Clarissa Curioso was supported through an appointment to the Research Participation Program at the U.S. Environmental Protection Agency administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA.

승인 (IRB # 08-1844, 노스 캐롤라이나, 채플 힐, 노스 캐롤라이나, 미국의 대학) 케론 비치, 푸에르토 리코,에서 beachgoers에서 자극 열구 타액 샘플의 수집에 대한 미국의 일환으로 임상 시험 심사위원회로부터 얻은 것 환경 보호청 (USEPA) 국립 역학 및 레크리에이션 (NEEAR) 물 연구 (23)의 환경 평가 수영 관련된 노출과 질병을 평가합니다. 연구 주제는 동의를 제공하고 훈련 USEPA 계약에 의해 타액 수집 장치의 사용에 지시했다. 타액 샘플을 수신하면, 그들이 원심 분리 4 바와 같이 -80 ° C에 저장된 얼음에 제공 하였다. 1. 구슬 활성화 <ol><li> 텍싱 20 초 동안 초음파 처리 및 마이크로 원심 튜브에 주식 비드 (400 μL)의 전송 약 5.0 × 10 (6)에 의해 재현 탁 비드 세트 주식. 참고 : T를그는 12.5 × 106 비즈 / ml의 농도로 공급된다 구슬. </li><li> 2 분 동안 10,000 XG에서 원심 분리하여 주식 비드 펠렛. </li><li> 20 초 동안 소용돌이와 초음파에 의해 증류수 (DH 2 O)를 뜨는을 제거하고 100 μL의 펠렛 비드를 재현 탁. 단계를 반복 1.2. </li><li> 상층 액을 제거하고 20 초 동안 소용돌이와 초음파 100 mM의 인산 나트륨 염기, 산도 6.2의 80 μL에 세척 된 비드를 재현 탁. </li><li> 즉시 사용하기 전에, 10 mg의 설포 NHS의 나누어지는 200 μL의 DH 2 O를 추가하여 50 ㎎ / ㎖ N -hydroxysulfosuccinimide (설포 NHS) 솔루션을합니다. 소용돌이로 섞는다. </li><li> 구슬에 50 ㎎ / ㎖ 설포 NHS의 10 μl를 추가합니다. 소용돌이로 섞는다. </li><li> 사용 직전에, 10 mg을 EDC 분취 200 μL를 증류수 (DH 2 O)을 첨가하여 50 ㎎ / ㎖ -1- 에틸 [3dimethylaminopropyl] 카르 보디이 미드 히드로 클로라이드 (EDC) 용액을 만든다. 소용돌이로 섞는다. </li><li> 50의 10 μl를 추가구슬로 ㎎ / ㎖ EDC 솔루션입니다. 소용돌이로 섞는다. 10 분 간격으로 와류에 의해 혼합하여, 어두운 곳에서 실온에서 20 분 동안 비드 부화. 2 분 동안 10,000 XG에서 microcentrifugation에 의해 활성화 된 구슬 펠렛. </li><li> 상층 액을 제거하고 20 초 동안 소용돌이와 초음파에 의해 50 mM의 2- [N -Morpholino] 에탄 설 폰산 (MES), 산도 5.0의 250 μL의 비드를 재현 탁. </li><li> 2 분 동안 10,000 XG에서 microcentrifugation에 의해 활성화 된 구슬 펠렛. </li><li> 반복 1.9 및 1.10 단계를 반복합니다. 참고 :이 50 MM의 MES, pH가 5.0이 세척의 총을 제공합니다. </li><li> 20 초 동안 소용돌이와 초음파 50 MM의 MES, pH 5.0으로 100 ㎕의 구슬을 재현 탁. </li></ol> 2. 구슬 커플 링 <ol><li> 몇 표 1에 도시 된 농도를 사용하여 비드 세트 항원. </li><li> 활성화 된 구슬 각 항원을 추가하고 50 MM의 MES, pH 5.0으로 500 μL에 총 볼륨을 가지고. 항원을 혼합소용돌이로 D 구슬. </li><li> 어둠 속에서 실온에서 회전 (~ 15 RPM)로 혼합하여 2 시간 동안 항원과 구슬을 품어. 2 분 동안 10,000 XG에서 microcentrifugation에 의해 결합 된 구슬 펠렛. </li><li> 상층 액을 제거하고 인산염 완충 생리 식염수 500 μL에 구슬을 재현 탁 (PBS) -bovine 혈청 알부민 (BSA) -polyoxyethylenesorbitan 모노 라우 레이트 (트윈 20) 소디움 아 지드 (PBS-TBN) 와류 및 초음파에 의해 pH가 7.4. 2 분 동안 10,000 XG에서 microcentrifugation으로 구슬을 펠렛과 뜨는을 제거합니다. 주의 : 아 지드 화 나트륨은 급성 독성 화학 물질이다. 섭취 또는 피부에 접촉에 들어갔을 경우는 치명적이다. 먼지 / 연기 / 가스 / 안개 / 증기 나 분무를 흡입하지 말 것. 적절한 개인 보호 장비 (PPE의) 처리 할 때 착용하고 적절한 법률에 따라 폐기. </li><li> 20 초 동안 소용돌이 및 초음파에 의해 PBS-TBN 1 ㎖의 구슬을 재현 탁. </li><li> 2 분 동안 10,000 XG에서 microcentrifugation으로 구슬을 펠렛. </li><li>반복 2.5 및 2.6 단계를 반복합니다. 참고 :이 PBS-TBN 두 세척 총을 제공합니다. </li><li> PBS 1 ml의 1 % BSA, 0.05 % 아 지드, pH 7.4의 결합에 세정 비드를 재현 탁. 어둠 속에서 2-8 ° C 형 냉장고에 결합 된 구슬을 저장합니다. </li></ol> 3. 구슬 개수 <ol><li> 물 또는 PBS 버퍼에 결합 된 구슬의 1:10 희석을 준비합니다. </li><li> 로드 샘플 도입 지점에서 혈구 상 비드 희석액 10 μL. </li><li> 4 × 4 코너 그리드 중 하나에서 볼 구슬을 계산합니다. 카운트 (4 × 4 격자의 모서리 1) × (1 × 104) × (희석 배수) 용액에 재현 탁 X 용적은 다음 식을 이용하여 비드에 결합의 총 수를 계산한다. </li></ol> 항원 커플 링 4. 확인 <ol><li> 20 초 동안 소용돌이와 초음파에 의해 관심의 항원에 결합 된 구슬의 주식 혼합물을 재현 탁. </li><li> 마지막으로 결합 된 비드 주식을 희석하여 작업 비드 혼합물을 제조PBS-1 % BSA 완충액 (PBS-1 % BSA, pH 7.4의)에서 설정 한 각 고유 비드 / 100 ㎕의 농도 비드. </li><li> 최소 7 PBS-1 % BSA 버퍼 96 웰 둥근 바닥 플레이트 제조업체의 권장 사항에 따라 IgG의 일차 항체 방지 종의 연속 희석을 두 배 준비합니다. </li><li> 세척 완충액 100 ㎕ 각 항원 결합 확인 시험 96 웰 필터 바닥 판의 별도의 8 잘 칼럼 (8 행) 사전 습식 진공에 의해 뜨는을 제거합니다. 사전 젖은 우물에 비드 혼합물 (항원 - 결합 구슬) 작업의 50 μl를 추가합니다. </li><li> 배경 우물 역할을 희석 ​​항체 대신에 팔을 행하기 위해 96 웰 필터 플레이트의 각 컬럼의 행 1-7과 PBS-1 % BSA 버퍼의 50 μL에 항체 희석의 50 μl를 추가합니다. 로 pipetting하여 5 회 다운 멀티 채널 피펫으로 혼합한다. 모든 항원 - 결합 된 비드 세트에 대해 동일한 절차를 수행하는 확인한다. </li><li> 덮고 실온에서 어둠 속에서 부화 할 수 있도록500 rpm에서 마이크로 통에 1 시간 동안 erature. 진공에 의해 뜨는을 제거합니다. </li><li> 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 1 배를 반복합니다. 멀티 채널 피펫에 PBS-1 % BSA 50 μL의 비드를 재현 탁. </li><li> PBS-1 % BSA에서 16 μg의 / ㎖ 비 오티 닐화 항에 종에게 특정 IgG를 이차 항체 검출을 희석. </li><li> 아래로 5 회를 피펫 팅에 의해 각 웰에 희석 차 항체의 50 μl를 추가합니다. </li><li> 필터 판을 포함하고, 플레이트 쉐이커상에서 실온에서 30 분 동안 어둠 속에서 배양 할 수 있습니다. 진공에 의해 뜨는을 제거합니다. 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 세척 1 배를 반복합니다. </li><li> 멀티 채널 피펫에 PBS-1 % BSA 50 μL의 비드를 재현 탁. </li><li> PBS-1 % BSA 24 μg의 / ㎖로 스트렙 타비 딘-R-피코 에리 트린 기자 (SAPE)를 희석. </li><li> 각 기자의 50 μl를 잘 추가로 pipetting에 의해 5 배 아래로 섞는다. </li><li> 기음필터 플레이트 위에 것은 플레이트 쉐이커상에서 실온에서 30 분 동안 어둠 속에서 배양 할 수 있습니다. 진공에 의해 뜨는을 제거합니다. 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 세척 1 배를 반복합니다. </li><li> 재현 탁의 PBS-1 % BSA 100 ㎕ 구슬 및 분석기 (29)를 사용하여 50 μl를 분석합니다. 참고 : 비드 기반 멀티 플렉스 면역의 결과는 중간 형광 강도 (MFI)로 측정된다. 오류를 방지하기 위해 가능한 경우 항상 소프트웨어 설명서의 최신 버전을 참조하십시오. </li></ol> 5. 타액 멀티 플렉스 면역 <ol><li> -80 ° C 냉장고에서 침을 제거하고 실온에서 해동 할 수 있습니다. </li><li> 재현 탁 항원은 20 초 동안 소용돌이와 초음파에 의해 비드 주식을 결합. </li><li> PBS-1 % BSA 버퍼 내의 각각의 고유 한 세트의 비드 / 100 μl의 비드의 최종 농도로 결합 된 비드 주식 희석 작업 비드 혼합물을 제조한다. </li><li> 타액의 4 희석 w : 1 준비PBS-1 % BSA의 96에서 버퍼 아니라, 깊은 우물 판 번째. </li><li> 사전 젖은 필터 세척 버퍼 100 ㎕와 함께 접시 진공에 의해 뜨는을 제거합니다. </li><li> 8 최종 희석 작업 비드 혼합물 1의 96 웰 필터 플레이트의 95 우물에 희석 타액의 같은 부피의 50 μl를 추가합니다. 멀티 채널 피펫으로 반응을 섞는다. 물론 하나의 제어에 50 μl의 항원 결합 비즈 플러스 (타액의 대체 등) PBS-1 % BSA 버퍼의 50 μl를 추가합니다. </li><li> 커버 500 rpm에서 마이크로 플레이트 진탕 기에서 실온에서 1 시간 동안 어둠 속에서 배양 할 수 있습니다. 진공에 의해 뜨는을 제거합니다. 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 세척 1 배를 반복합니다. </li><li> 멀티 채널 피펫 터로 PBS-1 % BSA 50 μL에 재현 탁 구슬. </li><li> PBS-1 % BSA에서 16 μg의 / ml의 바이오틴 화 된 염소 항 - 인간 IgG 이차 항체 검출을 희석. </li><li> 각 웰에 차 항체를 희석하여 50 μl를 추가로 내용을 혼합멀티 채널 피펫. </li><li> 필터 판을 포함하고, 플레이트 쉐이커상에서 실온에서 30 분 동안 어둠 속에서 배양 할 수 있습니다. 진공에 의해 뜨는을 제거합니다. 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 세척 1 배를 반복합니다. </li><li> 멀티 채널 피펫 터로 PBS-1 % BSA 50 μL에 재현 탁 구슬. </li><li> PBS-1 % BSA 24 μg의 / ㎖로 스트렙 타비 딘-R-피코 에리 트린 기자 (SAPE)를 희석. </li><li> 각 50 μL 기자를 잘 추가하고 멀티 채널 피펫과 함께 섞는다. </li><li> 필터 판을 포함하고, 플레이트 쉐이커상에서 실온에서 30 분 동안 어둠 속에서 배양 할 수 있습니다. 진공에 의해 뜨는을 제거합니다. 세척 완충액 100 ㎕로 우물을 세척하고 진공에 의해 뜨는을 제거합니다. 세척 1 배를 반복합니다. </li><li> 재현 탁의 PBS-1 % BSA 100 ㎕ 구슬 및 분석기 (29)를 사용하여 50 μl를 분석합니다. 참고 : 다중 면역의 결과는 중간 형광 강도 (MFI) 단위로 측정된다. 항상 참조소프트웨어 설명서의 최신 버전은, 가능하면하는 오류를 방지 할 수 있습니다. </li></ol>

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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+and+booster+salivary+antibody+responses+to+a+7-valent+pneumococcal+conjugate+vaccine+in+infants.">Primary and booster salivary antibody responses to a 7-valent pneumococcal conjugate vaccine in infants.</a> J Infect Dis. 182 (4), 1260-1263 (2000).</li><li>Tourinho, 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=Importance+of+the+cutoff+ratio+for+detecting+antibodies+against+hepatitis+A+virus+in+oral+fluids+by+enzyme+immunoassay.">Importance of the cutoff ratio for detecting antibodies against hepatitis A virus in oral fluids by enzyme immunoassay.</a> J Virol Methods. 173 (2), 169-174 (2011).</li><li>Moorthy, M., Daniel, H., Kurian, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+saliva+as+an+alternative+to+plasma+for+the+detection+of+hepatitis+C+virus+antibodies.">An evaluation of saliva as an alternative to plasma for the detection of hepatitis C virus antibodies.</a> Indian J Med Microbiol. 26 (4), 327-332 (2008).</li><li>Moe, C., Sair, A., Lindesmith, L., Estes, M., Jaykus, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+norwalk+virus+infection+by+indirect+enzyme+immunoassay+detection+of+salivary+antibodies+to+recombinant+norwalk+virus+antigen.">Diagnosis of norwalk virus infection by indirect enzyme immunoassay detection of salivary antibodies to recombinant norwalk virus antigen.</a> Clin Diagn Lab Immunol. 11 (6), 1028-1034 (2004).</li><li>Yap, G., Sil, B., Ng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+saliva+for+early+dengue+diagnosis.">Use of saliva for early dengue diagnosis.</a> PLoS Negl Trop Dis. 5 (5), e1046 (2011).</li><li>Schramm, W., Angulo, G., Torres, P., Burgess-Cassler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+saliva-based+test+for+detecting+antibodies+to+human+immunodeficiency+virus.">A simple saliva-based test for detecting antibodies to human immunodeficiency virus.</a> Clin Diagn Lab Immunol. 6 (4), 577-580 (1999).</li><li>Chart, H., Perry, N., Willshaw, G., Cheasty, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+saliva+for+antibodies+to+the+LPS+of+Escherichia+coli+O157+in+patients+with+serum+antibodies+to+E.+coli+O157+LPS.">Analysis of saliva for antibodies to the LPS of Escherichia coli O157 in patients with serum antibodies to E. coli O157 LPS.</a> J Med Microbiol. 52 (Pt 7), 569-572 (2003).</li><li>Ashbolt, N., Bruno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+and+refinement+of+the+WHO+risk+framework+for+recreational+waters+in+Sydney,+Australia.">Application and refinement of the WHO risk framework for recreational waters in Sydney, Australia.</a> J Water Health. 1 (3), 125-131 (2003).</li><li>Westrell, T., Schonning, C., Stenstrom, T., Ashbolt, N. <a target="_blank" 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+model+to+assess+microbial+health+risks+associated+with+beneficial+uses+of+biosolids.">A dynamic model to assess microbial health risks associated with beneficial uses of biosolids.</a> Risk Anal. 24, 221-236 (2004).</li><li>Wade, T. 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=.">.</a> Report on 2009 National Epidemiologic and Environmental Assessment of Recreational Water Epidemiology Studies, US EPA. US EPA Report Number: EPA/600/R-10/168: US EPA2011. , (2011).</li><li>Fujii, Y., 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=Serological+surveillance+development+for+tropical+infectious+diseases+using+simultaneous+microsphere-based+multiplex+assays+and+finite+mixture+models.">Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.</a> PLoS Negl Trop Dis. 8, e3040 (2014).</li><li>Rota, M., Massari, M., Gabutti, G., Guido, M., De Donno, A., Ciofi degli Atti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measles+serological+survey+in+the+Italian+population:+interpretation+of+results+using+mixture+model.">Measles serological survey in the Italian population: interpretation of results using mixture model.</a> Vaccine. 26 (34), 4403-4409 (2008).</li><li>Vyse, A., Gay, N., Hesketh, L., Pebody, R., Morgan-Capner, P., Miller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+serological+surveys+using+mixture+models:+the+seroepidemiology+of+measles,+mumps+and+rubella+in+England+and+Wales+at+the+beginning+of+the+21st+century.">Interpreting serological surveys using mixture models: the seroepidemiology of measles, mumps and rubella in England and Wales at the beginning of the 21st century.</a> Epidemiol Infect. 134 (6), 1303-1312 (2006).</li><li>Baughman, 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=Establishment+of+diagnostic+cutoff+points+for+levels+of+serum+antibodies+to+pertussis+toxin,+filamentous+hemagglutinin,+and+fimbriae+in+adolescents+and+adults+in+the+United+States.">Establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the United States.</a> Clin Diagn Lab Immunol. 11 (6), 1045-1053 (2004).</li><li>Clotilde, L., Bernard, C., Hartman, G., Lau, D., Carter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbead-based+immunoassay+for+simultaneous+detection+of+Shiga+toxins+and+isolation+of+Escherichia+coli+O157+in+foods.">Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.</a> J Food Prot. 74 (3), 373-379 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Luminex User Software Manual (RUO) xPONENT 3.1 Rev. 2. 3, 6-102 (2014).</li><li>Waterboer, T., Sehr, P., Pawlita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+non-specific+binding+in+serological+Luminex+assays.">Suppression of non-specific binding in serological Luminex assays.</a> J Immunol Methods. 309 (1-2), 200-204 (2006).</li></ol>

연구 개발의 오피스를 통해 미국 환경 보호국 (EPA)이 자금을 지원하고 여기에 설명 된 연구 관리. 이 기관의 관리 검토를 실시하고 게시 승인되었습니다. 상품명 또는 상용 제품에 대한 언급은 사용 승인 또는 추천을 구성하지 않습니다.

이러한 결과는 멀티 플렉스 면역 방법은 면역 양성 또는 immunonegative 있습니다 타액 샘플을 구별하는 데 유용 있음을 나타냅니다. 의 면역 반응을 결정하기 위해, 하나의 차단 점 평균 플러스 타액 샘플 모두에서 테스트 제어 비결 비드 로그 변환 MFI 응답의 세 개의 표준 편차를 계산하여 개발되었다. 컷 - 오프 포인트는 단일 또는 다수의 병원체에 노출 immunoprevalence을 평가하는 능력을 수득 하였다....

설사 관련 질병의 88 %가 전 세계적으로 약 150 만 명이 사망 원인이 오염 된 물에 대한 인체 노출 안전하지 않은 음식, 그리고 가난한 위생 / 위생과 연관되어, 대부분의 어린이 1입니다. 이는 공중 보건 관리 및 정책 결정자에 대한 관심의 주요 원인이다. 수성 및 기타 환경과 관련된 병원체 노출과 질병을 조사하기 위해, 우리는 사람의 샘플 2-4에 대한 항체를 측정하는 다중 면역 분석을 개발 하였다. 이 방법은 이러한 병원체의 인체 노출을 결정하기 위해 더 나은 immunoprevalence 및 사고 감염을 정의하는 역학 연구에 적용 할 수 있습니다. 침은 인간의 바이오 마커 연구를위한 혈청의 대안으로 상당한 약속을 보유하고있다. 타액의 장점 중에서는 비 침입 및 샘플 수집, 저비용의 용이성, 그리고 샘플 아이 쉽게 5-7 &lt;로부터 회수 할 수있다/ SUP&gt;. 혈청 및 타액 샘플 H.에 대한 항체에 대해 광범위하게 연구되어왔다 파이로리 2,3,8, 말라리아 원충 (9), Entamoeba의 아메바 (10),와 포자충 포자충 3,11, 연쇄상 구균 폐렴 (12), 간염 바이러스 A와 C 13-14, noroviruses 2-4,15, T. 포자충 2-4, 뎅기열 바이러스 16, 인간 면역 결핍 바이러스 (HIV) (17), 대장균 O157 : H7 18. 멀티 플렉스 면역 동시에 하나의 샘플 볼륨 내에서 단일주기 또는 실행 내에서 여러 분석의 분석이 가능합니다. C.에서 멀티 플렉스 항원 jejuni와, T. 포자충, H. 파이로리는 A 형 간염 바이러스와 두 noroviruses은을 사용하여 병원체 인간 IgG의 타액 2-4 IgA와 IgG의 3,4- 및 2,3- 플라즈마 항체 반응을 측정하는 데 사용했다비드 기반 다중 면역. 물, 토양 및 식품 미생물에 노출 역학적 연구와 함께 사용될 때, 본 연구에 기재된 분석의 유형은 환경 병원체에 의한 감염에 대한 이해를 향상시키기 위해 중요한 정보를 제공 할 수있다. 또한, 이러한 연구에서 수득 타액 항체 데이터는 위험 평가 모델 19-22을 향상 시키는데 사용될 수있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Electron Corporation</td>
			<td>75002446</td>
			<td>Used to centrifuge samples</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>G560</td>
			<td>Used to mix samples</td>
		</tr>
		<tr>
			<td>Sonicator (mini)</td>
			<td>Fisher Scientific</td>
			<td>15-337-22</td>
			<td>Used to separate beads</td>
		</tr>
		<tr>
			<td>Pipettors P10, P20, P100, P1000, 8 ch.</td>
			<td>Capp</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer (Bright Line)</td>
			<td>Housser Scientific&#160;</td>
			<td>3200</td>
			<td>Used to count coupled beads</td>
		</tr>
		<tr>
			<td>Multiscreen Vacuum Manifold</td>
			<td>Millipore</td>
			<td>MSVMHTS00</td>
			<td>Used in washing steps to remove supernatant</td>
		</tr>
		<tr>
			<td>MicroShaker</td>
			<td>VWR</td>
			<td>12620-926</td>
			<td>Used to agitate beads during incubations</td>
		</tr>
		<tr>
			<td>Tube rack (1.5mL and 0.5mL) (assorted)</td>
			<td>VWR</td>
			<td>30128-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Scale</td>
			<td>Mettler or other</td>
			<td></td>
			<td>Used to measure wash reagents for making buffers</td>
		</tr>
		<tr>
			<td>Dynabead Sample Mixer</td>
			<td>Invitrogen</td>
			<td>947-01</td>
			<td>Used during coupling incubation step</td>
		</tr>
		<tr>
			<td>MatLab (R2014b)</td>
			<td>The MathWorks, Inc.</td>
			<td></td>
			<td>Used to analyze antibody response data</td>
		</tr>
		<tr>
			<td>Microsoft Excel 2014</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td>Used to analyze antibody response data</td>
		</tr>
		<tr>
			<td>Luminex Analyzer with xPonent 3.1 software</td>
			<td>Luminex Corporation</td>
			<td>LX200-XPON3.1</td>
			<td>Instrument and software used to run assay</td>
		</tr>
		<tr>
			<td>Antigens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GI.1 Norwalk Virus : p-particle</td>
			<td>Xi Jiang (CCHMC)*</td>
			<td>NA</td>
			<td>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;g.</td>
		</tr>
		<tr>
			<td>GII.4 Norovirus VA387 : p-particle</td>
			<td>Xi Jiang (CCHMC)*</td>
			<td>NA</td>
			<td>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;g.</td>
		</tr>
		<tr>
			<td>Hepatitis A Virus : grade II concentrate from cell culture</td>
			<td>Meridian Life Sciences</td>
			<td>8505</td>
			<td>Antigen coupled at 100 &#181;g</td>
		</tr>
		<tr>
			<td>Helicobacter pylori : lysate</td>
			<td>Meridian Life Sciences</td>
			<td>R14101</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Toxoplasma gondii : recombinant p30 (SAG1)</td>
			<td>Meridian Life Sciences</td>
			<td>R18426</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Campylobacter jejuni : heat killed whole cells</td>
			<td>KPL</td>
			<td>50-92-93</td>
			<td>Antigen coupled at 50 &#181;g</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-Norovirus</td>
			<td>(CCHMC)*</td>
			<td>NA</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Mouse anti-Hepatitis A IgG</td>
			<td>Meridian Life Sciences</td>
			<td>C65885M</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Mouse anti-Hepatitis A IgG</td>
			<td>Meridian Life Sciences</td>
			<td>C65885M</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>BacTraceAffinity Purified Antibody to Helicobacter pylori</td>
			<td>KPL</td>
			<td>01-93-94</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Goat pAb to Toxoplasma gondii</td>
			<td>Abcam</td>
			<td>Ab23507</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>BacTrace Goat anti-Campylobacter species</td>
			<td>KPL</td>
			<td>01-92-93</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Secondary&#160; Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L)</td>
			<td>Jackson</td>
			<td>705-065-149</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Biotinylated Rabbit anti-Goat IgG (H+L)</td>
			<td>KPL</td>
			<td>16-13-06</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Biotinylated Goat anti-Mouse IgG (H+L)</td>
			<td>KPL</td>
			<td>16-18-06</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG(H+L)</td>
			<td>KPL</td>
			<td>176-1506</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(&#7518;)&#160;</td>
			<td>KPL</td>
			<td>16-10-02</td>
			<td>Used for Salivary Immunoassay</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL copolymer microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td>Used as low binding microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030050C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030080C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5130140C</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Various Vendors</td>
			<td></td>
			<td>Used keep beads in the dark during incubations</td>
		</tr>
		<tr>
			<td>Deep Well plates</td>
			<td>VWR</td>
			<td>40002-009</td>
			<td>Used for diluting saliva samples</td>
		</tr>
		<tr>
			<td>Multiscreen Filter Plates</td>
			<td>Millipore</td>
			<td>MABVN1250</td>
			<td>Used to run assays</td>
		</tr>
		<tr>
			<td>Oracol saliva collection system</td>
			<td>Malvern Medical Developments Limited</td>
			<td></td>
			<td>Used for saliva collection</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated microspheres (beads)</td>
			<td>Luminex Corporation</td>
			<td>Dependent on bead set</td>
			<td>Antigens are coupled to the microspheres</td>
		</tr>
		<tr>
			<td>EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride)</td>
			<td>Pierce</td>
			<td>77149 or 22980</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Pierce</td>
			<td>24510</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Steptavidin-R-phycoerythrin (1mg/mL)</td>
			<td>Molecular Probes</td>
			<td>S-866</td>
			<td>Used as reporter</td>
		</tr>
		<tr>
			<td>MES (2-[N-Morpholino]ethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>M-2933</td>
			<td>Used for coupling</td>
		</tr>
		<tr>
			<td>Tween-20 (Polyoxyethylenesorbitan monolaurate)</td>
			<td>Sigma</td>
			<td>P-9416</td>
			<td>Used in wash buffer to remove non-specific binding</td>
		</tr>
		<tr>
			<td>Protein Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)**</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3813</td>
			<td>138 mM NaCl, 2.7 mM KCl</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-7888</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P-9416</td>
			<td>0.2% (v/v)</td>
		</tr>
		<tr>
			<td>Sodium Azide (0.05% azide)**</td>
			<td>Sigma</td>
			<td>S-8032</td>
			<td>**Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE&#39;s. Dispose of according to applicable laws.</td>
		</tr>
		<tr>
			<td>MES/ Coupling Buffer (0.05 M MES, pH 5.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MES (2-[N-Morpholino]ethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td></td>
		</tr>
		<tr>
			<td>5 N NaOH</td>
			<td>Fisher</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer (PBS, 1% BSA, pH 7.4)&#160;</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, 1% BSA, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3688</td>
			<td>138 mM NaCl, 2.7 mM KCl, 1% BSA</td>
		</tr>
		<tr>
			<td>Activation Buffer (0.1 M NaH2PO4, pH 6.2)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaH2PO4 (Sodium phosphate, monobasic anhydrous)</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td>0.1M NaH2PO4</td>
		</tr>
		<tr>
			<td>5 N NaOH</td>
			<td>Fisher</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer (PBS, 0.05% Tween-20, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, 0.05% Tween-20, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3563</td>
			<td>138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN</td>
		</tr>
	</tbody>
</table>

developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens

병인 및 환경 병원체의 인체 노출의 영향에 따라서, 노출 및 감염을 사용하여 비용 효과적인, 높은 처리량 접근 방식을 평가하는 능력이 필수 불가결 한 것, 전세계 주요 관심사이고. 이 논문은 다수의 병원체를 동시에 인간 타액 항체의 존재를 측정 할 수있는 비드 기반 다중 면역 개발 및 해석을 설명한다. 그것은, 비 침습적 저렴하고 혈청보다 수집하기 쉽기 때문에 침이 응용 프로그램에서 특히 매력적이다. 환경 병원체로부터 항원 카르 미립자 (비드)에 커플 링 비드 기반 용액 상 분석을 이용하여 인간의 타액 샘플의 매우 작은 양의 항체를 측정하는데 사용 하였다. 구슬은 캄 필로 박터 jejuni와, 헬리코박터 파이로리, 톡소 포자충, noroviruses (G I.1과 G II.4)에서 항원과 A 형 간염 바이러스와 결합되었다. 항원이 충분히에 연결되었는지 확인하려면비드가 결합은 비 오티 닐화 항 - 종 차 검출 항체 및 스트렙 타비 딘 - 피코 에리 트린 R 기자 (SAPE)와 인큐베이션 한 후, 종 특이 동물 유래 기본 캡쳐 항체를 사용하여 확인 하였다. 그것은 임의의 항원에 결합되지 않은 것을 제외하고 비특이적 결합을 측정하기위한 대조군으로서, 하나의 비드 세트는 다른 동일하게 처리 하였다. 항원 - 결합 및 제어 비드는 유동 세포 계측법의 원리에 기초하여 높은 처리량 분석기상에서 측정 전진 수집 된 인간 타액 샘플과 함께 배양하고, 각 항원에 대한 항체의 존재는 중간 형광 세기 단위 (MFI)를 측정했다 . 다중화 면역 적은 샘플 이상의 데이터를 포함하는 장점들을 갖는다; 비용 절감과 노동; 과 능력은 관심이 많은 대상에 분석을 사용자 정의합니다. 결과 침 다중 면역 especia가 될 수있는 이전의 노출과 감염을 식별 할 수있는 것을 나타내는큰 인간 집단을 포함하는 감시 연구 에서야 베드로 유용합니다.

고유 한 비드 세트 다양성을 샘플링 비특이적 결합을 측정하기 위해 제어 및 시료로 하였다. 이러한 비드가 결합 단계에있는 항원과 함께 배양하지 않은 것을 제외하고는 항원 결합 비즈와 동일하게 처리 하였다. 상기 혈청에서 의심 오염으로 인해 분석하고, 나머지 응답은 분산 로그 된 모든 타액 샘플과 함께 배양 제어 비드에서 얻은 MFI 값&gt; (500)는 제거되었다. 잇몸이...

Watch this Scientific Journal Video about Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens at JoVE.com

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

타액 항체 멀티 플렉스 면역을 개발하는 것은 환경 병원체의 인체 노출을 측정하는

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

developing-salivary-antibody-multiplex-immunoassay-to-measure-human

Research

JoVE Journal

Immunology and Infection

8.7K 조회수.  U.S. Environmental Protection Agency. In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

비디오: Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

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

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

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

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

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

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

연구

면역학 및 감염병학

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Waterborne를 검색하는 접선 흐름 빈 섬유 Ultrafiltration 및 열 분해 단계를 사용 수정일 EPA 방법 1,623<em&gt; Cryptosporidium</em&gt;과<em&gt; Giardia SPP.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

사람 말초 혈액의 Immunophenotypic 분석에 대한 항체 칵테일을 준비하는 반 자동화 된 접근

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

신생 전염병 병원체 제거를위한 손씻기의 효능을 시험하는 방법

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

Cytokine Profiling Immunoassay Cytometric 구슬-기반 플랫폼을 사용 하 여 자극된 마우스 Splenocytes의 다중화

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

마우스와 차세대 시퀀싱에 의해 인간의 항 체 레파토리의 식별

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

병원 균, 공생, 그리고 호스트 창 자 상피 사이 상호 작용을 공부 하는 인간 Colonoid Monolayers

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

타액 항체 멀티 플렉스 면역을 개발하는 것은 환경 병원체의 인체 노출을 측정하는

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