Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00.000+00:00
Duration: 4 min 47 s
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

이 프로젝트는 자금을 일부 알레르기 국립 연구소와 전염병에서 연방 자금, 건강의 국가 학회, 학과의 보건 및 인적 서비스, CEIRS 아래 계약 P01AI097092-04S1 (P.E.L.).

이 원고에서 preformed 실험 윤리 및 규정의 다음 아이 칸의 학부의 기관 코드를 완료 했다.
1. 인플루엔자 바이러스 조류 Transfection 또는 감염의 표현
Transfection:
<ol><li>플레이트 인간 미 발달 신장 (HEK 293T) 셀 셀/잘 백색 조직 문화에서에서 2 x 104 의 밀도에서 96 잘 접시를 처리 하 고 (와 함께 5% CO2) 37 °C 배양 기에서 4 h 동안 앉아 보자.</li>
	<li>100 셀 transfect DNA 바이러스 조류 및 50 μ의 전체 볼륨에 잘 당 transfection 시 약 0.2 μ에 대 한 코딩의 ng. 참고: 모두 Opti-최소 필수 미디어 (MEM) 감소 된 혈 청 매체 x 1에 플라스 미드 그리고 transfectant 시 약 희석 하.</li>
	<li>플레이트 18 h 5% CO2 와 37 °C 배양 기에서 배양 후 transfection 미디어를 제거 합니다.</li>
</ol>2입니다. 감염
<ol><li>플레이트 A549 또는 Madin 다 비 개 신장 세포 세포/잘 백색 조직 문화에서에서 2 x 104 의 밀도에서 96 잘 접시를 처리 하 고 5% CO2와 37 °C 배양 기에 18 h 이상에 대 한 성장.</li>
	<li>1에서 희석 바이러스 MEM x 3, 1, 또는 5% CO2와 37 °C 배양 기에서 1 h 0.33의 감염의 다양성에서 세포를 감염 하 고. 총 볼륨 100 μ 동일 해야 합니다. 참고: 10 x 메모리 위에서 언급 한 바이러스 희석에 대 한 메모리 작업 재고 x 1을 물으로 희석.</li>
	<li>감염 후는 inoculum을 제거 하 고 추가 1의 100 μ trypsin의 부재에서 접시에 MEM x.</li>
	<li>5% CO2와 37 °C 배양 기에서 18 ~ 24 h에 대 한 감염 된 세포를 성장 한다.</li>
</ol>3. FcRg/NFAT 중재 활성화 단일 클론 항 체 또는 세라 Luciferase 활동의 평가
<ol><li>첫째, 25 µ L 분석 결과 미디어 (1 x RPMI 1640 4% (vol/vol) 보충 낮은 IgG 태아 소 세라)의 transfected 또는 감염 된 세포의 성장 미디어를 바꿉니다.</li>
	<li>별도 96 잘 접시에 분석 결과 미디어를 사용 하 여 30 μ g/mL에서 시작 하는 관심사의 항 체가의 4 배 희석을 수행 합니다. Triplicates에 희석을 수행 하 고 예제 플레이트 레이아웃은 그림 1A에 나타나. 배경 컨트롤 (혼자 분석 결과 버퍼) 뿐만 아니라 항 체 (항 체 제어)을 제외 하는 컨트롤을 포함 해야 합니다. 참고 사항: 모두 '아무 항 체'에 대 한 컨트롤 배경, 25 µ L 대신에 항 체 분석 결과 버퍼의 추가.</li>
	<li>Transfected 세포 또는 감염 된 세포의 접시에 단계 3.2 해당 우물을 연속적으로 희석된 한 항 체의 25 μ 전송.</li>
	<li>15 분 동안 (와 함께 5% CO2) 37 °C 배양 기에서 접시를 품 어.</li>
	<li>Murine FcgRIV 또는 인간 FcgRIIIa (셀/잘 75000) 표현 적절 한 수정된 이펙터 Jurkat 세포의 25 μ를 추가 합니다. 각각에 대 한 총 볼륨 잘 75 µ L 고 항 체의 시작 최종 농도 10 µ g/mL (그림 1B)에 이다. 참고: 배경 컨트롤에 대 한 분석 결과 버퍼 이펙터 셀 대신의 25 µ L 추가 합니다. 항 체 의존 세포 중재 먹어서 검사, 인간 FcgRIIa 표현 수정된 Jurkat 세포를 사용할 수 있습니다.</li>
	<li>플레이트 6 h 5% CO2 와 37 ° C 배양 기에서 품 어.</li>
	<li>해 동 하 고 사용 하기 전에 약 1 h는 37 °C 물에 따뜻한 luciferase 기판 버퍼. 참고: luciferase 기판 버퍼를 만들기 위해 제공 하는 분석 결과 버퍼에서 동결 건조 된 luciferase 분석 결과 기판 다시 구성. 재구성 된 기판 버퍼 최대 6 주 동안-30 °C ~-10 °C에 저장할 수 있습니다.</li>
	<li>배경 컨트롤을 제외한 모든 우물에 75 μ/우물 luciferase 기판 버퍼를 추가 합니다.</li>
	<li>읽기는 루미 노에.</li>
	<li>배 유도 다음 방정식으로 계산: 유도 배 = (유도 값-배경 값) / (mAb 값-배경 값 없음).</li>
</ol>

<ol>
	<li>Hobson, D., Curry, R. L., Beare, A. S., Ward-Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+serum+haemagglutination-inhibiting+antibody+in+protection+against+challenge+infection+with+influenza+A2+and+B+viruses.">The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses.</a> J Hygiene. 70 (04), 767-777 (1972).</li><li>Throsby, 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=Heterosubtypic+neutralizing+monoclonal+antibodies+cross-protective+against+H5N1+and+H1N1+recovered+from+human+IgM++memory+B+cells.">Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.</a> PloS One. 3 (12), e3942 (2008).</li><li>Ekiert, D. 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=Antibody+recognition+of+a+highly+conserved+influenza+virus+epitope.">Antibody recognition of a highly conserved influenza virus epitope.</a> Science. 324 (5924), 246-251 (2009).</li><li>Ekiert, D. 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=A+Highly+Conserved+Neutralizing+Epitope+on+Group+2+Influenza+A+Viruses.">A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses.</a> Science. 333 (6044), 843-850 (2011).</li><li>Tan, G. 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=A+pan-H1+anti-hemagglutinin+monoclonal+antibody+with+potent+broad-spectrum+efficacy+in+vivo.">A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo.</a> J Virol. 86 (11), 6179-6188 (2012).</li><li>Tan, G. 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=Characterization+of+a+Broadly+Neutralizing+Monoclonal+Antibody+That+Targets+the+Fusion+Domain+of+Group+2+Influenza+A+Virus+Hemagglutinin.">Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin.</a> J Virol. 88 (23), 13580-13592 (2014).</li><li>Friesen, R. H. 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=A+common+solution+to+group+2+influenza+virus+neutralization.">A common solution to group 2 influenza virus neutralization.</a> PNAS. 111 (1), 445-450 (2014).</li><li>Dreyfus, 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=Highly+Conserved+Protective+Epitopes+on+Influenza+B+Viruses.">Highly Conserved Protective Epitopes on Influenza B Viruses.</a> Science. 337 (6100), 1343-1348 (2012).</li><li>Krammer, F., Palese, P., Steel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Universal+Influenza+Virus+Vaccine+Design+and+Antibody+Mediated+Therapies+Based+on+Conserved+Regions+of+the+Hemagglutinin.">Advances in Universal Influenza Virus Vaccine Design and Antibody Mediated Therapies Based on Conserved Regions of the Hemagglutinin.</a> Curr Top Microb and Immunol. , (2014).</li><li>Impagliazzo, A., 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+stable+trimeric+influenza+hemagglutinin+stem+as+a+broadly+protective+immunogen.">A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.</a> Science. , (2015).</li><li>Nachbagauer, R., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Microbiology+and+Infection.">Clinical Microbiology and Infection.</a> Clin Microb Infect. , 1-7 (2017).</li><li>Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+universal+influenza+virus+vaccine+approaches.">Novel universal influenza virus vaccine approaches.</a> Current opinion in virology. 17, 95 (2016).</li><li>Steel, 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=Influenza+virus+vaccine+based+on+the+conserved+hemagglutinin+stalk+domain.">Influenza virus vaccine based on the conserved hemagglutinin stalk domain.</a> mBio. 1 (1), (2010).</li><li>Krammer, F., Pica, N., Hai, R., Margine, I., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+hemagglutinin+influenza+virus+vaccine+constructs+elicit+broadly+protective+stalk-specific+antibodies.">Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.</a> J Virol. 87 (12), 6542-6550 (2013).</li><li>Krammer, F., Pica, N., Hai, R., Tan, G. S., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+Stalk-Reactive+Antibodies+Are+Boosted+following+Sequential+Infection+with+Seasonal+and+Pandemic+H1N1+Influenza+Virus+in+Mice.">Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice.</a> J Virol. 86 (19), 10302-10307 (2012).</li><li>Hai, R., 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=Influenza+viruses+expressing+chimeric+hemagglutinins:+globular+head+and+stalk+domains+derived+from+different+subtypes.">Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.</a> J Virol. 86 (10), 5774-5781 (2012).</li><li>Dunand, C. J. H., 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=Both+Neutralizing+and+Non-Neutralizing+Human+H7N9+Influenza+Vaccine-Induced+Monoclonal+Antibodies+Confer+Protection.">Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.</a> Cell Host and Microbe. 19 (6), 800-813 (2016).</li><li>Tan, G. 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=Broadly-Reactive+Neutralizing+and+Non-neutralizing+Antibodies+Directed+against+the+H7+Influenza+Virus+Hemagglutinin+Reveal+Divergent+Mechanisms+of+Protection.">Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.</a> PLoS Path. 12 (4), e1005578 (2016).</li><li>DiLillo, D. J., Tan, G. S., Palese, P., 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=Broadly+neutralizing+hemagglutinin+stalk-specific+antibodies+require+Fc&#947;R+interactions+for+protection+against+influenza+virus+in+vivo.">Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc&#947;R interactions for protection against influenza virus in vivo.</a> Nat Med. 20 (2), 143-151 (2014).</li><li>DiLillo, D. J., Palese, P., Wilson, P. C., 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=Broadly+neutralizing+anti-influenza+antibodies+require+Fc+receptor+engagement+for+in+vivo+protection.">Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.</a> J Clin Invest. 126 (2), 605-610 (2016).</li><li>Potter, C. W., Oxford, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+immunity+to+influenza+infection+in+man.">Determinants of immunity to influenza infection in man.</a> Brit Med Bull. 35 (1), 69-75 (1979).</li><li>Memoli, M. 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=Evaluation+of+Antihemagglutinin+and+Antineuraminidase+Antibodies+as+Correlates+of+Protection+in+an+Influenza+A/H1N1+Virus+Healthy+Human+Challenge+Model.">Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model.</a> mBio. 7 (2), e00417 (2016).</li><li>Jegaskanda, 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=Age-associated+cross-reactive+antibody-dependent+cellular+cytotoxicity+toward+2009+pandemic+influenza+A+virus+subtype+H1N1.">Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.</a> J Infect Dis. 208 (7), 1051-1061 (2013).</li><li>Jegaskanda, 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=Cross-Reactive+Influenza-Specific+Antibody-Dependent+Cellular+Cytotoxicity+Antibodies+in+the+Absence+of+Neutralizing+Antibodies.">Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies.</a> J Immunol. 190 (4), 1837-1848 (2013).</li><li>Jegaskanda, S., Wheatley, A. K., Kent, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScienceDirectAntibody-dependent+cellular+cytotoxicity+and+influenza+virus.">ScienceDirectAntibody-dependent cellular cytotoxicity and influenza virus.</a> Curr Opin Virol. 22 (C), 89-96 (2017).</li><li>Srivastava, V., 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=Identification+of+Dominant+Antibody-Dependent+Cell-Mediated+Cytotoxicity+Epitopes+on+the+Hemagglutinin+Antigen+of+Pandemic+H1N1+Influenza+Virus.">Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus.</a> J Virol. 87 (10), 5831-5840 (2013).</li><li>He, 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=Epitope+specificity+plays+a+critical+role+in+regulating+antibody-dependent+cell-mediated+cytotoxicity+against+influenza+A+virus.">Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.</a> PNAS. 113 (42), 11931-11936 (2016).</li><li>Leon, P. 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=Optimal+activation+of+Fc-mediated+effector+functions+by+influenza+virus+hemagglutinin+antibodies+requires+two+points+of+contact.">Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact.</a> PNAS. , 201613225 (2016).</li><li>Benjamin, 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=A+Broadly+Neutralizing+Human+Monoclonal+Antibody+Directed+against+a+Novel+Conserved+Epitope+on+the+Influenza+Virus+H3+Hemagglutinin+Globular+Head.">A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head.</a> J Virol. 88 (12), 6743-6750 (2014).</li><li>Ekiert, D. C., Kashyap, A. 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=Cross-neutralization+of+influenza+A+viruses+mediated+by+a+single+antibody+loop.">Cross-neutralization of influenza A viruses mediated by a single antibody loop.</a> Nature. 488 (7417), 526-532 (2013).</li><li>He, 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=Broadly+Neutralizing+Anti-Influenza+Virus+Antibodies:+Enhancement+of+Neutralizing+Potency+in+Polyclonal+Mixtures+and+IgA+Backbones.">Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones.</a> J Virol. 89 (7), 3610 (2015).</li><li>Kristensen, A. 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=Antibody+Responses+with+Fc-Mediated+Functions+after+Vaccination+of+HIV-Infected+Subjects+with+Trivalent+Influenza+Vaccine.">Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine.</a> J Virol. 90 (12), 5724-5734 (2016).</li><li>Greenberg, S. B., Criswell, B. S., Six, H. R., Couch, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+cytotoxicity+to+influenza+virus-infected+cells.+II.+Requirement+for+antibody+and+non-T+lymphocytes.">Lymphocyte cytotoxicity to influenza virus-infected cells. II. Requirement for antibody and non-T lymphocytes.</a> J Immunol (Baltimore, Md. : 1950). 119 (6), 2100-2106 (1977).</li><li>Corrales-Aguilar, E., Trilling, 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=A+novel+assay+for+detecting+virus-specific+antibodies+triggering+activation+of+Fc&#947;+receptors.">A novel assay for detecting virus-specific antibodies triggering activation of Fc&#947; receptors.</a> J Immunol Meth. 387 (1-2), 21-35 (2013).</li><li>de Vries, R. 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=Influenza+virus-specific+antibody+dependent+cellular+cytoxicity+induced+by+vaccination+or+natural+infection.">Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection.</a> Vaccine. 35 (2), 238-247 (2017).</li><li>Cheng, Z., 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=Development+of+a+robust+reporter-based+ADCC+assay+with+frozen,+thaw-and-use+cells+to+measure+Fc+effector+function+of+therapeutic+antibodies.">Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.</a> J Immunol Meth. 414 (1), 69-81 (2014).</li></ol>

저자는 관심 없음 충돌 선언합니다.

여기에 설명 된 메서드를 사용 하면 murine FcγRIV 참여 하 특정 단일 클론 항 체의 능력을 측정 하 수 있습니다. 대상 항 인플루엔자 바이러스 조류 바이러스 또는 플라스 미드 DNA의 transfection 감염 후 세포의 표면에 표현 된다. 분석 결과 (인간 또는 murine) 다른 FcγRs와 함께에서 다른 표면 표현 바이러스 성 단백질을 사용 하 여 의무가 더 이다. 또한, 우리는 세라 샘플 사용 Fc effector 기능 (데이터 표시 ...

계절 독감 백신에서 제공 하는 면역은 전통적으로 hemagglutination 억제 (HI) 분석 결과1, 대상으로 하는 조류의 수용 체 바인딩 사이트 항 체의 존재를 측정 하 여 평가 되었습니다. 이러한이 활성 항 체 살 균 면역을 부여 수 있습니다 하지만 일반적으로 인플루엔자 바이러스의 단지 선택 몇 가지 계통에 면역을 제공 하는 보호의 그들의 범위에서 좁은. 격리 및 조류 (HA)를 인식 하는 광범위 하 게 반응 단일 클론 항 체의 특성화 제안 범용 인플루엔자 백신의 개발은 도달 시간2,,34, 5 , 6 , 7 , 8. 인플루엔자 바이러스9,10,,1112 의 보존된 지역으로 강한 항 체 응답을 유도 하는 것은 보편적인 인플루엔자 백신의 주요 목표 중 하나 , 13 , 14 , 15 , 16. 이러한 보존된 epitopes, 중화 및 비 중화 항 체17,18의 인식 반응 항 체 Fc-FcγR 상호 작용에 대 한 요구를 광범위 하 게 표시 되었습니다 최적의 vivo에서 보호 하 고 하이라이트 Fc 중재 면역 인플루엔자 바이러스19,20전반적인 면역 응답에의 기여.
보호의 관계가 중요 한 감염에 면역을 평가에 있습니다. 이러한 통계 과학자와 임상 백신의 효능, 데이터의 의미와 치료 과정을 추정 하실 수 있습니다. 인플루엔자 바이러스 감염에 대 한 보호의 유일한 설립된 상관 hemagglutination 저해 분석 결과 이다. 1시 40분의 titer 질병1,21 -위험 50% 감소와 관련 이며 바인딩 사이트는 HA의 구형 머리에 있는 수용 체를 대상으로 교착을 억제 하는 항 체의 존재를 반영 한다. 그러나, 그것은 또한 주목 해야한다 T 세포 응답 노인에서 인플루엔자 바이러스 감염에 대 한 보호의 더 나은 상관 있을 수 있습니다. 흥미롭게도, 최근 보고서 응집 저해 행위 면책 독감22의 더 나은 예언자를 있을 수 있습니다 제안 합니다. 다행히도, 일반적인 체 외에서 분석 실험 같은 중화 또는 응집 저해 분석 하 줄기 또는 응집 특정 항 체를 측정할 수 있습니다. 그러나 대부분 이러한 기존의 시험관에서 분석의,만 항 체의 항 원 바인딩 영역의 기능을 고려 하 고 Fc 영역의 역할을 측정 하지 않습니다. 또한, 비 중화 항 체 Fc 수용 체 참여에서 vivo에서 를 통해 보호 하는의 기여 감지17,18되지 않습니다. Fc 중재 면역 항 체 의존 세포 중재 세포 독성 (ADCC) 등을 유발 하는 항 체에 의해 여유 보호를 측정 하기 위하여 강력한 생체 외에서 시험이 필요 합니다.
아래 설명 하는 방법을 평가 수 murine 단일 클론 항 체의 Fc 중재 기능 활성화는 표현 유전자 변형된 Jurkat 세포 라인 사용 유도 murine 입력 1 FcR, FcγRIV. 항 체는 FcγR의 교전 세포내 신호 트리거 활성화 된 T 세포 중재 luciferase 활동의 핵 요인 transduces. 분석 결과 여러 장점이 격리 및 기본 효과 기 세포의 문화 및 cytometry Fc 중재 effector 기능19,23,24의 활성화를 감지를 사용 하 여 요구 하는 전통적인 기법 ,,2526. 첫째, 여기에 설명 된 프로토콜을 쉽게 통합 하는 다른 바이러스 성 대상, FcγRs, 및 다른 종 (인간과 murine)에서 항 체 적용할 수 있습니다. 둘째, 수정된 Jurkat 세포를 사용 하 여 표현 하는 활성화 된 T 세포 (NFAT)의 핵 요인 아래 luciferase 취재 원 유전자와 FcγR 발광 측정 플레이트 리더를 사용 하 여 쉽게 분석할 수 있는 대형 분석 결과 대 한 수 선. 여기, 기본 효과 기 세포의 전통적인 활성화 Jurkat 세포의 표면에 FcγR의 항 체 참여 시 NFAT luciferase의 유도로 대체 됩니다. 이 96 잘 접시 형식 분석 결과 7 희석-성가신 전통적인 기술을 사용 하 여 될 수 있는 샘플 수와 triplicates에서 최대 4 개의 서로 다른 샘플의 측정에 대 한 수 있습니다. 이 분석 결과 실험의 설계 및 데이터의 해석에 영향을 미칠 수 있습니다 고려해 야 할 추가 포인트가 있다. 바이러스 성 대상 항 체 인식에 세포 표면에 표현 해야 합니다. 이 줄이기 위해 대상 항 원 (예: 내부 바이러스 성 단백질)는 격판덮개의 표면에 직접 코팅 수 수 있습니다. 그러나, 이것은 아직 엄격 하 게 테스트 되지. 또한, 수정 된 Jurkat 세포 선 및 FcγR 활성화의 한 유형을 표현 1 차 셀 라인 표현 생리 적 맥락에서 모든 수용 체 어떤 금지 FcγRs을 표현 하지 않는다.
우리가 이전 polyclonal 컨텍스트에서 epitope 특이성 Fc 중재 effector 기능 조절에 중요 한 역할을 담당 하며 그 항 체-종속 셀 중재 응답의 최적 활성화의 두 가지 포인트를 설명 하기 위해이 분석 결과 사용 27,28문의. 여기, 우리가 참여 하 고 FcγRIV를 활성화 murine 활성화 줄기 특정 단일 클론 항 체의 능력을 평가 하는 방법을 설명 합니다. 예외29,30비록 광범위 하 게 반응 항 체의 많은 수는 조류의 antigenically 보존된 스토킹 지역 대상 고 따라서 범용 인플루엔자의 개발에 중요 한 역 백신입니다.

<table><tbody><tr><td>A549 cells&amp;nbsp;</td><td>ATCC</td><td>CCL-185</td><td>Adenocarcinomic human alveolar basal epithelial cells</td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Human embryonic kidney cells</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies&amp;nbsp;</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>1X Opti-MEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>51985-034</td><td></td></tr><tr><td>White tissue culture treated 96-well plate</td><td>Corning, Inc.&amp;nbsp;</td><td>3917</td><td>Assay plates</td></tr><tr><td>10X MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>Jurkat cell expressing murine FcgRIV</td><td>Promega</td><td>M1201</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIIa</td><td>Promega</td><td>G7010</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIa</td><td>Promega</td><td>G9901</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Luminometer</td><td>BioTek</td><td>Synergy H1 Multi-Mode reader</td><td>Luminescence plate reader</td></tr><tr><td>Bio-Glo Luciferase Assay System</td><td>Promega</td><td>G7940</td><td>Contains luciferase assay buffer and luciferase assay substrate</td></tr><tr><td>Madin Darby canine kidney cells</td><td>ATCC</td><td>CCL-34</td><td>Canine kidney cells</td></tr></tbody></table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

항 체 항 원 바인딩 도메인 Fc 영역을 통해 바이러스 성 병원 체에 대 한 타고 난 및 적응형 면역 응답 커플링에 중요 한 역할을 한다. 여기, 우리가 활성화를 측정 하는 방법에 설명 Fc의 단일 클론 항 체는 활성화 표현 유전자 조작된 Jurkat 세포 라인의 사용으로 인플루엔자 바이러스 조류를 대상으로 하 여 이펙터 기능 입력 1 Fc-FcγR. 전송 시각:이 메서드를 사용 합니다 특정 Fc-FcγR 상호 작용 면역 글로불린에 의해 수 여의 생체 외에서 분석 결과 사용 하 여 확인할 수 있습니다.

우리는 시연 스토킹 전용 mAb, 6F12, 하지만 하지 머리 전용 머리 전용 mAb, PY102, upregulating CD107a 및 인터페론 γ19에 의해 기본 자연 킬러 세포를 활성화할 수 있었습니다. 모델링 기본 인간 NK 세포를 활성화 하는 스토킹 특정 항 체의 능력, 우리 스토킹 관련 mAb, 6F12, 튼튼하게 의해 murine FcγRIV 표현 수정된 Jurkat 세포를 활성화할 수 있습니다 보여 ~ 14-fold. 다른 한편으로, 머리 전용 mAb, P...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

항 체에 의해 그 대상 인플루엔자 바이러스 조류 Fc 중재 effector 기능 활성화를 측정 하는 방법을 설명 합니다. 이 분석 결과 또한 Fc 중재 면역 유도의 단일 클론 항 체 또는 polyclonal 세라 다른 바이러스 표면 glycoproteins를 대상으로 능력을 평가 하기 위해 적응 수 있습니다.

독감 조류 특정 항 체에 의해 유도 된 Fc 중재 Effector 기능을 평가 하는 방법

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

a-method-to-assess-fc-mediated-effector-functions-induced-influenza

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.6K 조회수.  Icahn School of Medicine at Mount Sinai. 항 체에 의해 그 대상 인플루엔자 바이러스 조류 Fc 중재 effector 기능 활성화를 측정 하는 방법을 설명 합니다. 이 분석 결과 또한 Fc 중재 면역 유도의 단일 클론 항 체 또는 polyclonal 세라 다른 바이러스 표면 glycoproteins를 대상으로 능력을 평가 하기 위해 적응 수 있습니다.

비디오: A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

모든 독감의 양적 분석 간단한 슬롯 얼룩 Assays에서 유니버설 항체를 사용하는 바이러스성 Hemagglutinins 및 Neuraminidases를 입력하십시오

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

연구

면역학 및 감염병학

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

효소 결합 렉틴 분석에 의해 인플루엔자 뉴 라미니다 아제 억제 항체가를 측정

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

감염 마우스 모델에서 인플루엔자 A 바이러스 연구

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

인플루엔자 바이러스 단일 클론 항 체를 중화의 탈출 변종의 생성

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

사용 하 여 MDCK SIAT1 세포 분석 실험 측정 인플루엔자 항 체 응답 Microneutralization에 의해 인간의 세라에 A(H3N2) 바이러스를 중화

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

인플루엔자-특정 항 체 Titers 계량에 최적화 된 Hemagglutination 억제 (HI) 분석 결과

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

의학

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

인플루엔자 A 바이러스 감염 중 T 여포 도우미 세포 및 발아 센터 반응 평가

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

HA 항체에 의한 Fc 매개 효과기 기능의 활성화 측정

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

중화 항체에 대한 인플루엔자 바이러스 탈출 변이의 생성

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

표적 항원에 대한 혈청 항체를 검출하기 위한 Hemagglutination Inhibition Assay

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...