우리는 Virochip 프로토콜의 초기 개발 및 최적화에 대한 데이비드 왕, 아나톨리 Urisman, 에이미 키슬러, 카엘 피셔, 패트릭 당나라, 그리고 알렉산더 Greninger 감사드립니다. 이 작품은 NIH K08 부여와 애보트 디스커버리 수상 (CC)을하고 하워드 휴스 의학 연구소 (JLD)를 지원합니다.

Virochip 분석의 단계는 임상 샘플에서 (1) 핵산 추출, 추출 RNA (2) 반대로 전사 및 2 차 - 가닥 cDNA 합성, 무작위로 준비하는 cDNA (3) PCR 증폭, (3) Cy3 형광 염료의 정관을 포함 , Virochip microarray로 표시된 자료 (4) 하이브 리다이 제이션, 그리고 (5) 검사 및 분석 (그림 1). 아래의 프로토콜은 독감과 같은 질병을 가진 아이에서 비강 면봉 샘플에 Virochip 분석의 사용을 보여줍니다. 프리머 시퀀스 프라이머 5&#39; - GTTTCCCAGTCACGATA - (N 9) -3 &#39; 프라이머 B 5&#39; - GTTTCCCAGTCACGATA - 3 &#39; 1. 핵산 추출 <ol><li> 이 샘플은 잠재적으로 전염성이 있으므로 임상 시료에서 핵산 추출은 Biosafety 레벨 2 (BSL - 2) 이상의 조건​​ 하에서 수행되어야합니다. </li><li> 비강 면봉 샘플 바이러스성 지주 매체 수송이다. 1.5 ML Eppendorf의 microcentrifuge 관에 샘플 나누어지는 200 μL. </li><li> 옵션 : 0.22 μm의 필터 (바이러스성 입자 정화)을 통과 샘플 </li><li> 선택 사항 : 제조 업체의 지침에 따라 터보 DNAse 키트 (앰비온)를 사용 DNAse (호스트 게놈 DNA를 저하하고 보호 encapsidated 바이러스 핵산에 대한 정화)와 pretreat 샘플 </li><li> Zymo ZR 바이러스성 RNA 추출 키트 또는 제조 업체의 지침에 따라 Qiagen Ultrasens 바이러스 키트를 사용하여 샘​​플을 추출합니다. </li></ol> 2. 역방향 전사 및 2 차 - 스트랜드 CDNA 합성 ( &quot;라운드&quot;) <ol><li> RNA를 추출 4 UL 1 UL 40 μL / pmol 프리머을 추가합니다. 65 열 ° C thermocycler (예 : GeneAmp PCR 시스템 9700) 후 5 분 방 온도 X 5 분 식지. </li><li> 2 μL 배 RT 버퍼, 1 μL 12.5 MM dNTP 1 μL 물, 0.5 μL 0.1M DTT, 0.5 μL 및 SSIII RT (Invitrogen)로 구성된 마스터 믹스 5 μL를 추가합니다. 42 품어 ° C X 60분. </li><li> 프로그램 thermocycler에서 94에 열을 ° 10로 냉각 C X 2 분 (중지 리버스 transcriptase 반응), ° C, 펄스 원심 분리기, 다음 1 μL 배 Sequenase 버퍼, 3.8 μL 물로 구성된 5 μL Sequenase 믹스를 추가 그리고 0.15 μL Sequenase (USB 공사). 2 차 스트랜드 합성 들어, 10 ° 37 C ° C 8 분 이상, 94에 다음 8 분, 열을 보유 ° C X 2 분 (Sequenase를 inactivate)을 10 ° C (보유)에 냉각. 최대 램프 </li><li> 옵션 : 앳는 cDNA 샘플은 -20 ° C.에이 시점에서 저장할 수 있습니다 </li></ol> 3. 의 PCR 증폭이 임의로 준비하는 CDNA ( &quot;라운드 B&quot;) <ol><li> 5 μL 10X PCR 버퍼, 1 μL 12.5 MM dNTP 1 μL 100 pmol / μL 뇌관의 B, 1 μL KlenTaq LA (시그마), 37 μL 물로 구성된 마스터 믹스 45 μL에 드르 5 UL 샘플을 추가합니다. </li><li> 다음과 같이 PCR 프로토콜을 실행 94 ° C, 2 분 → (94의 25주기 ° C, 30 S / 50 ° C, 45 S / 72 ° C, 1 분) → 72 ° C, 5 분 → 10 ° C ( 개최) </li><li> 1.5 % 아가로 오스 전기 영동 젤에 드르 B 샘플을 확인하십시오. 약 200의 얼룩 - 1000 BP는 시각되어야합니다 (그림 2A) </li></ol> 4. Cy3 형광 염료의 설립 <ol><li> 옵션 : 다른 샘플이 Cy5 형광 염색으로 표시하고 동일한 microarray에 hybridized 수 있습니다. </li><li> AA - dNTP를 통합하기 위해, 5 UL 10X PCR 버퍼, 1 UL 12.5 MM AA - dNTP (Invitrogen), 1 UL 100 pmol / UL 뇌관 B, 1로 구성된 마스터 믹스 45 UL에 드르 B 샘플 5 UL을 추가 1 UL KlenTaq LA (시그마), 37 UL 물. PCR 프로토콜 다음과 같이 프로그램을 실행 : 94 ° C, 2 분 → (94의 15주기 ° C, 30 S / 50 ° C, 45 S / 72 ° C, 1 분) → 72 ° C, 5 분 → 10 ° C ( 개최) </li><li> 제조 업체의 지침에 따라 DNA 청소 및 농축기 - 5 키트 (Zymo 연구)로 앳 C 예제를 청소합니다. 10 μL에 Elute. </li><li> 1M 중탄산염 1 UL (시그마)와 앳 C 예제 1 UL Cy5 (GE 헬스케어)를 추가합니다. </li><li> 어둠 속에 품어 X 1 시간. </li><li> 제조 업체의 지침에 따라 DNA 청소 및 농축기 - 5 키트 (Zymo 연구)와 Cy3 - 라벨 샘플을 청소하십시오. 12 μL에 Elute. </li></ol> 5. Virochip Microarray에 하이브 리다이 제이션 <ol><li> 옵션 : (샘플의 정상화를위한) Nanodrop 분광 광도계에 통합 cDNA 농도와 염료의 양을 확인 1.5 μL를 사용하여 </li><li> PCR 스트립 관에 25 μL의 총 볼륨 (애질런트) 1 μL 25X 조각 버퍼, 5 μL 배 차단 버퍼, Cy3 - 라벨 샘플의 9.5 μL (또는 정상화 금액) 및 물을 추가 </li><li> 2X GEx의 하이브리드화 버퍼 (애질런트) 25 μL를 추가합니다. 거품을 제거하기 위해 간략하게 스핀 다운. </li><li> 가스켓 슬라이드에있는 예제 40 μL를로드합니다. </li><li> 장소 애질런트 - 인쇄 Virochip 배열 (Californi 대학샌프란 시스코 / 애질런트) (아래 &quot;애질런트&quot;표시된 쪽) 개스킷 슬라이드 위에 단단히하고 나사를 고정. </li><li> 65 밤새 잡종 ° C 오븐, 10 RPM 속도 설정. </li><li> 37 ° C.에 배열, 앞서 열을 가하다 버퍼 2 (애질런트)를 씻어 버퍼 1 (애질런트)의 개스킷 슬라이드 / 배열 샌드위치 수중 갈라놓을. 버퍼 1 X 1 분 씻으십시오. preheated 버퍼 2 X 1 분에 씻으십시오. 천천히 (멀리 직접 배열의 적극적인 측면을 만지지 않도록주의하고 여분의 수분 윅에 kimwipe 사용) 거품을 피하기 위해주의하고, 버퍼 2 슬라이드를 제거 </li><li> 슬라이드 홀더에 슬라이드를 삽입하고 배열 ​​스캐너에 넣습니다. </li></ol> 6. Virochip 검색 및 분석 <ol><li> 스캔 Cy3 - 표시된 배열 악기의 표준 스캔 프로토콜 (그림 2B)에 따라 5 μm의 또는 2 μm의에서. </li><li> 육안 검사, 클러스터 분석, 또는 자동 Virochip 분석 프로그램에 의해 Virochip의 microarrays를 분석, 11 E - 예측했다. 이러한 세 가지 방법 중 각각으로 인플루엔자 바이러스가 쉽게 비강 면봉 샘플 (독감과 같은 질병을 가진 아이에서) (그림 2C)에 감지 </li></ol> 7. 대표 결과 : 프로토콜이 제대로되면 얼룩이 드르 B 단계 (그림 2A) 이후의 아가로 오스 겔 전기 영동에서 볼 수 있습니다. 애질런트 플랫폼에 Virochip microarray는 (그림 2B) 시각 분석하기 어려울 수 있지만, 바이러스가 존재한다면 그것은 쉽게 육안 검사, 클러스터 분석, 및 / 또는 E - 예측 (그림 1C)에 의해 식별되어야합니다. 검정에 대한 긍정적인 제어 역할을하는, 샘플은 알려진 바이러스 (박테리오 파지 MS2 예 : 담배 모자이크 바이러스)에서 핵산의 측정할 수량를 타서 수 있습니다. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="그림 1" /> 그림 1. Virochip microarray 처리 및 분석의 개략도. 임상 시료에서 핵산을 추출은 무작위 증폭 형광 염료로 표시하고, Virochip microarray에 hybridized입니다. Microarrays 2 μm의 해상도에서 스캔 및 전자 예측을 포함하여, 전산 다양한 도구를 사용하여 분석하고 있습니다. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="그림 2" /> 그림 2. Virochip 분석의 단계는 임의의 PCR, 200 얼룩에 의해 증폭 후 -. 1000 BP 겔 전기 영동에 의해 시각 수 (A) (B) 8 배열 / 유리 슬라이드의 세 Virochip의 microarrays 아웃은 작은 영역으로 표시됩니다. 고장 접속 오른쪽 하단 모서리에있는 삽입된 페이지에 한 microarray니다. (C) 자동 microarray 바이러스 분석을 사용하여 임상 샘플에서 독감 바이러스의 존재를 드러내 E를 예측했다. 높은 유사성 점수 (S = 0.55)은 이처럼 매우 중요한 예측하고, 제로에 가까운 P - 값에 해당합니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

여기에 설명한 Virochip 프로토콜은 복잡하고 세심한과 숙련된 연구 기술자가 필요합니다. 정확한 시약의 농도와 PCR, 라벨에 대한 조건과 하이브 리다이 제이션이 중요합니다. Virochip 프로토콜은 쉽게 같은 핵산 추출에 대한 TRIzol (Invitrogen)와 같은 조직 추출 방법의 사용에 의해 질병 조직의 분석을 수용하기 위해 수정할 수 있습니다. 프로브를 추가하거나 원하는 스펙트럼 및 범위의 광범를 얻을 필요 삭제할 수 있습니다, 예를 들어 &quot;- the - fly 방식에서&quot;같은 박테리아 및 곰팡이 등 nonviral 타겟의 검출을위한 프로브가 설계 될 수 있으며 추가됩니다. 현재 개발중인 Virochip 분석의 일부 응용 프로그램은 임상 실험실, 확산 조사, 약물 및 백신의 순도 검사, 그리고 새로운 바이러스성 병원체 발견에 광범위한 스펙트럼 바이러스 진단을 포함합니다.

<table border="1"><tbody><tr><th> 시약 / 재료의 이름 </th><th> 시약 / 재료의 이름 </th></tr><tr><td> 프리메라 </td><td> 5&#39; - GTTTCCCAGTCACGATA - (N9) -3 &#39; </td></tr><tr><td> 프리머 B </td><td> 5&#39; - GTTTCCCAGTCACGATA - 3 &#39; </td></tr><tr><td rowspan="5"> RT 마스터 믹스 (5 μL) </td><td> 2 μL 배 RT 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 물 </td></tr><tr><td> 0.5 μL 0.1M DTT </td></tr><tr><td> 0.5 μL SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase 믹스 (5 μL) </td><td> 1 μL 배 Sequenase 버퍼 </td></tr><tr><td> 3.8 μL 물 </td></tr><tr><td> 0.15 μL Sequenase (USB 공사) </td></tr><tr><td rowspan="5"> 차 B PCR 마스터 믹스 (45 μL) </td><td> 5 μL 10X PCR 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 100 μL / pmol 프리머 B </td></tr><tr><td> 1 μL KlenTaq LA (시그마) </td></tr><tr><td> 37 μL 물 </td></tr><tr><td rowspan="5"> 앳 C PCR 마스터 믹스 (45 μL) </td><td> 5 UL 10X PCR 버퍼 </td></tr><tr><td> 1 μL 12.5 MM dNTP (Invitrogen) </td></tr><tr><td> 1 μL 100 μL / pmol 프리머 B </td></tr><tr><td> 1 μL KlenTaq LA (시그마) </td></tr><tr><td> 37 μL 물 </td></tr><tr><td rowspan="4"> 하이브 리다이 제이션 버퍼 (25 μL) </td><td> 1 μL 25X 조각 버퍼 (애질런트) </td></tr><tr><td> 5 μL 5 배 차단 버퍼 (애질런트) </td></tr><tr><td> Cy3 - 라벨 샘플의 9.5 μL (또는 정상화 금액) </td></tr><tr><td> 25 μL의 총 볼륨 물을 추가 </td></tr><tr><td> 애질런트 Virochip microarray </td><td> 애기 실험실, UCSF에서 제공; 보류 라이센스 </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

많은 전염성 질병의 바이러스 원인의 진단은 바이러스의 고유한 시퀀스 다양성뿐만 아니라 전통적인 방법에 의해 감지되지 SARS coronavirus 2009 유행성 H1N1 독감 바이러스로 소설 바이러스 병원균의 지속적인 출현으로 인해 어렵습니다. 이러한 문제를 해결하기 위해, 우리는 이전에 개발하고 보존 순서 상동 1을 바탕으로 모든 알려진 바이러스뿐 아니라 소설 변종을 감지할 수있는 능력과 Virochip라는 판 - 바이러스 microarray 플랫폼을 확인했습니다. Virochip를 사용하여, 우리는 동등하거나 기존의 임상 테스트를 2-5로 뛰어난 감도로 입원한 환자에 설명할 수없는 중요한 질병의 경우를 포함한 호흡기 감염,과 관련된 바이러스의 전체 스펙트럼을 확인했습니다. Virochip 또한 SARS coronavirus 6,7, 소설 리노 바이러스의 clade 5, XMRV (전립선암에 연결된 레트로 바이러스) 8, 조류 bornavirus (앵무새의 낭비 질병의 원인) 9 포함한 새로운 바이러스를 식별하는 데 사용되었습니다 호흡기 질환과 diarrheal 10 어린이와 소설 cardiovirus. Virochip의 현재 버전은 애질런트 microarray 플랫폼에 포팅 2009 년 12 월 현재 GenBank에 ~ 이상 1500 바이러스로부터 파생된 ~ 36,000 프로브로 구성되어있다. 여기 샘플 핵산 추출, 임의 primers를 사용하여 PCR 증폭, 형광 염료 정관 및 microarray의 하이브리드화, 검사 및 분석을 포함하여 (~ 24 시간 처리 시간) 처음부터 끝까지 Virochip 분석 처리에 관련된 단계를 보여줍니다.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

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

Virochip 동시에 보존 순서 상동에 기초하여 모든 알려진 바이러스뿐 아니라 소설의 바이러스를 검출하기위한 판 - 바이러스 microarray입니다. 여기 알려진 알려지지 않은 두 바이러스의 존재에 대한 임상 샘플을 분석 Virochip 분석을 실행하는 방법을 보여줍니다.

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 ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

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

18.7K 조회수.  University of California, San Francisco. Virochip 동시에 보존 순서 상동에 기초하여 모든 알려진 바이러스뿐 아니라 소설의 바이러스를 검출하기위한 판 - 바이러스 microarray입니다. 여기 알려진 알려지지 않은 두 바이러스의 존재에 대한 임상 샘플을 분석 Virochip 분석을 실행하는 방법을 보여줍니다.

비디오: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral 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 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 ...

연구

면역학 및 감염병학

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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 ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Biocontainment 내 바이러스 샘플의 음성 염색을위한 캡슐의 활용

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic 살해 분석 결과 세균성 병원 체에 대 한 면역학 응답을 평가 하

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

루시퍼라제 기반 성장 분석을 사용하여 세포내 톡소플라즈마 곤디성장에 대한 화학적 억제제 효율 결정

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

유동 세포측정 기반 분석체에 의한 플라스모듐 팔시파룸 기생충에 자연적으로 획득된 식세포증-유도 항체 측정

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

등온 증폭을 이용한 인간 호흡기 세균 감염 진단

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

저자 스포트라이트: Advancing Rapid detection of respiratory Pathogens Using Microfluidic Chip(미세유체 칩을 사용한 호흡기 병원체의 신속한 검출 발전) 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Microfluidic-Chip-based Loop-Mediated Isothermal Amplification을 통해 하기도 감염을 일으키는 세균성 병원체의 신속한 검출

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...