했다. <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="그림 1"/> 그림 1. 분석 품질 관리. 1A, HIV 부정적인 주제 (블랙 추적)와 HIV 양성 과목 (회색 추적)에서 항체 샘플에 대한 phagocytosis의 유동세포계측법의 histograms.1B : 샘플 항원에 대한 최적의 비드 코팅 조건의 실험적 결정. 제어 항체 (삼각형)이 더 phagocytic 활동을 보여주는없는 동안 항원 특정 단클론 항체 (원형, 사각형)는,&gt; 2 μg 항원 / 구슬 μl로 코팅 구슬의 최대한의 phagocytosis를 보여줍니다. <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="그림 2"/> 그림 2. Phagocytosis 투여량 - 반응 곡선. differentially HIV 양성 (처리, 치료, 및 안티 retroviral 치료의 부재에서 바이러스 복제를 제어할 전시)와 gp120의 HIV 부정적인 과목 드라이브 phagocytosis (HIV 봉투) 코팅 비즈에서 임상 항체 샘플. <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="그림 3"/> 그림 3. 효율적인 국제화. 비 opsonized 구슬이 솔에 남아있는 동안 현미경은 항체 - opsonized 비즈 (녹색)의 국제화를 확인ution (63x 배율). 영화 1. 항체 주도 phagocytosis의 시간 경과 현미경은 높은 처리량 분석에 활용 THP - 1 세포의 phagocytic 활동의 시각화 14 시간 코스 이상을 수행하고 있습니다되었습니다. 녹색 형광 구슬이 항체 opsonized 있으며, 빨간색 형광 구슬은 부정적인 컨트롤을 제공합니다. <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">영화를보고하려면 여기를 클릭하십시오.</a>

<ol>
	<li>Cartron, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+activity+of+humanized+anti-CD20+monoclonal+antibody+and+polymorphism+in+IgG+Fc+receptor+FcgammaRIIIa+gene.">Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene.</a> Blood. 99, 754-758 (2002).</li><li>Ahmad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+correlation+between+antibody-dependent+cellular+cytotoxicity-mediating+anti-HIV-1+antibodies+and+prognostic+predictors+of+HIV+infection.">Evidence for a correlation between antibody-dependent cellular cytotoxicity-mediating anti-HIV-1 antibodies and prognostic predictors of HIV infection.</a> J. Clin. Immunol. 21, 227-233 (2001).</li><li>Hashimoto, G., Wright, P. F., Karzon, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-dependent+cell-mediated+cytotoxicity+against+influenza+virus-infected+cells.">Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells.</a> J. Infect Dis. 148, 785-794 (1983).</li><li>Anthony, R. M., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th	.">Th	.</a> Curr. Opin. Organ Transplant. , (2010).</li><li>Takai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptors+and+their+role+in+immune+regulation+and+autoimmunity.">Fc receptors and their role in immune regulation and autoimmunity.</a> J. Clin. Immunol. 25, 1-18 (2005).</li><li>Jefferis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+therapeutics:+isotype+and+glycoform+selection.">Antibody therapeutics: isotype and glycoform selection.</a> Expert. Opin. Biol. Ther. 7, 1401-1413 (2007).</li><li>Halstead, S. B., O'Rourke, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+viruses+and+mononuclear+phagocytes.+I.+Infection+enhancement+by+non-neutralizing+antibody.">Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody.</a> J. Exp. Med. 146, 201-217 (1977).</li><li>Subramaniam, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+absence+of+serum+IgM+enhances+the+susceptibility+of+mice+to+pulmonary+challenge+with+Cryptococcus+neoformans.">The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans.</a> J. Immunol. 184, 5755-5767 (2010).</li><li>Ackerman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust,+high-throughput+assay+to+determine+the+phagocytic+activity+of+clinical+antibody+samples.">A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.</a> J. Immunol. Methods. 366, 8-19 (2011).</li><li>Tsuchiya, 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+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1.">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1.</a> Int. J. Cancer. 26, 171-176 (1980).</li><li>Ward, E. S., Ghetie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effector+functions+of+immunoglobulins:+implications+for+therapy.">The effector functions of immunoglobulins: implications for therapy.</a> Ther. Immunol. 2, 77-94 (1995).</li><li>Winiarska, M., Glodkowska-Mrowka, E., Bil, J., Golab, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+antitumor+effects+of+anti-CD20+antibodies.">Molecular mechanisms of the antitumor effects of anti-CD20 antibodies.</a> Front. Biosci. 16, 277-306 (2011).</li><li>Takai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptors:+their+diverse+functions+in+immunity+and+immune+disorders.">Fc receptors: their diverse functions in immunity and immune disorders.</a> Springer Semin. 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>

관심 없음 충돌 선언하지 않습니다.

검정 여기에서 설명한 오래 phagocytic 프로세스 10 판을 기반으로 자동 유동세포계측법 연구를 이용 monocytic 셀 라인을 이용하여 임상 항체 샘플의 phagocytic 활동의 높은 처리량 분석을 수 있습니다. 이 분석뿐만 아니라 다른 주제에서 질병 특정 항체에 의해 주도 phagocytosis의 차이 연구 있도록 정확하게 항원 특정 항체 하위 집합을 특성화하기 위해 능력 넘을 유익뿐만 아니라 동일한 주제 내에서 여러 항원 specificities의 연구. monocytes와 다른 항원 제시 세포를 포함하여 본래 효과기 세포의 항체 모집 강하게 질병 결과 11-13 영향을, 그리고 FC 도메인 14-16의 Asparagine297에서 항체 기하학, IgG의 하위 클래스, 그리고 글리코 실화에 따라 생물 학적 변수이기 때문에,이 방법에 대한 제공 행동의 중요한 항체 메커니즘 평가. 또한, 항체 - 메디 때문에ated phagocytosis가 감염 7,17,18 과정 중에 몇 가지 병원균에 의해 악용,이 분석은 항체에 의존 향상 과정의 평가에 약속을 보유하고, 잠재적으로 보호뿐만 아니라, 잠재적으로 해로운로 phagocytosis의 이중 성격을 강조 항체 활동. 설명한 분석은 환자 인구의 임상 샘플에서 항체를 분석 활용뿐만 아니라 vaccinees, 그리고 오히려 정화 IgG보다 혈청 활용 적응 수 있습니다. 또한, phagocytosis에 대한 응답으로 시토킨 분비는 문화 뜨는에서 분석 수 있으며, 특정 FcR의 영향은 결정 phagocytic 힘뿐만 아니라 차이를 수 있도록 FcR 차단 항체를 사용하여 결정 될 수 있지만, 하류 통찰​​력과 이벤트를 신호 메커니즘으로,이 면역 메커니즘의 이해를 해결하고 수정하는 데 도움이 될 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> EZ - 링크 마이크로 Sulfo - NHS - LC - Biotinylation 키트 </td><td> 써모 Scientfic </td><td> 21,935 </td><td></td></tr><tr><td> Ultracel - 50 막과 Amicon 울트라 - 15 원심 필터 유닛 </td><td> Millipore </td><td> UFC905024 </td><td> 필터 유닛 크기는 항원의 크기에 따라 다를 수 있습니다 </td></tr><tr><td> FluoSpheres NeutrAvidin은 microspheres, 1.0 μm의, 황록색 형광 (515분의 505)를 표시 </td><td> Invitrogen </td><td> F - 8776 </td><td> 제조 업체들은 다양한 색상이 제품을 생산 </td></tr><tr><td> 멜론 젤 IgG의 정제 키트 </td><td> 써모 </td><td> 45,212 </td><td> 관리는 SDS 페이지로 AB 샘플의 순도를 확인하기 위해 이동해야합니다. </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

항체 기반의 phagocytosis는 전문 phagocytes에서 FC 수용체의 참여를 통해 유도하고, 질병의 모두 허가뿐만 아니라 병리에 기여할 수 있습니다. 항체의 변수 도메인의 속성이 긴 생체내 기능에 치명적인 것으로 간주되고있는 반면, 그들의 FC 도메인을 통해 타고난 면역 세포를 모집 항체의 능력은 모두 재조합의 설정에서, 그들의 효험의 주요 요소로 점점 더 높이 평가되고있다 단클론 항체 치료뿐만 아니라, 자연 감염이나 예방 접종 1-3의 과정 인치 중요한 것은 지속적인 도메인으로의 명칭에도 불구하고, 항체 FC 도메인 일정 기능을 가지고 있고, IgG의 하위 클래스 (IgG1 - 4) 및 아스파라긴에서 글리코 실화 297 4-6로 강하게 변조되지 않습니다. 따라서,이 방법은 phagocytic 냄비의 상관 관계를 촉진합니다 임상 샘플에서 항원 - 항체 특정의 기능 차이를 연구하기질병 상태, 감염에 자화율, 진행, 또는 임상 결과에 항체의 ential. 또한,이 이펙터 기능 FC 수용체 중심의 phagocytosis 7을 통해 호스트 세포에 병원균에 대한 액세스를 제공하여 감염을 향상시키기 위해 항체의 문서화 능력에 비추어 특히 중요합니다. 또한, 면역 단지의 phagocytic 이해가 면역 반응 8 Th1/Th2 분극에 영향을 미칠 수있는 몇 가지 증거가있다. 여기, 우리는 차동 IgG의 하위 클래스, Asn297에서 glycan 구조뿐만 아니라, 높은 처리량 방식으로 항원 특정 항체의 면역 단지를 형성하는 능력으로 인해 발생할 수 있습니다 항체 유발 phagocytosis의 차이를 감지하기위한 분석을 설명 . 이를 위해, 1 μm의 형광 구슬은 다음 형광 항원 특정 면역 단지를 생성, 임상 항체 샘플 incubated, 항원과 코팅입니다. 이 antibo다이 - opsonized 구슬은 다음 억제와 활성화 모두를 포함하여 여러 FcγRs을 표현 monocytic 세포 라인 incubated 있습니다. 분석 출력 phagocytic 활동, 시토킨 분비 및 FcγRs 사용량 패턴을 포함할 수 있으며,이 감염 및 백신 - 중재 보호 구 모두에서이 항체 의존 이펙터 함수의 해석 차이에 대한 높은 유용한 시스템을 만들고, 표준 방식으로 결정됩니다.

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

우리는 - 수용체 제공 여러 FC의 표현 형광 항원 코팅 구슬과 monocytic 세포 라인을 활용하여 표준의 수용체 사용량과 phagocytic 활동 결정하고, 임상 샘플에서 항원에 특정한 항체의 phagocytic 활동을 결정하기 위해 높은 처리량 흐름 cytometric 분석을 제시 관심의 항원에 대한 재현할 패션.

임상 항체 샘플 Phagocytic 활동 결정

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

determining-the-phagocytic-activity-of-clinical-antibody-samples

Research

JoVE Journal

Immunology and Infection

23.3K 조회수.  Ragon Institute of MGH, MIT, and Harvard. 우리는 - 수용체 제공 여러 FC의 표현 형광 항원 코팅 구슬과 monocytic 세포 라인을 활용하여 표준의 수용체 사용량과 phagocytic 활동 결정하고, 임상 샘플에서 항원에 특정한 항체의 phagocytic 활동을 결정하기 위해 높은 처리량 흐름 cytometric 분석을 제시 관심의 항원에 대한 재현할 패션.

비디오: Determining the Phagocytic Activity of Clinical Antibody Samples

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination 분석을 사용하여 혈청의 반응성 및 Titre를 결정

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

연구

면역학 및 감염병학

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

바이러스성 병원균에 대한 임상 샘플을 화면에 판 - 바이러스성 Microarray 분석 (Virochip)를 사용

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Oligonucleotide - 결합 형광등 Microspheres를 사용하여 복잡한 임상 및 환경 샘플에서 박테리아의 멀티 플렉스 감지

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

xCELLigence 시스템으로 CR51 릴리스 분석을 비교하여 인간 Her2neu 특정한 CD8 T 세포의 최적 세포 독성 활동 결정

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

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

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

포유 동물 세포와 관련있는 박테리아의 생존을 확인하는 방법에 대한 형광 현미경 방법

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

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

파지 - 표시 합성 항체 라이브러리의 확장 성 높은 처리량 선택

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

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략<em&gt; Vermamoeba의 vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

백혈구가 풍부한 혈액 샘플의 할로겐 퍼 옥시 다제 활동의 신속하고 구체적인 평가

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...