We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

윤리 선언문 : 사용 왼쪽 위에 임상 검체 승인 기념 슬로안 케터링 암 센터 임상 시험 심사위원회의 가이드 라인을 준수합니다. 1. 분석을 실행하기 전에 참고 : 인플루엔자 A와 B의 분석은 비 인두 면봉 표본과 바이러스 성 전송 매체에 저장 인두 면봉 승인. 면봉은 키트에 포함되어 있으며 최적의 성능을 위해 사용되어야한다. 그러나, 레이온, 발포체, 또는 폴리 에스테르 솜 비강 면봉 또한 비강 면봉 샘플을 수집하기 위해 사용될 수있다 플록. <ol><li> 비강 면봉 샘플을 수집하려면, 가장 눈에 띄는 배수가 또는 가장 혼잡 콧 구멍에 면봉을 삽입합니다. 저항이 충족 될 때까지 부드럽게 회전으로, 콧 구멍에 면봉을 누르고 천천히 콧 구멍에서 제거하기 전에 비강 벽에 여러 번 회전합니다. 면봉을 저장하고 바이러스 전송 매체 (VTM) 3 ㎖를 포함하는 유리 병에서 그들을 수송. 참고 :주의 깊은 관심 appropr하기늦었 표본 수집은 잘못된 결과를 얻을 수 있습니다 부적절한 표본 컬렉션으로주의해야한다. 참고 : 면봉 표본이 수집 한 후 가능한 한 빨리 테스트해야합니다. 단, 최대 2 시간 동안 실온에서 저장 또는 검사가 바로 사용할 수없는 경우에는 최대 24 시간 동안 2-8 ℃로 냉각 될 수있다. 갓 수집 된 표본은 최적의 테스트 성능에 이상적이다. 부적절한 시료 처리, 저장 및 / 또는 전송 잘못된 결과를 산출 할 수있다. 참고 : 예방 조치로,이 테스트를 수행하는 개인 표본을 처리 할 때 보편적 인 예방 조치를 수행하여 잠재적으로 감염 모든 표본을 처리해야합니다. </li><li> 시험 전에 실온에 모든 샘플을 가져와. 주 : 모든 시험 성분을 하나만 사용하기위한 다수의 테스트를 수행하는 데 사용되어서는 안된다. 다른 로트 번호의 키트 구성 요소를 함께 사용하지 마십시오. 자신의 유효 기간이지나 키트 시약을 사용하지 마십시오. </li><li> ROO에 파란색 샘플 수신기를 가져와시험 전에 m 온도. 오렌지 시험 기재를 실온으로 가온 할 필요없이 테스트 될 수있다. </li><li> 때까지 즉시 사용하기 전에 호 일 포장에있는 모든 시험편을 둡니다. 악기의 측면에있는 전원 버튼을 눌러 기기를 켭니다. 사용자 ID 입력하고 Enter 키를 누릅니다 &#39;OK를.&#39; </li></ol> 2. 테스트 실행 <ol><li> 테스트 프로세스를 시작하려면, 기기 화면에 &#39;테스트 실행&#39;을 누릅니다. 온 스크린 키보드 또는 바코드 스캐너를 눌러 사용하여 환자 ID를 입력 후 확인을. &#39; 뚜껑을 열고 오렌지 시험베이스 홀더에 오렌지 시험베이스를 삽입합니다. </li><li> 정확한 테스트가 화면을 누릅니다에 표시되어 있는지 확인 &#39;OK.&#39; 파란색 샘플 수신기 홀더에 파란색 샘플 수신기를 삽입합니다. 참고 :이 올바른 작동 온도에 도달 용출 버퍼를 방지하고 테스트 성능에 영향을 미칠 수 있으므로 기기에 배치하기 전에 샘플 수신기를 열지 마십시오. </li&gt; <li> 샘플 수신기가 준비가 될 때까지 기다립니다. 악기에 연결되면, 호일 씰을 제거하고 샘플 수신기로 시험 할 환자 면봉을 배치합니다. 참고 : 호일을 제거 할 때 제자리에 남아 있는지 확인하기 위해 샘플 수신기의 가장자리에 두 손가락을 놓습니다. </li><li> 적극적으로는 면봉의 표본을 제거하는 데 도움을 혼합으로 샘플 수신기가 측면에 면봉 머리를 누르면 10 초 동안 액체에 면봉을 섞는다. 를 눌러 &#39;확인&#39;을 진행한다. 바이러스 운반 매체에 저장된 테스트 면봉은 10 초 동안 와동 VTM 경우 샘플 수신기 200 μL를 추가한다. </li><li> 파란색 샘플 수신기에 흰색 전송 카트리지를 누릅니다. 주황색 표시등이 상승 할 때까지 샘플 수신기 눌러 계속합니다. </li><li> 들어 올린 다음 테스트베이스에 전송 카트리지를 연결합니다. 카트리지 전송이 올바르게 연결되면 주황색 표시 등을 관찰 내려. 뚜껑을 닫고 &quot;테스트 완료&quot;될 때까지 열지 마십시오메시지가 화면에 나타납니다. </li></ol> 3. 품질 관리 <ol><li> 홈 화면에서 &#39;실행 QC 테스트&#39;를 터치합니다. 품질 관리 테스트를 선택 실행합니다. &#39;확인&#39;을 터치하고 화면에 테스트를 완료하라는 메시지에 따라 시험을위한 품질 관리 샘플과 일치하도록 시험 유형을 확인합니다. </li></ol> 4. 결과 해석 <ol><li> 테스트 실행이 완료된 후, 인플루엔자 A, B, 알 아형의 존재를 해석하여 화면에 표시된 시험 결과를 관찰한다. 유효하지 않은 결과를 테스트를 반복합니다. </li></ol> 5. 유지 보수 및 청소 <ol><li> 악기와 70 % 에탄올 또는 10 % 표백제 용액으로 매일 주변 벤치 영역을 청소합니다. 청소 젖은 보풀이없는 천으로 세정 용액을 스프레이. 악기에 직접 솔루션을 붓지 마십시오. </li></ol>

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The authors have nothing to disclose.

인플루엔자 바이러스는 이환율과 사망률의 중요한 세계적으로 원인입니다. 인플루엔자의 신속하고 정확한 진단은 호흡기 시즌 동안 독감 발생을 관리하는 주요 열쇠 중 하나입니다. 기타 항원 - 기반 테스트 신속하고 수행하기 쉽다; 그러나, 낮은 민감도 (13)를 갖는다. 한편, 기존의 분자 테스트는 감도를 향상,하지만 수행하고 더 비용이 많이 드는 더 많은 경험 실험실 기술자?...

인플루엔자 바이러스 감염은 질병률과 사망률 매년 1, 2, 3 상당량 초래한다. 복잡하지 않은 인플루엔자는 발열, 근육통, 두통, 비 생산성 기침 4, 5로 헌법과 호흡기 증상을 특징으로한다. 기본 동반 질환과 이전 개인, 어린이, 면역 저하 환자, 환자는 폐렴, 심근염, 중추 신경계 질환 또는 사망 6, 7과 같은 심각한 합병증에 대한 높은 위험이 있습니다. 인플루엔자 감염은 질병 심각도와 길이 8을 줄일 수 있습니다 증상 발현 48 시간 이내에 항 바이러스 치료의 메시지 관리에 다른 호흡기 바이러스에서 유일하다. 인플루엔자의 신속한 식별도있다불필요한 항생제 (9) (10)의 사용을 줄이기 위해 도시. 또한, 인플루엔자 감염 입원 환자는 적절한 감염 관리 예방 조치와 격리 된 방에 배치해야합니다. 임상 인플루엔자 구별하기 그러나, 비 인플루엔자 바이러스로 인한 호흡기 질환이 어려울 수있다. 이러한 이유로, 인플루엔자 신속하고 정확한 진단 시험은 임상 환자의 관리를 위해 매우 중요하다. 여러 분석은 검출 및 인플루엔자 바이러스의 식별을 위해 사용할 수 있습니다. 그들이 사용하고, 15 분 11 12 30 내 결과를 제공하는 간단한 때문에 신속한 인플루엔자 항원 검출 검사 (RIDTs)은 광범위 치료 시점 시험으로 임상에서 사용된다; 그러나, 그 감도가 크게 (10-80%) 제조사 및 인구에 따라 시험되는 변화와 인플루엔자 타입 subtyPE 13, 14, 15. 직접 형광 분석법 (DFAS)는 RIDTs 위에 우수한 감도를 제공하지만, 처리 시간이 더 (~ 3 시간)이며, 숙련 기술자 (16), (17)에 의해 완료되어야한다. 바이러스 성 문화는 인플루엔자 진단을위한 황금 표준되었습니다 모두 RIDTs 및 DFAS 18 감도를 향상되었습니다. 그러나, 인플루엔자 바이러스 성 문화는 환자 관리 19 원조에 자사의 유틸리티를 감소, 완료 2-14 D에서 걸릴 수 있습니다. 마지막으로, 핵산 증폭 검사 (NAAT)는 인플루엔자 진단에 새로운 골드 표준으로 배양 기술을 대체하고있다. NAAT는 몇 시간 인플루엔자를 검출하는 가장 큰 감도를 갖는 것으로 간주된다. 그러나 NAAT 가장 비싼 분석하고 5를 수행하는 특수 장비 및 기술자가 필요 <s업&gt; 20, 21, 22, 23, 24, 25. 여기에 설명 된 인플루엔자 A와 B 분석은 쉽게 사용할 수있는 첫 번째 CLIA-면제 분자 빠른 독감 테스트입니다. 이 분석은 분자 표지 프로브와 표적 탐지 뒤에 인플루엔자 특이 적 프라이머와 등온 증폭을 사용하는 칼자국을내는 효소 증폭 반응 (NEAR)을 이용하여 작동합니다. 이 분석은, B에서 인플루엔자 A 차별화 설정하고 하나의 샘플을 처리하는 데 2 ​​분을 필요로하며, 완료하는 데 15 분 총이 필요합니다. 여기, 우리는 인플루엔자 A &amp; B 분석을위한 프로토콜을 제시한다. 또, 샘플 데이터 (바이러스 전송 매체에 저장 보관 된 시편 인두 면봉 (NPS)의 상기 인플루엔자 A 및 B의 성능 분석을 비교 VT 세트 제공다른 호흡기 병원체 패널 분석에 M).

<table border="0" cellpadding="0" cellspacing="0" width="660">
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			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
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			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
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			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
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			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
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			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
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			<td height="20" style="height:20px;">Viral transport medium</td>
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rapid molecular detection and differentiation of influenza viruses a and b

인플루엔자는 인간 인플루엔자 바이러스 A 및 B에 의한 전염성 호흡기 질환이며 질병률과 사망률 매년 상당한시킨다. 인플루엔자 A 및 B의 분석은 제 CLIA - 면제 분자 빠른 감기 시험 유효했다. 인플루엔자 A와 B 테스트는 분자 표지 프로브와 표적 탐지 뒤에 인플루엔자 특이 프라이머를 등온 증폭을 사용하여 작동합니다. 여기에, 냉동에 인플루엔자 (A)의 성능과 B의 분석은, 호흡 패널 분석 비교 하였다 표본 바이러스 수송 배지 (VTM)에 저장된 비 인두 면봉 (NPS)를 보관. 인플루엔자 A 및 B 시험의 성능은 호흡 패널 기준있어서 결과를 비교함으로써 평가 하였다. 총 인플루엔자 바이러스 A에 대한 민감도는 67.5 % (95 % CI (CI), 56.6-78.5)이었고, 특이도는 86.9 % (CI, 71.0-100)를했다. 인플루엔자 바이러스 B 테스트에 대한 민감도 및 특이도는 90.2 %였다 (CI, 68.5-100각각)와 98.8 % (CI, 68.5-100). 이 시스템은 완벽하게 통합 된, 폐쇄, 소형 풋 프린트 시스템에서 실행되는 다른 현재 사용 가능한 분자 분석 및 간단한, 피펫없는 절차보다 훨씬 짧은 테스트 시간의 이점이있다. 전반적으로,이 연구에서 평가 된 인플루엔자 A와 B의 분석은 현장 진료 신속 인플루엔자 진단 검사의 역할을 할 가능성이있다.

본 연구에서는 보관 NPS 표본은 2013 NPS 시편 3 ㎖에 제출 된 2012년 12월 15일 3 월 1 일 사이에 인플루엔자 발생시 메모리얼 슬론 - 케터링 암 센터 (MSKCC)에서 독감 같은 증상으로 제시 입원 환자에서 수집 VTM의 및 연구 기간 동안 인플루엔자 A, A-1, A-3, B를 포함하는 호흡기 바이러스 (RP)의 패널을 검출하는 분자 분석과 일상적인 임상 실습의 일환으로 테스트 3,675 NPS 표본은 제출...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

우리는 분자 기반 인플루엔자 A, 신속하고 B 분석에 대해 설명합니다. 인플루엔자 분석은 분자 표지 프로브와 표적 탐지 뒤에 인플루엔자 특이 프라이머를 등온 증폭을 이용하여 15 분 이내에 각 대상을 감지합니다. 인플루엔자 A와 B의 분석은 사용자 친화적이고 수행 할 최소한의 실습 시간이 필요합니다.

빠른 분자 탐지 및 인플루엔자 바이러스 A와 B의 차별화

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

rapid-molecular-detection-differentiation-influenza-viruses

Research

JoVE Journal

Immunology and Infection

15.5K 조회수.  Memorial Sloan Kettering Cancer Center.  우리는 분자 기반 인플루엔자 A, 신속하고 B 분석에 대해 설명합니다. 인플루엔자 분석은 분자 표지 프로브와 표적 탐지 뒤에 인플루엔자 특이 프라이머를 등온 증폭을 이용하여 15 분 이내에 각 대상을 감지합니다. 인플루엔자 A와 B의 분석은 사용자 친화적이고 수행 할 최소한의 실습 시간이 필요합니다. 

비디오: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

인플루엔자에의 DNA Microarray에 ampliPHOX Colorimetric 검색

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

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, ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

진정한 검출 마우스 B 리니지 세포를 IgE 항체가 발현

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

돼지에서 인플루엔자 바이러스 탐지 및 격리를위한 코 닦음

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

표현형 및 기능 분석에 대한 검색 및 희귀의 농축 항원 특이 적 B 세포

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...