SZ와 DW는 Viropharma의 무제한 허가를받을 수 있습니다.

1. 브로 멜라 인 처리 적혈구의 준비 <ol><li> 세척 0 형식의 적혈구는 1X PBS (2 ~ 5 분 664 XG에 원심 분리)으로 3 배. </li><li> 37 ℃에서 10 분 동안 0.5 % 브로 멜라 인 솔루션의 두 개의 볼륨으로 포장 적혈구의 1 볼륨을 품다 </li><li> 세포는 1X PBS로 3 배 씻으십시오. </li><li> 세포는 1 주일 이상 1X PBS에서 39 ° C에서 3 % 용액으로 저장 될 수있다. </li></ol> 2. FACS에 의한 적혈구에 C3와 C4 증착 분석 <ol><li> 56 ℃에서 30 분 동안 샘플을 가열함으로써 환자 혈청의 필요량 (또는 다른 안티 RBC 항체 소스) 열 불 활성화 </li><li> (5 mM의 Veronal, 150 mM의 염화나트륨, 0.05 %의 젤라틴의 pH 7.4)과 VBG는 + +에서 그들을 재현 탁 - VBG에 브로 멜라 인 처리 적혈구를 세 번 씻으십시오 (VBG - + 2 mM의 MgCl2를 2 + 10 mM의 염화칼슘 2)에게주는 0.5 % 용액. </li><li> (F 유리 진주 둥근 바닥 판에 다음과 같은 구성 요소를 피펫또는 적절한 혼합) : 25 ㎕의 0.5 % 적혈구, 37.5 ㎕의 신선한 AB 혈청 (보수 원) 37.5 ㎕를 200 ㎍ / ㎖의 항-C5. 안티-C5는 비싼 구하기 어려울 수 있습니다. 하나는 C5-/C6-/C7- 또는 C8-결핍 혈청 대신 AB 혈청 및 안티-C5를 사용하여 고려할 수 있습니다. </li><li> 0.1-33 %의 최종 농도에 잘 당 환자의 혈청을 추가합니다. 올바른 열 불 활성화 AB 혈청을 사용으로 인해 잘 당 혈청 농도의 차이. 물론 150 μL /의 최종 볼륨을 얻기 위해 VBG + +를 ​​추가합니다. ELISA 호일 닫기 판. </li><li> 흔들리는 동안 37 ° C에서 1.5 시간을 품어. </li><li> PBS + 0.5 % BSA (2 분 664 XG에 원심 분리)과 적혈구 세 번 씻으십시오. </li><li> 추가 형광으로 표지 된 항-C3 및 항-C4 단클론 항체 또는 Fab 단편은 1 ㎍ / ml의 최종 농도로 PBS + 0.5 % BSA에서 (응집을 감소시키기 위하여). 여기에 사용자 정의 앤티 C3-알렉사 플 루어 488 개발 및 안티-C4-알렉사 플 루어 647 단일 클론 항체를 사용, 살전의 선택전자 형광 라벨 FACS에 의해 C3과 C4의 동시 탐지 할 수 있습니다. </li><li> 부드러운 흔들림과 실온에서 30 ~ 45 분을 품어. </li><li> PBS + 0.5 % BSA와 함께 세척 적혈구 배. </li><li> 재현 탁 적혈구 (150) PBS +0.5 %의 BSA의 μL과 (FACS 분석을하기 전에 유리 진주를 제거하는) 새 접시에 그들을 피펫. </li><li> FACS 분석을 수행합니다. 분산하려 FSC-A의 이중선에서 분리 한 세포는, 하나의 적혈구에 게이트를 설정, 형광 신호 (예 : FITC 및 APC)에 대한 적절한 감지 채널을 선택합니다. </li><li> 평균 형광 강도를 사용하여 결과를 정량화. </li></ol> 주 : 이소 타입 컨트롤 (예, 항-IL6 형광 쥐의 IgG1 표지 된)을 사용하는 것이 가능하다. 그러나, 환자의 혈청 (만 AB 혈청)없이 또는 보완 소스 (열 불 활성화 AB 혈청 및 열 - 불 활성화 된 환자의 혈청)없이 적혈구를 배양하여 예를 들어 진정한 부정적인 컨트롤을 포함하는 것이 좋습니다D 다음 같은 항-C3-알렉사 플 루어 488 및 안티-C4-알렉사 플 루어 647 항체 이러한 얼룩. 이러한 상이한 조절은 일반적으로 동일한, 부정적인 결과를주고 있지만 이것은, 적혈구 (12)의 경우, 이소 제어보다 더 유효하게 제어 할 수 있습니다. 3. 양 용혈성 분석 <ol><li> 56 ° C에서 샘플 30 분 가열하여 환자의 혈청 필요한 금액 (또는 다른 항 적혈구 항체 소스) 열 불 활성화 </li><li> VBG로 세척 적혈구 배 - 한 번 VBG와 + +. </li><li> (적절한 혼합을위한) 유리 진주 둥근 바닥 판에 다음과 같은 구성 요소를 피펫 35 μL 3 % 적혈구, 25 ㎕의 신선한 AB 혈청 (보완 소스) 및 1~50% 환자의 혈청. 열 - 불 활성화 된 AB 혈청을 이용한 혈청 농도의 차이를 보정. 물론 150 μL /의 최종 볼륨을 얻기 위해 VBG + +를 ​​추가합니다. ELISA 호일 닫기 판. </li><li> 진탕 1.5 시간 동안 37 ° C에서 알을 품다. </li><li> C감소 감속 (2 ~ 3 분에 완전 정지 등 감속)와 5 분 664 XG에 entrifuge 판. </li><li> 조심스럽게 새로운 마이크로 플레이트에 90 ㎕의 상층 액을 피펫. 이 기포를 방지하고 세포 손상을 따라 취할 것을 방지하기 위해이 단계에서 중요하다. </li><li> 적당한 분광 광도계에 A414/690을 측정합니다. </li><li> 순수로 샘플 적혈구의 용해의 비율로 용해를 표현한다. </li></ol>

<ol>
	<li>Zeerleder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+haemolytic+anaemia+-+a+practical+guide+to+cope+with+a+diagnostic+and+therapeutic+challenge.">Autoimmune haemolytic anaemia - a practical guide to cope with a diagnostic and therapeutic challenge.</a> Neth. J. Med. 69 (4), 177-184 (2011).</li><li>Reardon, J. E., Marques, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+and+transfusion+support+of+patients+with+autoimmune+hemolytic+anemia.">Laboratory evaluation and transfusion support of patients with autoimmune hemolytic anemia.</a> Am. J. Clin. Pathol. 125, 71-77 (2006).</li><li>Lai, M., Leone, G., Landolfi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hemolytic+anemia+with+gel-based+immunohematology+tests.">Autoimmune hemolytic anemia with gel-based immunohematology tests.</a> Am. J. Clin. Pathol. 139 (4), 457-463 (2013).</li><li>Zantek, N. D., Koepsell, S. A., Tharp Jr, ., R, D., Cohn, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+direct+antiglobulin+test:+A+critical+step+in+the+evaluation+of+hemolysis.">The direct antiglobulin test: A critical step in the evaluation of hemolysis.</a> Am. J. Hematol. 87 (7), 707-709 (2012).</li><li>Natukunda, B., Brand, A., Schonewille, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+alloimmunization+from+an+African+perspective.">Red blood cell alloimmunization from an African perspective.</a> Curr. Opin. Hematol. 17 (6), 565-570 (2010).</li><li>Packman, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+anemia+due+to+warm+autoantibodies.">Hemolytic anemia due to warm autoantibodies.</a> Blood Rev. 22 (1), 17-31 (2008).</li><li>Berentsen, S., Tj&#248;nnfjord, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+cold+agglutinin+mediated+autoimmune+hemolytic+anemia.">Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia.</a> Blood Rev. 26 (3), 107-115 (2012).</li><li>Petz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+antibody+autoimmune+hemolytic+anemia.">Cold antibody autoimmune hemolytic anemia.</a> Blood Rev. 22 (1), 1-15 (2008).</li><li>Barros, M. M. O., Blajchman, M. A., Bordin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warm+autoimmune+hemolytic+anemia:+recent+progress+in+understanding+the+immunobiology+and+the+treatment.">Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment.</a> Transfus. Med. Rev. 24 (3), 195-210 (2010).</li><li>Endoh, T., 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=Optimal+prewarming+conditions+for+Rh+antibody+testing.">Optimal prewarming conditions for Rh antibody testing.</a> Transfusion. 46 (9), 1521-1525 (2006).</li><li>Wouters, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C1-esterase+inhibitor+concentrate+rescues+erythrocytes+from+complement-mediated+destruction+in+autoimmune+hemolytic+anemia.">C1-esterase inhibitor concentrate rescues erythrocytes from complement-mediated destruction in autoimmune hemolytic anemia.</a> Blood. 121 (7), 1242-1244 (2013).</li><li>Arndt, P. A., Garratty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Review+of+Published+Methods+for+Analysis+of+Red+Cell+Antigen-Antibody+Reactions+by+Flow+Cytometry,+and+Approaches+for+Resolving+Problems+with+Red+Cell+Agglutination.">A Critical Review of Published Methods for Analysis of Red Cell Antigen-Antibody Reactions by Flow Cytometry, and Approaches for Resolving Problems with Red Cell Agglutination.</a> Transfus. Med. Rev. 24 (3), 172-194 (2010).</li></ol>

저자가 공개하는 게 없다.

위의 분석을 재현하고 강력한 있습니다. 그들은 수행하기 쉽고 그것이 (96 - 웰 플레이트에서) 동시에 많은 샘플들을 수행 할 수있다. 따라서,이 방법은 ELISA 로봇 시스템 예를 들어, 완전 자동화 된 시스템에 적합하다. 현재 사용되는 기술과 대조적으로, 이러한 분석은 정량적이며 이것은 보체 억제제 효과의 연구에서 예를 도울 것이다. 또한, 해석은 현재 사용되는 분석법에 비해 개?...

적혈구 (적혈구)에 대한 항체는받는 사람의 적혈구에 존재하지 않는 항원을 발현하는 적혈구의 수혈에 의해 유발 될 수있다. 이러한 알로 - 항체는 다음과 같은 수혈 5에 활성화를 보완에 의한 중증 급성 용혈성 수혈 반응을 일으킬 수 있습니다. 자가 면역 용혈성 빈혈 (AIHA), 환자는 자신의 적혈구에 대한 자동 항체가있다. 이것은 비장 및 / 또는 간 6,7 보체 수용체 활성화를 통해 보완 할 수 자동 항체의 경우 식세포상의 Fcγ 수용체에 결합하여 적혈구의 IgG의 상호 작용을 통해 세포의 가속 간극에 이르게한다. 혈관 내 용혈의 결과로 전격 보체 활성화는 드물지만 종종 치명적이다. 가속, 급성 빈혈 알로 또는 항체 결과, 따라서 잠재적으로 치명적인 조직의 저산소증 중 하나에 의해 유도 된 매개 RBC 파괴를 보완. AIHA의 자동 항체 dependin, 따뜻하고 차가운 항체로 분류된다그들은 적혈구 (각각 37 ° C 이하)에 바인딩 최적의 온도에 g. 따뜻한 항체의 IgG 아이소 타입의 일반적이며, IgM 항체의 차가운 항체는 8,9 이소. AIHA 예 : lymphoprolyferative 장애, 결합 조직 질환, 고형 종양, 감염이나 약물에 보조 할 수 있지만, 50 %의 경우에 AIHA 특발성 9입니다. gammaglobulins (예.의 IgG 또는 IgM 항체)의 검출 및 환자의 적혈구에 바인딩 보완은 반 정량적 직접 글로불린 (쿰스) 시험 (DAT)에 의해 수행된다. DAT에서 환자의 적혈구가 항-IgG의 또는 항-C3D와 함께 배양된다. RBC의 응집의 발생이 부착 보완 구성 요소 나 IgG의 바인딩의 존재를 보여줍니다. 알로 - 또는 환자의 혈청 내 항체의 검출은 간접 글로불린 테스트 (IAT)에 의해 수행된다. IAT에서, 브로 멜라 인 처리 테스트 적혈구는 환자의 혈청 배양 세척 한 후 항-H와 함께 배양하는IgG의 우만. 경우 적혈구 방지 RBC의 IgG 환자 혈청 응집에 존재 발생에 민감하게되었습니다. 적혈구에 대한 IgM 항체는 직접 환자의 혈청과 브로 멜라 인 처리 테스트 적혈구의 배양에 응집으로 이어질 것입니다. 직접 쿰스 시험이나 IAT에있는 RBC의 응집은 시각적으로 테스트 튜브에 눈 또는 크기 (1)에 의해 응집 및 단일 적혈구를 분리하는 작은 세파 덱스 컬럼에 샘플을로드하여 하나 평가한다. 적혈구의 보체 활성화를 측정하는 또 다른 자주 사용되는 기술은 브로 멜라 처리 적혈구 10의 (보완 중재) 용혈을 유도하는 환자 혈청의 능력이 평가되는 용혈성 분석 한 것이다. 원심 분리 후 상층 액 인해 출시 헤모글로빈에 빨간색 염색 무색 남아있는 경우 부정적인 것으로 간주 될 때 시험은 긍정적으로 기록됩니다. 글로불린 테스트 및 용혈성 분석 둘, 반 정량적이기 때문에 가장 높은 빼m은 희석 시험 여전히 양성되는 표시된다. 글로불린 테스트 및 용혈성 분석은 정기적으로 진단에 사용되는 강력한 분석이다. 이러한 분석은 반 정량적 및 보완 억제제의 효능을 평가할 때 필요에 따라 그들은 적혈구에 보체 활성화의 미묘한 차이를 연구하기에 적합하지 않은 분석을 수행하는 기술자의 경험에 의존하기 때문이다. 따라서, 우리는 우리가이 글에서 설명 할 것이다 적혈구로 (자동) 항체가 보체 활성화를 결정하는 두 개의 정량 분석​​법을 개발했다. 첫째, 우리는 적혈구에 보체 C3과 C4의 활성화 조각의 증착 (그림 1A)를 측정 할 수있는 분석을 개발했다. 이 분석에서, 인간의 브로 멜라 인 처리 유형 0 적혈구는 열 불 활성화 된 환자의 혈청 (항 적혈구 항체 원), 신선한 AB 혈청 (보완 소스) 및 안티-C5의 단일 클론 항체 (Eculizumab)와 함께 배양된다. 이 INC 중환자 혈청 안티 RBC 항체를 활성화 보완이 포함 된 경우 ubation, C3와 C4 증착이 발생합니다. 하류 보체 활성화하여 RBC 용해를 방지하기 위해 차단 항-C5 단클론 항체를 첨가한다. 이어서, 적혈구의 C3 및 C4 증착은 각각 C3 및 C4와 반응시켜 형광 표지 된 모노클로 날 항체의 Fab-단편을 사용하여 FACS에 의해 검출된다. 하나의 적혈구에 게이팅 결과의 신뢰성을 확보하는 것이 중요합니다. 이 기술의 이점은 환자 물질의 소량이, 캐스케이드의 조기 활성화를 보완이 평가된다 필요하고 상기 방법은 재현성 및 정량적 인 것을 포함한다. C3와 C4 모두보고의 또 다른 장점은 구별이 클래식과 렉틴 경로 활성화 (C3와 C4 증착 모두) 및 대안 경로 활성화 (만 C3 증착) 사이에 할 수 있다는 것입니다. 대신 치료 적혈구 브로 멜라 인 처리 적혈구의 사용으로 감도를 증가말한다. 두 번째 분석은 현재 사용되는 용혈성 분석 (그림 1B)를 기반으로합니다. 인간 브로 멜라 인 처리 유형 0 적혈구는 열 불 활성화 된 환자의 혈청 (항 적혈구 항체 소스)와 신선한 AB 혈청 (보완 소스)와 함께 배양된다. 보완이 환자 안티 RBC 항체에 의해 활성화되는 경우, 농도 의존적​​ RBC의 용해는 헤모글로빈의 방출로 이어지는, 발생합니다. 릴리스 된 헤모글로빈의 양이 본래와 단편화 적혈구 회전을 멈추지 후 상등액 414 nm에서 흡광도를 측정함으로써 정량한다. 흡광도가 발생 용혈 량과 상관. 현재 사용 된 분석과는 대조적으로,이 프로토콜은 대물, 재현성 및 분석을 해석하는 사람에 의존하지 용혈의 양적인 값을 허용한다. 이러한 방법의 애플리케이션은 AIHA에서 보체 억제제로서 C1-억제제의 잠재적 인 사용을 해제이었다 (11)에 기재 한 내용tudied.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:195px;">Fresenius Kabi</td>
			<td style="width:119px;"></td>
			<td style="width:316px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B-4287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0261</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0263</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0470.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0238.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 488 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">46402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 650 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">62265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#945;C5 (Eculizumab)</td>
			<td style="width:195px;">Alexion Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS (Canto)</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;"></td>
			<td>Any FACS can be used that has the appropiate lasers.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer (e.g. Multiskan spectrum)</td>
			<td style="width:195px;">Thermo Labsystems</td>
			<td style="width:119px;">1500-193</td>
			<td>Any spectrophotometer with the right wavelength range can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSDiva software v 6.1.2</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">643629</td>
			<td>Any compatible FACS analysis software can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bromelain</td>
			<td style="width:195px;">Sanquin</td>
			<td style="width:119px;">K1121</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for quantitative detection of antibody-induced complement activation on red blood cells

적혈구 (적혈구)에 대한 항체는 간 (혈관 외 용혈) 또는 적혈구의 혈관 내 용해에 이르는 보체 수용체를 통해 가속 정리의 결과로 활성화를 보완하기 위해가 발생할 수 있습니다. 혈관 내 용해 선도 특히 - - 활성화를 보완하기 위해 선도적 인 동종 항체 (자가 면역 용혈성 빈혈, AIHA에서 볼 수있는 것처럼) (예 ABO) 또는 적혈구 항원에 대한자가 항체가 잠재적으로 유해 할 수 있으며 치명적인 1. 현재 인해 적혈구에 (자동) - 항체 활성화를 보완은 RBC 또는 RBC 용해 1-4을 반영하는 nonquantitative 용혈성 분석에 의해 보완 증착을 반영하는 쿰스 테스트를 사용하여 체외에서 평가된다. 그러나, 보체 억제제의 효능을 평가하기 위해, 그것은 정량적 기법을 가지고 필수적이다. 여기에서 우리는 이러한 두 기술에 대해 설명합니다. 첫째, 환자의 혈청에서의 항체에 의해 유도되는 적혈구에 C3 및 C4 증착을 검출하는 분석법이다 사전하고 처리하며. 이를 위해, FACS 분석은 형광 표지 된 항-C3 또는 항-C4 항체로 사용된다. 이어서, 정량 분석​​ 용혈성 설명한다. 환자의 혈청에 의해 유도 된이 분석에 보완 - 중재 용혈이 발표 헤모글로빈의 분광 검출 만들기 사용을 측정한다. 이러한 분석은 모두 항체에 의한 보체 활성화의 연구를 촉진, 재현성과 정량이다.

그림 2A는 적혈구의 대표 산점도를 보여줍니다. FSC-A 플롯 (P1) - 하나의 적혈구에 적합한 게이트 드 FSC-W에서 볼 수 있습니다. 적혈구의 보통 약 95 %는이 P1 게이트 (단일 세포) 내에 있지만, 환자 혈청의 높은 비율을 사용하는 경우 안티 RBC IgM 항체가 환자의 혈청에 존재 특히,이 70 ~ 80 %로 떨어 뜨릴 수 있습니다. C4 증착에 AIHA 환자 혈청의 효과에 대한 대표적인 결과...

Watch this Scientific Journal Video about Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells at JoVE.com

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

여기에서 우리는 적혈구에 대한 항체에 의해 유도 된 보체 활성화를 측정하기위한 두 가지 분석을 설명합니다. 현재의 분석을 통해 가장 큰 장점은 양적 및 쉬운 해석하는 성격이다.

적혈구에 대한 항체에 의한 보체 활성의 정량 검출하는 방법

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

methods-for-quantitative-detection-antibody-induced-complement

Research

JoVE Journal

Immunology and Infection

30.4K 조회수.  University of Amsterdam.  여기에서 우리는 적혈구에 대한 항체에 의해 유도 된 보체 활성화를 측정하기위한 두 가지 분석을 설명합니다. 현재의 분석을 통해 가장 큰 장점은 양적 및 쉬운 해석하는 성격이다. 

비디오: Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

마우스 복막 구멍 세포의 분리

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

수신자 같은 수용체 활성화 레굴라팅에 Neu1 Sialidase 활동 감지

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

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 ...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

재조합 RBD 기반 SARS 백신에 대한 프로토콜 : 단백질 준비, 동물 예방 접종 및 중화 감지

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

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, ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...