없음입니다.

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+seasonal+influenza+with+vaccines.+Recommendations+of+the+Advisory+Committee+on+Immunization+Practices--United+States,+2013-2014.">Prevention and control of seasonal influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices--United States, 2013-2014.</a> MMWR Recomm Rep. 62, 1-43 (2013).</li><li>Dominguez-Cherit, G., 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=Critically+Ill+patients+with+2009+influenza+A(H1N1)+in+Mexico.">Critically Ill patients with 2009 influenza A(H1N1) in Mexico.</a> JAMA. 302 (17), 1880-1887 (2009).</li><li>Fox, B. 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=Pandemic+influenza+(H1N1):+impact+on+lung+transplant+recipients+and+candidates.">Pandemic influenza (H1N1): impact on lung transplant recipients and candidates.</a> J Heart Lung Transplant. 29 (9), 1034-1038 (2010).</li><li>Piercy, J., Miles, A., Krankheiten, S. B. f. G. S. V., Values, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Economic Impact of Influenza in Switzerland: Interpandemic Situation. , (2003).</li><li>Barclay, V. 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=Positive+network+assortativity+of+influenza+vaccination+at+a+high+school:+implications+for+outbreak+risk+and+herd+immunity.">Positive network assortativity of influenza vaccination at a high school: implications for outbreak risk and herd immunity.</a> PLoS One. 9 (2), 87042 (2014).</li><li>Baluch, 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=Randomized+controlled+trial+of+high-dose+intradermal+versus+standard-dose+intramuscular+influenza+vaccine+in+organ+transplant+recipients.">Randomized controlled trial of high-dose intradermal versus standard-dose intramuscular influenza vaccine in organ transplant recipients.</a> Am J Transplant. 13 (4), 1026-1033 (2013).</li><li>Haralambieva, I. 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=The+Impact+of+Immunosenescence+on+Humoral+Immune+Response+Variation+after+Influenza+A/H1N1+Vaccination+in+Older+Subjects.">The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects.</a> PLoS One. 10 (3), 0122282 (2015).</li><li>Egli, 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=Vaccine+adjuvants--understanding+molecular+mechanisms+to+improve+vaccines.">Vaccine adjuvants--understanding molecular mechanisms to improve vaccines.</a> Swiss Med Wkly. 144, 13940 (2014).</li><li>O'Shea, D., Widmer, L. A., Stelling, J., Egli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+face+of+vaccination+in+immunocompromised+hosts.">Changing face of vaccination in immunocompromised hosts.</a> Curr Infect Dis Rep. 16 (9), 420 (2014).</li><li>Meulemans, G., Carlier, M. C., Gonze, M., Petit, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hemagglutination-inhibition,+agar+gel+precipitin,+and+enzyme-linked+immunosorbent+assay+for+measuring+antibodies+against+influenza+viruses+in+chickens.">Comparison of hemagglutination-inhibition, agar gel precipitin, and enzyme-linked immunosorbent assay for measuring antibodies against influenza viruses in chickens.</a> Avian Dis. 31 (3), 560-563 (1987).</li><li>Martins, T. B. <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+internal+controls+for+the+Luminex+instrument+as+part+of+a+multiplex+seven-analyte+viral+respiratory+antibody+profile.">Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.</a> Clin Diagn Lab Immunol. 9 (1), 41-45 (2002).</li><li>Webster, R., Cox, N., St&#246;hr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+Animal+Influenza+Manual.">WHO Animal Influenza Manual.</a> WHO/CDS/CSR/NCS. 2002.5, 1-99 (2002).</li><li>Mittelholzer, C. 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=Human+cell+lines+used+in+a+micro+neutralization+test+for+measuring+influenza-neutralizing+antibodies.">Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.</a> Scand J Immunol. 63 (4), 257-263 (2006).</li><li>Hamilton, R. S., Gravell, M., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+antibody+titers+determined+by+hemagglutination+inhibition+and+enzyme+immunoassay+for+JC+virus+and+BK+virus.">Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus.</a> J Clin Microbiol. 38 (1), 105-109 (2000).</li><li>Kumakura, 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=Comparison+of+hemagglutination+inhibition+assay+and+enzyme+immunoassay+for+determination+of+mumps+and+rubella+immune+status+in+health+care+personnel.">Comparison of hemagglutination inhibition assay and enzyme immunoassay for determination of mumps and rubella immune status in health care personnel.</a> J Clin Lab Anal. 27 (5), 418-421 (2013).</li><li>Ogundiji, O. T., Okonko, I. O., Adu, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+measles+hemagglutination+inhibiting+antibody+levels+among+school+children+in+Ibadan,+Nigeria.">Determination of measles hemagglutination inhibiting antibody levels among school children in Ibadan, Nigeria.</a> J Immunoassay Immunochem. 34 (2), 208-217 (2013).</li><li>Cwach, K. T., Sandbulte, H. R., Klonoski, J. M., Huber, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+murine+innate+serum+inhibitors+toward+interference+within+influenza+virus+immune+assay.">Contribution of murine innate serum inhibitors toward interference within influenza virus immune assay.</a> Influenza Other Respir Viruses. 6 (2), 127-135 (2012).</li><li>Lee, P. 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=Receptor+mimicry+by+antibody+F045-092+facilitates+universal+binding+to+the+H3+subtype+of+influenza+virus.">Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus.</a> Nat Commun. 5, 3614 (2014).</li><li>Blumel, 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=Age-related+prevalence+of+cross-reactive+antibodies+against+influenza+A(H3N2)+variant+virus,+Germany,+2003+to+2010.">Age-related prevalence of cross-reactive antibodies against influenza A(H3N2) variant virus, Germany, 2003 to 2010.</a> Euro Surveill. 20 (32), 16-24 (2015).</li><li>Reber, A. 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=Seasonal+Influenza+Vaccination+of+Children+Induces+Humoral+and+Cell-Mediated+Immunity+Beyond+the+Current+Season:+Cross-reactivity+with+Past+and+Future+Strains.">Seasonal Influenza Vaccination of Children Induces Humoral and Cell-Mediated Immunity Beyond the Current Season: Cross-reactivity with Past and Future Strains.</a> J Infect Dis. , (2016).</li></ol>

A.E.에 의해 지원 되었다 연구 보조금 (PZ00P3_154709), "SNSF Ambizione 점수" 프로그램에서 "Forschungsfond, Förderung strategischer Projekte" 바젤의 대학, Stiftungsinfektionskrankheiten 바젤, 및 Bangeter Rhyner 재단. L.K. 기술 대학의 그라츠, 오스트리아의 교부 금에 의해 지원 되었다. : J.L. 시스템 생물학 프로그램 (9번째 전화)에서 SystemsX.ch 이니셔티브의 iPhD 친교에 의해 지원을 인정합니다.

사전 및 사후 예방 접종 독감 바이러스 titers 특정 항 체의 정량화 백신 연구에 필요한 중요 한 도구입니다. Seroprotection 같은 바이러스 감염에 대 한 보호의 대리 측정에 따라 (> 1시 40분) 또는 혈 (4-fold titer 증가), 예방 접종 전략 수9에 최적화 된. 제공 된 프로토콜을 사용 하 여 확인할 수 있습니다: (i) 특정 바이러스, 및 (ii) 항 체 titers 관심의 바이러스에 대 한 hemagglutination 잠재력....

인플루엔자 바이러스 감염 상당한 질병 률, 사망률, 및 높은 의료 비용1,2,,34와 연결 됩니다. 특히, 신생아, 임산부, 노인과 만성 질병을 가진 환자는 더 심한 임상 결과 대 한 위험이 있습니다. 따라서, 순환 하는 인플루엔자 바이러스 변종에 대 한 예방 접종이 고 위험도 인구에 있는 질병의 부담을 줄이기 위하여 기본 척도 이다. 예방 접종, 예를 들면, 보호 임계값 이상 인플루엔자-특정 항 체 후 개별 면역 반응의 증가 감염의 개별 위험과 일반 인구 내에서 바이러스 성 전송의 가능성을 감소 5. 백신 유도 체액 면역 반응의 다른 인구에 다양 한 연령층에 걸쳐 자세한 이해는 중요 한 임상 질문6,,78 대답 하는 핵심 요소 , 9와 같은: 왜 일부 노인 환자 이전 예방 접종에도 불구 하 고 감염을 해야 합니까? "좋은"와 "충분 한" 백신 유도 보호는 무엇입니까? 얼마나 자주 백신 보호 titers 도달 immunosuppressed 환자에 게 적용 한다? 가장 효과적인 복용량 무엇입니까? 예방 접종 후 항 체 titers에 소설 보조의 영향 이란 무엇입니까? 백신 관련 항 체 생산의 측정 이러한 중요 한 질문에 대답 하 고 예방 접종 결과 개선 하는 데 도움이 있습니다.
바이러스 특정 항 체 titers의 정량화는 다양 한 면역 방법으로 수행할 수 있습니다. 이 고체 상10 또는 비드 기반 ELISA11 분석 실험,이 분석 결과12및 중화 분석 실험13포함 됩니다. ELISA 기반 메서드는 상대적으로 많은 양의 다양 한 항 원에 대 한 혈 청 샘플의 검사를 수 있습니다. 또한, 병원 체 관련 면역 글로불린 (Ig) M와 IgG 수 수 별도로 탐험. 항 원, 예를 들면, 선형 아미노산 시퀀스 또는 바이러스와 같은 입자의 특성 항 체의 바인딩을 영향을 미칠 수 있습니다, 비록 잠재적인 epitopes의 스펙트럼은 매우 광범위 하 고 여부 항 체에 정보를 제공 하지 않습니다. 응답 기능 관련이 있다.
반면, 중화 시험 기능 세포의 감염을 억제 하는 항 체의 잠재력을 결정 하 고 따라서 잠재적인 중립화를 반영 한다. 그러나,이 방법은 매우 노동 집약 이다, 라인 세포 및 바이러스, 라이브 특정의 경작 그리고 그러므로, 그것은 소모, 비싼, 특별 한 장비가 필요 합니다 필요 합니다.
이 문서에서는 인플루엔자-특정 항 체 titers 척도를 세계 보건 기구 WHO 기반이 프로토콜12 는 단계별 설명. Hemagglutination은 적혈구의 교착을 선도 하는 일부 바이러스의 특성 효과 이다. 환자 혈 청이이 효과의 저해 중화 효과 반영 하는 억제 항 체 농도의 측정을 허용 한다.
우리는 동시에 여러 샘플의 효율적 처리 및 필요한 시간을 줄일 수 있도록 WHO 프로토콜의 워크플로 수정 했습니다. 첫 번째 프로토콜 특정 인플루엔자 항 원의 교착 잠재력의 결정을 설명합니다. 이 과정에서 두 번째 프로토콜에 대 한 올바른 인플루엔자 항 원 농도 결정 됩니다. 이 부분은 혈액의 각 배치 뿐 아니라 모든 새로운 바이러스 성 항 원, 반복 한다.
두 번째 프로토콜 인플루엔자-특정 항 체 titers 결정을 설명합니다. 그러나 제시 프로토콜 인플루엔자 바이러스와 인간 혈 청 샘플의 수사를 위해 최적화 된, 그것은 또한 적용할 수 마우스 혈 청 샘플 또는 셀 문화 supernatants 자극된 면역 세포, 예를 들어, 바이러스 특정 B 세포에서. 절대 측정된 titers로 결과 확인할 수 있습니다. 많은 백신 연구,은 titers 및 95% 신뢰 구간에는 각 특정 인구 표시 됩니다. 해석, seroprotection 또는 혈에 대 한 특정 바이러스 인구의 민감성을 설명 하기 위해 자주 사용 됩니다. Seroprotection 이상 4-fold titer 두 시간 점 (가장 일반적으로 사전 예방 접종과 30 일 사후 예방 접종에 사용 되는) 사이 seroprotective titers 달성과 증가의 ≥1:40, 그리고 혈 titer로 정의 됩니다.
두 프로토콜은 사용 하기 쉽게 하 고 그들은 연구 질문의 광범위 한 범위에 적용할 수 있습니다. 특히, 안정적이 고 신속 하 게 결정 하기 위해 사용할 수 있습니다 hemagglutination, 홍 역, polyomaviruses, 유행 성 이하선염, 또는 풍 진14,15,16 등에 대 한 용량을 가진 다른 다양 한 바이러스에 대 한 항 체 titers .

<table><tbody><tr><td>25 ml Disposable Multichannel Pipette Reservoirs</td><td>Integra</td><td>4312</td><td></td></tr><tr><td>8-well PCR tubes</td><td>Brand GMBH</td><td>781332</td><td>For serum aliquots</td></tr><tr><td>96-well microtiter plate, U-shaped</td><td>TPP</td><td>92097</td><td>For HI assay when using mammalian RBCs</td></tr><tr><td>96-well microtiter plate, V-shaped</td><td>Corning Costar</td><td>3897</td><td>For HI assay when using avian RBCs</td></tr><tr><td>Aqua ad iniect. Steril</td><td>Bichsel AG</td><td>1000004</td><td>For preparing influenza antigen and cholera filtrate solutions</td></tr><tr><td>Chicken RBC (10%)</td><td>Cedarlane</td><td>CLC8800</td><td>10% suspension of chicken red blood cells in Alsever&#039;s solution</td></tr><tr><td>Cholera filtrate</td><td>Sigma-Aldrich</td><td>C8772</td><td>Used as receptor destroying enzyme (RDE)</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>For diluting the serum samples, RBCs and antigens</td></tr><tr><td>Eppendorf Multichannel pipette, 12-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683949</td><td></td></tr><tr><td>Eppendorf Multichannel pipette, 8-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683930</td><td></td></tr><tr><td>Guinea Pig RBC (10%)</td><td>Cedarlane</td><td>CLC1800</td><td>10% suspension of guinea pig red blood cells in Alsever&#039;s solution</td></tr><tr><td>Influenza Anti-A/California/7/09 HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/134&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Switzerland/9715293/2013-like HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/272</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Texas/50/2012-Like HA Serum&amp;nbsp;</td><td>NIBSC</td><td>13/178</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Brisbane/60/2008-HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/254&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Massachusetts/02/2012 HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/182</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza antigen A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp;</td><td>NIBSC</td><td>12/168</td><td>Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp; virus (ca. 46&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Switzerland/9715293/2013 (NIB88)</td><td>NIBSC</td><td>14/254</td><td>Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223)</td><td>NIBSC</td><td>13/112</td><td>Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Brisbane/60/2008</td><td>NIBSC</td><td>13/234</td><td>Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Massachusetts/02/2012</td><td>NIBSC</td><td>13/134</td><td>Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35&amp;micro;gHA/ml)</td></tr><tr><td>Serum-Tubes</td><td>S-Monovette, Sardstedt</td><td>01.1601.100</td><td>For serum extraction with clotting activator</td></tr><tr><td>Single Donor Human RBC, Type 0</td><td>Innovative Research</td><td>IPLA-WB3&amp;nbsp;</td><td>Suspension of single donor human red blood cells in Alsever&#039;s solution (ca. 26%)</td></tr><tr><td>Turkey RBC (10%)</td><td>Cedarlane</td><td>CLC1180</td><td>10% suspension of turkey red blood cells in Alsever&#039;s solution</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td></td><td></td></tr></tbody></table>

an optimized hemagglutination inhibition (hi) assay to quantify influenza-specific antibody titers

항 체 titers 인플루엔자와 다른 병원 체에 대 한 혈 청 학적인 보호에 대 한 대리 표식으로 일반적으로 사용 됩니다. 사전 및 사후 예방 접종 항 체 생산의 상세한 지식은 백신 유도 면역을 이해 해야 합니다. 이 문서에서는 인플루엔자-특정 항 체 titers를 확인 하는 신뢰할 수 있는 포인트 프로토콜을 설명 합니다. 첫 번째 프로토콜 hemagglutination, 두 번째 프로토콜 (hemagglutination 분석 결과, 분석 결과 HA)에 후속 사용 농도 표준화에 필요한 항 원 금액을 지정 하는 방법을 설명 합니다. 두 번째 프로토콜 인간의 혈 청 또는 셀 문화 supernatants (hemagglutination 저해 분석 결과,이 분석 결과)의 직렬 희석을 사용 하 여 다른 바이러스 성 긴장에 대 한 인플루엔자-특정 항 체 titers의 정량화를 설명 합니다.
적용 예를 들어, 우리는 trivalent inactivated 인플루엔자 백신을 접종 하는 건강 한 코 호트의 항 체 응답을 보여줍니다. 또한, 다른 인플루엔자 바이러스 사이의 분 표시 되 고 동물 적혈구 (Rbc)의 다른 종류를 사용 하 여 분을 최소화 하는 방법을 설명 했다. 토론 하이라이트 장점과 제시 분석의 단점 및 어떻게 인플루엔자-특정 항 체 titers 결정 백신 관련 면역의 이해를 향상 시킬 수 있습니다.

인플루엔자 A H3N2에 대 한 사전 및 사후 예방 접종 유도 항 체 응답 백신 유발 항 체 응답 26 건강 한 자원 봉사자는 사 3가 소 단위 독감 백신을 접종, 포함 된 인플루엔자 A/H1N1/캘리포니아/2009 A/H3N2/텍사스/2012, B/매사 추세 츠/02/2012는 2014 이전에 평가 되었다 / 2015 독감 시즌입니다. 그림 6 2 백신 수령인의 대표적인 예를 보여 줍니다. 흥미...

Watch this Scientific Journal Video about An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers at JoVE.com

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

제시 프로토콜 인플루엔자 백신 받는 사람 혈 청 샘플에서 인플루엔자-특정 항 체 titers 척도를 hemagglutination 억제 시험을 수행 하는 방법을 설명 합니다. 첫 번째 분석 결과 hemagglutination에 의해 최적의 바이러스 성 항 원 농도를 결정합니다. 두 번째 분석 결과 hemagglutination 저해 하 여 인플루엔자-특정 항 체 titers 단정.

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

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

35.8K 조회수.  University of Basel. 제시 프로토콜 인플루엔자 백신 받는 사람 혈 청 샘플에서 인플루엔자-특정 항 체 titers 척도를 hemagglutination 억제 시험을 수행 하는 방법을 설명 합니다. 첫 번째 분석 결과 hemagglutination에 의해 최적의 바이러스 성 항 원 농도를 결정합니다. 두 번째 분석 결과 hemagglutination 저해 하 여 인플루엔자-특정 항 체 titers 단정.

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

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

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

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 ...

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 ...

<meta charset="utf8"></head><body>정확한 NDV 항체 검출을 위한 간소화된 4-HAU 항원 준비

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

<meta charset="utf8"></head><body>뉴캐슬 질병 바이러스 혈청학의 혈구 응집 억제 분석을 위한 항원 준비의 최적화

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

뉴캐슬 질병 바이러스 혈청학의 혈구 응집 억제 분석을 위한 항원 준비의 최적화

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article ...

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 ...

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

요약

더 많은 비디오 탐색

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

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

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

인플루엔자 바이러스 단일 클론 항 체를 중화의 탈출 변종의 생성

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

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

뉴캐슬 질병 바이러스 혈청학의 혈구 응집 억제 분석을 위한 항원 준비의 최적화

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