우리이 원고 준비 박사 Xiuhua Lu, 박사 펭 류, 및 그들의 중요 한 검토 및 지원에 대 한 질병 통제 센터의 독감 부문에서 양 애슐리 버로우즈 감사합니다. 우리는 그림 3의 그래픽을 준비 하 고 그의 도움에 대 한 질병 통제 센터의 독감 부문에서 박사 아드리안 Reber 감사. 마지막으로, 우리는 박사 M. Matrosovich, Marburg, 독일 MDCK SIAT1 세포를 제공 하기 위한 감사 합니다.

<ol><li>Reber, A., Katz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunological+assessment+of+influenza+vaccines+and+immune+correlates+of+protection%5BTitle%5D)+AND+%22Expert+Rev+of+Vaccines%22%5BJournal%5D)">Immunological assessment of influenza vaccines and immune correlates of protection.</a> Expert Rev of Vaccines. 12, 519-536 (2013).</li>
<li>Li, C. K., Rappuoli, R., Xu, X. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlates+of+protection+against+influenza+infection+in+humans--on+the+path+to+a+universal+vaccine%3F%5BTitle%5D)+AND+%22Curr+Opin+in+Immunol%22%5BJournal%5D)">Correlates of protection against influenza infection in humans--on the path to a universal vaccine?</a> Curr Opin in Immunol. 25, 470-476 (2013).</li>
<li>WHO Global Influenza Surveillance Network. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Manual+for+the+labratory+diagnosis+and+virological+surveillence+of+influenza%22">Manual for the labratory diagnosis and virological surveillence of influenza</a>. , 
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<li>WHO. Recommended composition of influenza virus vaccines for use in the northern hemisphere influenza season. , 
 <a target="_blank" href="http://www.who.int/influenza/vaccines/virus/recommendations/201703_recommendation.pdf?ua=1">http://www.who.int/influenza/vaccines/virus/recommendations/201703_recommendation.pdf?ua=1</a> 2017-2018 (2017).</li>
<li>Levine, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutralizing+Antibody+Responses+to+Antigenically+Drifted+Influenza+A%28H3N2%29+Viruses+among+Children+and+Adolescents+following+2014-2015+Inactivated+and+Live+Attenuated+Influenza+Vaccination%5BTitle%5D)+AND+%22Clin+Vaccine+Immunol+%3A+CVI%22%5BJournal%5D)">Neutralizing Antibody Responses to Antigenically Drifted Influenza A(H3N2) Viruses among Children and Adolescents following 2014-2015 Inactivated and Live Attenuated Influenza Vaccination.</a> Clin Vaccine Immunol : CVI. 23, 831-839 (2016).</li>
<li>Lin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+characteristics+and+antigenic+properties+of+recently+emerged+subclade+3C.3a+and+3C.2a+human+influenza+A%28H3N2%29+viruses+passaged+in+MDCK+cells%5BTitle%5D)+AND+%22Influenza+Other+Respir+Viruses%22%5BJournal%5D)">The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells.</a> Influenza Other Respir Viruses. , (2017).</li>
<li>Lin, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuraminidase+receptor+binding+variants+of+human+influenza+A%28H3N2%29+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site%3A+a+role+in+virus+attachment%3F%5BTitle%5D)+AND+%22J+of+Virol%22%5BJournal%5D)">Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?</a> J of Virol. 84, 6769-6781 (2010).</li>
<li>Skowronski, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+acquired+during+cell+culture+isolation+may+affect+antigenic+characterisation+of+influenza+A%28H3N2%29+clade+3C.2a+viruses%5BTitle%5D)+AND+%22Euro+Surveillance%22%5BJournal%5D)">Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses.</a> Euro Surveillance. 21, 30112(2016).</li>
<li>Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N. A., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+the+alpha-2%2C6-sialyltransferase+in+MDCK+cells+increases+influenza+virus+sensitivity+to+neuraminidase+inhibitors%5BTitle%5D)+AND+%22J+of+Virol%22%5BJournal%5D)">Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.</a> J of Virol. 77, 8418-8425 (2003).</li>
<li>Oh, D. Y., Barr, I. G., Mosse, J. A., Laurie, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MDCK-SIAT1+cells+show+improved+isolation+rates+for+recent+human+influenza+viruses+compared+to+conventional+MDCK+cells%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells.</a> J Clin Microbiol. 46, 2189-2194 (2008).</li>
<li>US Department of Health and Human Services. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Biosafety+in+Microbiological+and+Biomedical+Laboratories%22">Biosafety in Microbiological and Biomedical Laboratories</a>. , (2009).</li>
<li>Reed, H. M. <a target="_blank" href="https://academic.oup.com/aje/article-abstract/27/3/493/99616/A-SIMPLE-METHOD-OF-ESTIMATING-FIFTY-PER-CENT?redirectedFrom=PDF">A simple method of estimating fifty percent endpoints.</a> The American Journal of Hygiene. 27, (1938).</li>
<li>Laurie, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((International+Laboratory+Comparison+of+Influenza+Microneutralization+Assays+for+A%28H1N1%29pdm09%2C+A%28H3N2%29%2C+and+A%28H5N1%29+Influenza+Viruses+by+CONSISE%5BTitle%5D)+AND+%22Clinical+Vaccine+Immunol+%3A+CVI%22%5BJournal%5D)">International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE.</a> Clinical Vaccine Immunol : CVI. 22, 957-964 (2015).</li>
<li>Lin, Y., Gu, Y., McCauley, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+a+Quantitative+Micro-neutralization+Assay%5BTitle%5D)+AND+%22J+Vis+Exp+%3A+JoVE%22%5BJournal%5D)">Optimization of a Quantitative Micro-neutralization Assay.</a> J Vis Exp : JoVE. , (2016).</li>
<li>van Baalen, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ViroSpot+microneutralization+assay+for+antigenic+characterization+of+human+influenza+viruses%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.</a> Vaccine. 35, 46-52 (2017).</li>
<li>WHO. Recommended composition of infleunza virus vaccines for use in the 2015-2016 northern heamisphere infleunza season. , 
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</ol>

저자는 충돌의 관심을 보고. 결과 및 결론을이 책에서 저자는 고 반드시 센터의 질병 통제 및 예방 및 자금 지원 기관에 대 한 대표 하지 않습니다.

MN 분석 결과 인플루엔자 혈 청 학에 대 한 인플루엔자 감염이 나 예방 접종을 다음과 같은 항 체 응답을 검색 하는 데 사용 하는 주요 분석 실험 중 하나입니다. 많은 인플루엔자 seroepidemiology 연구의 주요 결과 titers MN 분석 실험에서 생성 된 자주 사용 됩니다. MN 분석 sero 진단 및 백신 immunogenicity의 평가 또한 널리 사용 됩니다. 국제 간 연구소 연구14여러 실험실 수행 하는 미네소?...

인플루엔자 바이러스는 인간의 인구 매년 사망률 및 사망 원인을 계속. 하는 인플루엔자 바이러스의 주요 표면 당단백질. HA를 대상으로 하는 중화 항 체는 인플루엔자 감염1,2의 보호와 상관 관계가 주요 면역 메커니즘입니다. Hemagglutination 억제 (HI) 분석 실험 및 MN 분석 널리 인플루엔자 감염이 나 예방 접종3후 인간의 혈 청에 항 체 응답을 측정 하는 데 사용 하는 두 가지 방법이 있습니다. 이 분석 결과 붉은 혈액 세포의 바이러스 hemagglutination의 항 체 억제를 측정 하 고 분석 결과 대리 하는 것으로 간주 됩니다. 안녕, 달리 미네소타 분석 결과 직접 인간의 세라 셀 문화에서 인플루엔자 감염을 중화 하는 항 체의 수치를 측정할 수 있습니다. MDCK 셀 인플루엔자 바이러스 분리에 자주 사용 하 고 미네소타 분석 실험4.
인플루엔자 바이러스는 지속적으로 antigenic 드리프트와 shift, 바이러스의 수용 체 의무적인 특이성을 변경할 수 있는 HA 단백질에 돌연변이 인수 받 다. 2014 년 이후 A(H3N2) 바이러스의 새로운 클러스터 등장 하 고 현재 시즌까지 회람 하기 위하여 계속 했다. 이 바이러스의 대부분은 유전 그룹 3C.2a와 3C.3a HA 단백질의 계통 발생 분석에 기반에 속한다. 순환 3C.2a 바이러스의 많은 hemagglutinate 붉은 혈액 세포 수를 감소 했다 고이 분석 실험5에 의해 특성화 될 수 없습니다. 중립화 분석6hemagglutinate 하지 않는 이러한 바이러스에 항 체 응답을 측정 하기 위해 사용 되어야 한다. 또한, 연구는 이러한 현대 A(H3N2) 바이러스 수용 체 바인딩 속성 이전 A(H3N2) 바이러스에 비해 변경 및 문화 적응 돌연변이 및 다형성 체 외에서 세포에 passaged 때 축적 하는 경향이 나타났습니다. 문화7,,89. 기존의 MDCK 세포와 비교 하면, MDCK-SIAT1는 Matrosovich 그 외 여러분 에 의해 개발 된 셀 라인 인간의 α2, 6-sialtransferase (SIAT1)의 cDNA와 MDCK 세포의 안정 되어 있는 transfection을 통해 이 셀 라인 α2, 6 sialic 갈 락 토스 moieties 및 α2, MDCK 셀10부모 보다 3 sialic acid moieties 감소 금액의 증가 양을 표현 한다. MDCK-SIAT1 세포 MDCK 세포11에 비해 A(H3N2) 바이러스에 대 한 격리 속도 향상 시킬 나타났습니다. 최근, 린 외. 보고는 새로 등장 3C.2a 및 3C.3a 인간 A(H3N2) 인플루엔자 바이러스, 더 충실 한 바이러스 복제와 더 나은 바이러스 infectivity 달성 했다 바이러스 MDCK SIAT1 셀 라인 MDCK 셀7와 비교에 교양 있었다. 따라서, MDCK-SIAT1 세포는 더 미네소타 A(H3N2) 바이러스의 최근 클러스터에 대 한 항 체 응답 특성 분석에 적합 합니다.
여기 현대 3C.2a 및 3C.3a A(H3N2) 바이러스 인간 혈 청에 항 체 응답을 측정 하기 위해 MDCK SIAT1 셀을 사용 하 여 MN 분석 결과 설명 합니다. 이 분석 결과에 계란 또는 세포에서 바이러스를 사용할 수 있습니다. 열 비활성화 세라 중화 항 체를 포함 하는 먼저 순차적으로 희석, 다음 100 TCID50/인플루엔자 바이러스에 바인딩할 세라에 항 체를 수 있도록 A(H3N2)의 인 큐베이 팅. MDCK-SIAT1 셀 다음 바이러스 항 체 혼합물에 추가 하 고 37 ° C, 5% CO2 A(H3N2) 바이러스 MDCK SIAT1 세포를 감염 하 고 복제를 허용 하도록에서 18-20 h에 대 한 인 큐베이 팅. 야간 보육, 후 판을 고정 하 고는 ELISA 항 인플루엔자 A NP 단일 클론 항 체를 사용 하 여 각 우물에서 바이러스의 양을 계량은. NP의 감지 바이러스 감염의 존재와 항 체를 중화의 부재를 나타냅니다. 중화 항 체 titer 바이러스 infectivity ≥ 50% 억제를 제공 하는 높은 혈 청 희석의 상호로 정의 됩니다.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM) with high Glucose</td><td>Life Science</td><td>11965</td><td>A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) Fraction V, Protase Free</td><td>Sigma-Aldrich</td><td>3117332001</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Science</td><td>25030-081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Science</td><td>11360-070</td><td></td></tr><tr><td>Geneticin G-418 disulfate salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A1720-5G&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>HEPES</td><td>Life Science</td><td>15630-080</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Science</td><td>15140-122</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>67-64-1</td><td>Used at an 80% concentration</td></tr><tr><td>Phosphate-Citrate Buffer with Sodium Perborate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SLBF2806V</td><td></td></tr><tr><td>O-Phenylenediamine Dihydrochloride tablet</td><td>Sigma-Aldrich</td><td>SLBQ1086V</td><td>1 tablet per 100ml of cell culture grade water</td></tr><tr><td>Sulfuric Acid</td><td>Fisher Scientific</td><td>A510-P500</td><td>Used 0.5M final concentration</td></tr><tr><td>Ethanol, Denatured, 4L</td><td>VWR</td><td>EM-AX0422-3</td><td>Used at an 70% concentration</td></tr><tr><td>Trypsin-EDTA</td><td>Life Science</td><td>1748048</td><td></td></tr><tr><td>RDE II &quot;Seiken&quot;</td><td>Denka Seiken</td><td>370013</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379-500ml</td><td></td></tr><tr><td>Anti-NP mouse monoclonal Ab</td><td>Millipore pool</td><td>MAB 8257 MAB 8258</td><td></td></tr><tr><td>Anti-mouse IgG HRP&amp;nbsp;</td><td>KPL</td><td>074-1802</td><td></td></tr><tr><td>96-well flat-bottom plates</td><td>Thermo Scientific</td><td>3455</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Device</td><td>Spectromax 384 plus</td><td></td></tr><tr><td>Cell Culture Flask 162 cm2/Vent Cap</td><td>Corning/VWR</td><td>3151</td><td></td></tr></tbody></table>

measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells

조류 독감 바이러스 (HA)에 대 한 중화 항 체는 인플루엔자 감염에 대 한 보호와 상관 관계가 주요 면역 메커니즘으로 간주 됩니다. Microneutralization (미네소타) 분석 실험 자주 독감 예방 접종 또는 감염 후 인간의 혈 청의 중화 항 체 응답을 측정 하는 데 사용 됩니다. Madine 다 비 개 신장 (MDCK) 세포는 미네소타 분석 실험에 대 한 일반적으로 사용 되 셀 기판. 그러나, 현재 3C.2a 및 3C.3a A(H3N2) 인플루엔자 바이러스 획득 순환 수용 체 바인딩 특이성 변경. 표면에 증가 된 α-2, 6 sialic 갈 락 토스 moieties로 MDCK SIAT1 셀 라인은 이러한 현대 A(H3N2) 바이러스에 대 한 향상 된 infectivity 기존의 MDCK 셀 보다 더 충실 한 복제를 제공을 입증 했다. 여기, 우리는 미네소타 분석 결과 MDCK SIAT1 셀을 사용 하 여 이러한 현대 A(H3N2) 바이러스 중화 항 체 titers 계량 하도록 최적화 된 설명 합니다. 이 프로토콜 열 비활성화 세라 중화 항 체를 포함 하는 먼저 순차적으로 희석, 다음 100 TCID50/인플루엔자 바이러스에 바인딩할 세라에 항 체를 수 있도록 A(H3N2)의 인 큐베이 팅. MDCK-SIAT1 셀 다음 바이러스 항 체 혼합물에 추가 하 고 37 ° C, 5% CO2 A(H3N2) 바이러스 MDCK SIAT1 세포를 감염 시킬 수 있도록에서 18-20 h에 대 한 인 큐베이 팅. 하룻밤 배양 후 플레이트 고정 되 고 각 바이러스의 양을 잘 효소 연결 된 immunosorbent 분석 결과 (ELISA) 항 인플루엔자를 사용 하 여 계량은 nucleoprotein (NP) 단일 클론 항 체. 중화 항 체 titer 바이러스 infectivity의 50% 억제를 제공 하는 높은 혈 청 희석의 상호로 정의 됩니다.

바이러스 주식의 infectivity의 결정만 분석 결과 첫 번째 단계입니다. 그림 2 에서는 접시 레이아웃을 바이러스 주식의 TCID50 결정을 보여 줍니다. 알 수 없는 infectivity 바이러스 주식, 대 한 바이러스 바이러스의 infectivity 계산을 최고의 적정 곡선을 캡처하기 위해 여러 사전 희석, 예 10-2 와 10-3에서 적정 수 있습니다. MN 분석 결?...

Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

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

인플루엔자 중화 항 체는 인플루엔자 감염의 보호와 연관. Microneutralization 분석 실험 인간 혈 청의 중화 항 체를 측정 하 고 자주 독감 인간의 혈 청 학에 대 한 사용 됩니다. 인플루엔자 예방 접종 또는 감염 다음 현대 3C.2a 및 3C.3a A(H3N2) 바이러스 중화 항 체 titers 측정 MDCK-SIAT1 세포를 사용 하 여 microneutralization 분석 결과 설명 합니다.

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 ...

measuring-influenza-neutralizing-antibody-responses-to-h3n2-viruses

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

18.0K 조회수.  Centers for Disease Control and Prevention. 인플루엔자 중화 항 체는 인플루엔자 감염의 보호와 연관. Microneutralization 분석 실험 인간 혈 청의 중화 항 체를 측정 하 고 자주 독감 인간의 혈 청 학에 대 한 사용 됩니다. 인플루엔자 예방 접종 또는 감염 다음 현대 3C.2a 및 3C.3a A(H3N2) 바이러스 중화 항 체 titers 측정 MDCK-SIAT1 세포를 사용 하 여 microneutralization 분석 결과 설명 합니다.

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

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 ...

연구

면역학 및 감염병학

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

정량적 마이크로 중화 분석의 최적화

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

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 ...

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

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

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 ...

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

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Calcium Phosphate transfection 방법을 이용한 pseudo-typed H5 조류 인플루엔자 바이러스의 제조 및 항체 중화 활성 측정

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

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 ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

인플루엔자 뉴라미니다아제 억제 항체 역가를 측정하기 위한 분석

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...

Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

Source: Lin, Y., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54897">Optimization of a Quantitative Micro-neutralization Assay.</a> J. Vis. Exp. (2016)
This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

이미지 기반 미세중화 분석을 사용한 바이러스 간의 항원 관계 분석

1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) ...