SZ ve DW VIROPHARMA sınırsız bir hibe alırsınız.

1.. Bromelain ile tedavi edilen kırmızı kan hücrelerinin hazırlanması <ol><li> Yıkayın 0-yazdığınız kırmızı kan hücreleri 1x PBS (2-5 dakika boyunca 664 x g santrifüj) ile 3x. </li><li> 37 ° C&#39;de 10 dakika boyunca% 0.5 bromelain çözeltisi iki hacim ile dolu eritrositlerde 1 hacim inkübe </li><li> Hücreler 1x PBS ile 3 kez yıkayın. </li><li> Hücreler en az bir hafta için 1x PBS içinde 4 ° C&#39;de% 3 çözelti halinde saklanabilir. </li></ol> 2. FACS RBCs C3 ve C4 Birikme Analizi <ol><li> 56 ° C&#39;de 30 dakika boyunca ısıtılarak örnek hasta serumu gerekli miktarda (ya da diğer anti-RBC antikoru kaynağı) Isı-inaktif </li><li> (5 mM Veronal, 150 mM NaCl,% 0.05 jelatin pH 7.4) ve VBG + + bunları tekrar süspansiyon - VBG in bromelain ile tedavi edilen eritrosit üç kez yıkayın (VBG - + 2 mM MgCI2, 10 mM CaCl2) vermek üzere bir % 0.5 çözelti. </li><li> (F cam inci ile bir yuvarlak tabanlı bir plaka içine, aşağıdaki bileşenleri Pipetveya uygun karıştırma): 25 ul% 0.5 eritrosit, 37.5 ul taze AB serumu (kompleman kaynak) ve 37.5 ul 200 ug / ml anti-C5. Anti-C5 pahalı ve elde etmek zor olabilir. Bir C5-/C6-/C7- veya C8-eksik serum yerine AB serumu ve anti-C5 kullanarak düşünebiliriz. </li><li> 0,1-33% bir nihai konsantrasyona kadar oyuk başına hasta serum ekleyin. Doğru ısı ile inaktive edilmiş AB serum kullanılarak dolayı, oyuk başına serum konsantrasyon farkları için. De 150 ul / lik bir son hacim elde etmek için VBG + + ekleyin. ELISA folyo ile yakın plaka. </li><li> Çalkalarken 37 ° C &#39;de 1.5-2 saat inkübe edin. </li><li> PBS +% 0.5 BSA (2 dakika boyunca 664 x g&#39;de santrifüj) ile RBC üç kez yıkayın. </li><li> Ekle floresan etiketli anti-C3 ve anti-C4 monoklonal antikorlar ya da bunların fragmanları, Fab 1 ug / ml &#39;lik bir son konsantrasyona PBS +% 0.5 BSA içinde (aglütinasyon azaltmak için). Burada, özel anti-C3-Alexa Fluor 488 geliştirilmiş ve anti-C4-Alexa Fluor 647 monoklonal antikorlar kullanılır; thes seçimie floresan etiketler FACS ile C3 ve C4 arasında aynı anda algılanmasını sağlar. </li><li> Hafif sallama ile oda sıcaklığında 30-45 dakika kuluçkalayın. </li><li> PBS +% 0.5 BSA ile yıkayın eritrosit 3x. </li><li> Süspanse alyuvarlar 150 PBS +0.5% BSA ul ve (FACS analiz öncesi cam inci kurtulmak için), yeni bir plaka içine pipetle. </li><li> FACS analizi yapın. Bir dağılım komplo FSC-A çifti ayrı tek hücreleri; tek eritrositler üzerindeki kapıyı ayarlayın; floresan sinyallerin (örn. FITC ve APC) için uygun algılama kanalı seçin. </li><li> Medyan floresan yoğunluğunu kullanarak sonuçlarını ölçmek. </li></ol> Not: Bu, bir izotip kontrolü (örn., flüoresan anti-IL-6 fare IgG1 etiketli) kullanmak mümkündür. Bununla birlikte, hasta serumu (sadece AB serum olmadan) ya da bir tamamlayıcı kaynağı (ısı ile inaktive AB serum ve ısı ile inaktive edilmiş hasta serum olmadan) eritrositlerde inkübe edilmesi ile, örneğin, gerçek bir negatif kontrol dahil edilmesi önerilmektedir bird sonra aynı anti-C3-Alexa Fluor 488 ve anti-C4-Alexa Fluor 647 antikorları, bu leke. Bu farklı kontroller genellikle aynı, negatif sonuç veren, ancak bu, kırmızı kan hücrelerinin 12 durumunda, bir izotip kontrolü daha geçerli bir kontrol sağlar. 3. Kantitatif Hemolitik Deneyi <ol><li> 56 ° C &#39;de, numune 30 dakika ısıtılarak hasta serasının gerekli miktarda (ya da diğer anti-RBC antikoru kaynağı) Isı-inaktif </li><li> VBG ile yıkayın RBC&#39;ler 3x - ve bir kez VBG + + ile. </li><li> (Uygun karıştırma için) bir cam inci ile bir yuvarlak tabanlı bir plaka içine aşağıdaki bileşenleri Pipet: 35 ul% 3 kırmızı kan hücreleri, 25 ul taze AB serumu (tamamlayıcı kaynak) ve% 1-50 hastanın serum. Isı ile inaktive edilmiş AB serum kullanılarak serum konsantrasyon farkları için doğru. De 150 ul / lik bir son hacim elde etmek için VBG + + ekleyin. ELISA folyo ile yakın plaka. </li><li> Çalkalanarak 1.5-2 saat boyunca 37 ° C&#39;de inkübe edilir. </li><li> Cindirgenmiş yavaşlama (2-3 dakika içinde tam durdurmak için örneğin yavaşlama) ile 5 dakika boyunca 664 x g entrifuge plakası. </li><li> Dikkatli bir şekilde yeni bir mikrotiter plakası içinde 90 ul üstte yüzen madde pipet. Bu hava kabarcıkları önlemek ve sağlam hücrelerin yanınızda önlemek için bu aşamada önemlidir. </li><li> Uygun bir spektrofotometre içinde A414/690 ölçün. </li><li> Saf su ile bir örnek eritrosit lizis yüzdesi olarak liziz ifade eder. </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>

Yazarlar ifşa hiçbir şey yok.

Yukarıda bahsedilen deneyler, tekrarlanabilir ve sağlamdır. Bunlar, uygulaması kolay olan ve (a-96 oyuklu bir plaka içerisinde) aynı anda birçok örnekleri ile gerçekleştirmek mümkündür. Dolayısıyla, bu yaklaşım aynı zamanda bir ELISA robot sistemi, örneğin, tam otomatik bir sistem için uygun olacaktır. Şu anda kullanılan tekniklerin aksine, bu deneyler, nicel ve bu tamamlayıcı önleyicileri etkisinin çalışmaya örneğin yardımcı olacaktır. Ayrıca, yorumlama anda kullan?...

Kırmızı kan hücrelerinin (RBC) karşı antikorlar, alıcı eritrositlerde üzerinde mevcut olmayan bir antijeni eksprese eritrositlerde transfüzyonu ile indüklenebilir. Bu allo-antikorlar aşağıdaki transfüzyon 5 üzerinde aktivasyonu tamamlamak nedeniyle ciddi akut hemolitik transfüzyon reaksiyonlara neden olabilir. Oto-immün hemolitik anemi (AIHA), hastalar kendi RBCs karşı oto-antikorlar var. Bu durum, dalak ve / veya karaciğer 6,7 tamamlayıcı reseptörleri yoluyla komplemanı aktive edebilmek oto-antikorların durumda fagositoz on-Fcy reseptörleri ile eritrosit bağlı IgG etkileşimi yolu ile hücrelerin hızlandırılmış açıklık yol açar. Intravasküler hemoliz sonuçlanan fulminan kompleman aktivasyonu nadir fakat sıklıkla ölümcüldür. Hızlandırılmış akut anemi allo-veya otoantikorlar sonuçlar ve bu nedenle potansiyel olarak ölümcül bir dokunun hipoksi ile uyarılan ya da aracılık ettiği RBC imha tamamlar. AIHA in oto-antikorlar dependin, sıcak ve soğuk antikorlar sınıflandırılırBu eritrositlerde (sırasıyla 37 ° C ya da daha düşük) bağlanan en uygun sıcaklık g. Sıcak antikorlar IgG izotipinde genellikle ve IgM soğuk antikorlar 8,9 izotip. AIHA örneğin lymphoprolyferative bozukluğu, bağ dokusu hastalıkları, solid tümörler, enfeksiyonlar veya ilaçlara ikincil olabilir, ancak vakaların% 50 AIHA idiyopatik 9&#39;dur. Gammaglobulini (örneğin,. IgG ya da IgM) tespiti ve hastanın eritrosit için bağlı tamamlayıcı bir yarı niceliksel direkt antiglobulin (Coombs) testi (DAT) vasıtasıyla gerçekleştirilir. DAT olarak hasta eritrosit anti-IgG veya anti-C3d ile inkübe edilir. RBC aglutinasyon oluşumu bağlı tamamlayıcı bileşenleri veya IgG bağlanma mevcudiyetini gösterir. Allo-ya da hastanın serum içinde antikorların tespiti dolaylı antiglobulin testi (IAT) vasıtasıyla gerçekleştirilir. TBD de, bromelain ile muamele edilen deney eritrosit, hasta serumu ile kuluçkalandı, yıkandı ve daha sonra anti-h ile kuluçkalanırIgG Uman. Durumda eritrositler anti-RBC IgG hasta serum yapışmasına mevcut oluşacak ile duyarlı olmuştur. RbcS IgM antikorlar doğrudan hasta serumu ile bromelain ile muamele edilen deney eritrositlerde inkübasyon üzerine aglütinasyon yol açacaktır. Doğrudan Coombs testinde veya TBD de RBC aglütinasyon görsel bir test tüpü içinde göz ya da boyutu 1 ile aglutinant ve tek eritrositlerde ayıran küçük bir Sephadex kolonu üzerinde örnek yükleme yoluyla değerlendirilir. Eritrosit kompleman aktivasyonu ölçmek için sıkça kullanılan bir başka teknik, bromelain ile tedavi edilen eritrosit 10 (tamamlayıcı-aracılı) hemoliz indükleme hasta serumunun kapasitesi değerlendirilmiştir edildiği hemolitik deney 1 &#39;dir. Santrifüjlemeden sonra süpernatan nedeniyle serbest hemoglobin kırmızı lekeli ve renksiz kalırsa, negatif olarak kabul edilir zaman test pozitif olarak not edilir. Antiglobulin test ve hemolitik tahlil, hem yarısayısaldır yana en yüksek serum seyreltme testin hala pozitif olduğu belirtilmiştir. Antiglobulin test ve hemolitik tahlil rutin teşhis kullanılan sağlam tahliller vardır. Bu analizler semikantitatif ve kompleman inhibitörlerinin etkinliğini değerlendirirken gerektiğinde onlar, eritrositler kompleman aktivasyon ince farklılıklarını incelemek için uygun değildir tahlil gerçekleştiren teknisyen deneyimine bağlı olduğundan. Bu nedenle, biz bu yazıda anlatacağım RBCs, (otomatik-) antikorlar kompleman aktivasyonunu belirlemek için iki nicel analizler geliştirdi. İlk olarak, eritrositlerde kompleman C3 ve C4 aktivasyonu fragmanlarının birikimi (Şekil 1A) ölçülmesi için bir tahlil geliştirilmiştir. Bu deneyde, insan bromelain ile tedavi edilen tip-0 eritrosit ısı ile inaktive edilmiş hasta serumu (anti-RBC antikoru kaynağı), taze AB serumu (tamamlayıcı kaynak) ve anti-C5 monoklonal antikor (Eculizumab) ile inkübe edilir. Bu inc sırasındaHastanın serum anti-RBC antikorları harekete tamamlayacak içeriyorsa ubation, C3 ve C4 birikimi oluşacaktır. Alt kompleman aktivasyonu ile RBC liziz önlenmesi için bir bloke edici anti-C5 monoklonal antikoru ilave edilir. Daha sonra, eritrositlerde C3 ve C4 çökelme sırasıyla, C3 ve C4 ile reaksiyona floresan etiketli monoklonal antikorlar ya da Fab-fragmanları kullanılarak FACS ile tespit edilir. Tek RBCs üzerinde yolluk sonuçların güvenilirliğini sağlamak için önemlidir. Bu tekniğin avantajları, hasta bir malzeme küçük hacimli, kaskadının erken bir aşamasında aktive edilmeyi tamamlayan değerlendirilir gereklidir ve yöntem, yeniden üretilebilir ve nicel olduğu bulunur. C3 ve C4 hem de ideal bir diğer avantajı, bir ayrım klasik ve lektin yolak aktivasyonu (C3 ve C4 biriktirme her ikisi de) ve alternatif bir yol, aktive (sadece biriktirme C3) arasında yapılabilir olmasıdır. Yerine işlenmemiş RBCs bromelain-tedavi RBCs kullanımı gibi hassasiyetini artırırsöylüyorlar. İkinci deney şu anda kullanılan hemolitik tahlilinde (Şekil 1 B) dayanır. İnsan bromelain ile tedavi edilen tip-0 eritrosit ısı ile inaktive edilmiş hasta serumu (anti-RBC antikoru kaynağı) ve taze AB serumu (tamamlayıcı kaynak) ile inkübe edilir. Kompleman hasta anti-RBC antikorlar tarafından aktive edilirse, doza bağımlı RBC parçalama hemoglobin serbest yol, meydana gelecektir. Serbest hemoglobin miktarı sağlam ve parçalanmış eritrositlerde aşağı eğirme işleminden sonra süpernatan içinde 414 nm&#39;de absorbans ölçümü ile belirlenir. Absorbans meydana hemoliz miktarı ile ilişkilidir. Mevcut deneyde tersine, bu protokol, bir hedef, çok tekrarlanabilir ve değerlendirilirken kişiye bağımlı değildir hemoliz kantitatif değeri sağlar. Bu yöntemlerin bir uygulama AIHA kompleman önleyici olarak C1-inhibitörünün potansiyel kullanımı olduğu, 11 s &#39;de tarif edilmiştirtudied.

<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

Kırmızı kan hücrelerinin (RBC) karşı antikorlar, karaciğer (ekstravasküler hemoliz) ya da damar içi eritrositlerde lizise neden olarak tamamlayıcı reseptörleri ile hızlandırılmış bir boşlukla sonuçlanan kompleman aktivasyonu yol açabilir. Damar içi lizise neden - özellikle - kompleman aktivasyonu neden alloantikorları (oto-bağışıklık hemolitik anemi, AIHA görüldüğü gibi) (örn. ABO) veya RBC antijenlere karşı antikorlar, potansiyel olarak zararlı olan ve ölümcül olabilir 1. Şu anda, eritrositlerde nedeniyle on (otomatik)-antikorları için kompleman aktivasyonu RBC üzerinde veya RBC parçalama 1-4 yansıtan bir nonquantitative hemolitik deneyi ile tamamlayıcı çökelmesini yansıtan Coombs testi kullanılarak in vitro tayin edilmiştir. Ancak, kompleman inhibitörleri etkinliğini değerlendirmek için, nicel teknikleri olması zorunludur. Burada iki gibi teknikler tarif eder. İlk olarak, hasta serumunda antikorlar indüklenir kırmızı kan hücreleri üzerinde C3 ve C4 birikmesini tespit etmek için bir deney öncesi olduğusunulmuş. Bunun için, FACS analizi, floresan etiketli anti-C3 ya da anti-C4 antikorları ile birlikte kullanılır. Bundan sonra, bir miktar hemolitik tahlili tarif edilmektedir. Hasta serumu ile indüklenen Bu deneyde, tamamlayıcı aracılı hemoliz serbest hemoglobin spektrofotometrik tespit yapma kullanımı ölçülür. Bu tahlillerin ikisi de antikorun neden olduğu kompleman aktivasyon çalışmalar sağlayarak yeniden üretilebilen ve kantitatif bulunmaktadır.

Şekil 2A RBCs için temsili bir dağılım grafiği göstermektedir. FSC-A arsa (P1) - Tek RBCs için uygun yolluk de FSC-W görülebilir. RBCs genellikle% 95 civarında bu P1 kapısı (tek hücre) içinde kalan, ancak hasta serumunda yüksek oranda kullanıldığında anti-RBC IgM hasta serumunda mevcut olduğunu, özellikle bu,% 70-80 düşüş olabilir. C4 birikimi ile ilgili AIHA hasta serumunun etkisi için Örnek sonuçlar Şekil 2B&#39;de ve ...

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

Burada, kırmızı kan hücrelerine karşı antikorlar tarafından neden olunan kompleman aktivasyon ölçmek için iki tahlil tarif eder. Mevcut deneylerde üzerinde en büyük avantajı, nicel ve kolay yorumlamak doğasıdır.

Kırmızı kan hücreleri üzerinde antikor kaynaklı kompleman aktivasyon sayısal tespiti için Yöntemler

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 Görüntüleme.  University of Amsterdam.  Burada, kırmızı kan hücrelerine karşı antikorlar tarafından neden olunan kompleman aktivasyon ölçmek için iki tahlil tarif eder. Mevcut deneylerde üzerinde en büyük avantajı, nicel ve kolay yorumlamak doğasıdır. 

Video: 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.

Kompleman tükenmesi Özel Hücre Populasyonlarının tükenmesi

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

İmmünoloji ve Enfeksiyon

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.

Fare Periton Boşluk Hücreleri İzolasyon

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.

Murin Model B hücreleri CD40 aktivasyonu

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.

Toll-like reseptör Etkinleştirme Düzenlenmesi Neu1 Sialidase Faaliyet Algılama

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).

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

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.

Rekombinant RBD tabanlı SARS Aşılar için Protokol: Protein hazırlanması, Hayvan Aşılama ve Nötralizasyon Algılama

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.

Gribi için yüksek verimlilik Tespit Yöntemi

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.

Antijenler Kompakt Hyperbranched Polyglycerols Of Aşılama Membran By Fonksiyonel Kırmızı Kan Hücreleri Korumalı

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.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

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 fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

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