Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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Bu çalışma Tunus Sağlık Bakanlığı ve Tunus Yüksek Öğretim ve Bilimsel Araştırma Bakanlığı tarafından finanse edilmiştir.

Bu çalışma, Tunus Pasteur Enstitüsü etik kurulu tarafından oluşturulan etik yönlere uygun olarak yürütülmüştür.
1. ELISA öncesi adımlar: Önkoşullar ve ön işleme
<ol><li>Bakteri suşları ve büyüme ortamı NOT: Bu çalışmada kullanılan Mollicutes türleri ve duvarlı bakteriler ile bunların büyüme ortamları Tablo 1'de listelenmiştir.<ol><li>Mollicutes türlerinin büyümesi<ol><li>Ortamın 1.800 μL'sinde her türün gliserol ...

<ol>
	<li>Razin, S., Yogev, D., Naot, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+and+pathogenicity+of+mycoplasmas.">Molecular biology and pathogenicity of mycoplasmas.</a> Microbiology and Molecular Biology Reviews. 62 (4), 1094-1156 (1998).</li><li>Chanock, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+infections+of+man.">Mycoplasma infections of man.</a> The New England Journal of Medicine. 273 (22), 1199-1206 (1965).</li><li>Braun, G. S., Wagner, K. S., Huttner, B. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+enzyme-linked+immunosorbent+assay+for+the+early+diagnosis+of+Mycoplasma+pneumoniae+infection.">Use of the enzyme-linked immunosorbent assay for the early diagnosis of Mycoplasma pneumoniae infection.</a> European Journal of Clinical Microbiology. 3 (2), 116-121 (1984).</li><li>Raisanen, S. M., Suni, J. 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P., Shaw, E. J., Lemcke, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+analysis+of+Mycoplasma+pneumoniae:+3.+Separation+and+chemical+identification+of+serological+active+lipids.">Immunochemical analysis of Mycoplasma pneumoniae: 3. Separation and chemical identification of serological active lipids.</a> Australian Journal of Experimental Biology and Medical Science. 47 (2), 171-195 (1969).</li><li>Ponka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+and+clinical+picture+of+serologically+verified+Mycoplasma+pneumoniae+infections+with+emphasis+on+central+nervous+system,+cardiac+and+joint+manifestations.">The occurrence and clinical picture of serologically verified Mycoplasma pneumoniae infections with emphasis on central nervous system, cardiac and joint manifestations.</a> Annals of Clinical Research. 11, 1-60 (1979).</li><li>Bradford, M. 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Procedure and some applications.</a> Proceedings of National Academy of Sciences. 76 (9), 4350-4354 (1979).</li><li>Ben Abdelmoumen, B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-linked+immunosorbent+assay+for+detection+of+avian+mycoplasmas+in+culture.">An enzyme-linked immunosorbent assay for detection of avian mycoplasmas in culture.</a> Avian Diseases. 39, 85-93 (1995).</li><li>Fawcett, P. T., O'Brien, A. E., Doughty, R. 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D., Clough, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extragenital+Mycoplasma+hominis+infections+in+adults:+emphasis+on+immunosuppression.">Extragenital Mycoplasma hominis infections in adults: emphasis on immunosuppression.</a> Clinical Infectious Diseases. 17, 243-249 (1993).</li><li>Pitcher, D. G., Nicholas, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+host+specificity:+Fact+or+fiction.">Mycoplasma host specificity: Fact or fiction.</a> Veterinary Journal. 170 (3), 300-306 (2005).</li><li>Lierz, M., Jansen, A., Hafez, H. 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L., Madoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mycoplasma+species+from+a+patient+with+seal+finger.">Isolation of Mycoplasma species from a patient with seal finger.</a> Clinical Infectious Diseases. 27 (5), 1168-1170 (1998).</li><li>Slack, M., P, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+role+of+Haemophilus+influenzae+in+community-acquired+pneumonia.">A review of the role of Haemophilus influenzae in community-acquired pneumonia.</a> Pneumonia. 6 (1), 26-43 (2015).</li><li>Janira Avire, N., Whiley, H., Ross, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+Streptococcus+pyogenes:+public+health+risk+factors,+prevention+and+control.">A review of Streptococcus pyogenes: public health risk factors, prevention and control.</a> Pathogens. 10 (2), 248 (2021).</li><li>Steinitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+the+blocking+effect+of+Tween+20+and+bovine+serum+albumin+in+ELISA+microwells.">Quantitation of the blocking effect of Tween 20 and bovine serum albumin in ELISA microwells.</a> Analytical Biochemistry. 282 (2), 232-238 (2000).</li><li>Tully, J. G., Whitcomb, R. F., Clark, H. F., Williamson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+mycoplasmas:+cultivation+and+vertebrate+pathogenicity+of+a+new+spiroplasma.">Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma.</a> Science. 195 (4281), 892-894 (1977).</li><li>Frey, M. L., Hanson, R. P., Andrson, D. 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Bu makale, esas olarak spesifik taramayı sağlamak için geliştirilen kurum içi ELISA'nın genel bir tanımını sunmaktadır. M. pneumoniae enfeksiyon. ELISA testinin kendisinin protokolü ve bazı işlem öncesi ve sonrası adımlar hakkında ayrıntılar sağlanmaktadır. Bu tahlilin özgüllüğü adsorpsiyon tekniği ile sağlanır. Bu prosedür daha önce insan ve kuş tanısı için geliştirilen ELISA testlerinde tanımlanmıştır. Mycoplasmas17,<sup class=...

Mikoplazmalar bilinen en küçük ve en basit prokaryotlar arasındadır. Esas olarak bir hücre duvarı yapısının olmaması ile diğer bakterilerden ayırt edilirler. Böylece, Mikoplazmalar Mollicutes1 adlı ayrı bir sınıfta sınıflandırıldı. Hücre duvarı eksikliği, bazı antimikrobiyal ajanlara karşı bu mikroorganizmalara içsel direnç kazandırır ve polimorfizmlerinden büyük ölçüde sorumludur. Mikoplazmalar , metabolik ve biyosentetik yeteneklerini sınırlayan ve parazitik ve saprofitik doğalarını açıklayan küçük genoma ve küçültülmüş boyuta sahiptir1.
Mycoplasma pneumoniae, insanı enfekte eden Mikoplazmalardan biridir ve en virülan2 olduğu düşünülmektedir. M. pneumoniae, üst solunum yollarını kolonize ederek çocuklarda ve genç erişkinlerde atipik pnömoniye yol açar. M. pneumoniae enfeksiyonunun yol açtığı klinik bulgular grip benzeri, baş ağrısı, ateş ve öksürük3'tür. M. pneumoniae'nin konakçı hücrelere olan sitaderansına, P1 majör adezyonu ve birkaç aksesuar proteini içeren bir ataşman organeli aracılık eder 4,5. M. pneumoniae'nin hava yolu mukozasına yakın yapışmasından kaynaklanan lokal inflamasyon ve konakçı bağışıklık sisteminin uyarılması nedeniyle daha fazla klinik bulgu ortaya çıkabilir6. Pnömoni, M. pneumoniae enfeksiyonunun ayırt edici bir özelliği olmasına rağmen, bu bakteri ile enfeksiyonun, merkezi sinir sistemi, kalp, cilt ve eklemler gibi farklı anatomik bölgelerde geniş bir pulmoner olmayan bulgular spektrumundan da sorumlu olabileceği ortaya konmuştur7.
Tüm Mycoplasma türlerine gelince, M. pneumoniae'nin teşhisi zordur. Mikoplazmozu çağrıştıran klinik bulgular çoğunlukla görünmez ve karakteristik değildir8. M. pneumoniae enfeksiyonunu sadece klinik bulgulara ve semptomlara dayanarak teşhis etmek çok zor olduğundan, laboratuvar taraması özellikle ilgi çekicidir9. M. pneumoniae kolonilerinin kültür yoluyla saptanması doğru tanı için altın standart yöntemdir. Bununla birlikte, titiz büyüme gereksinimleri ve kesin sonuçların verilmesi için gereken uzun süre (1-2 hafta) kültürü karmaşıklaştırır ve bu nedenle rutin tanı için nadiren kullanıldığı anlamına gelir10. Nükleik asit amplifikasyon teknolojileri hız ve verimlilik açısından doğrulanmıştır, ancak bazı sağlık tesislerinde nispeten yüksek maliyetleri ve kullanılamamaları nedeniyle, bu moleküler teknikler birinci basamak tanı testleri olarak kabul edilmemektedir. Ticari PCR testlerinin M. pneumoniae enfeksiyonlarını teşhis etmek için yaygın olarak kullanıldığı doğrudur, ancak yine de serolojinin yerini alamazlar. Ayrıca, hem yanlış negatif hem de yanlış pozitif sonuçların sık görülmesi, PCR9'un kullanımını sınırlamıştır. Rutin olarak, seroloji, M. pneumoniae enfeksiyonunun teşhisi için laboratuvarlarda en çok uygulanan yöntem olmaya devam etmektedir. Soğuk hemaglutininler, kompleman fiksasyon testi11, dolaylı hemaglutinasyon testi 12, immünofloresan 13 ve ilk olarak 1980'lerin başında mikoplazma serolojisine uygulanan ELISA teknolojisi14,15,16 gibi çeşitli seroloji yaklaşımları on yıllardır bildirilmiştir. M. pneumoniae enfeksiyonunun ELISA serodiagnostiği yapılırken karşılaşılan en önemli sorunlardan biri, tekniğin özgüllüğünü önemli ölçüde azaltan çapraz reaksiyonlardır. İnsan serumlarının M. pneumoniae antijenleri ile spesifik olmayan adsorpsiyonu daha önce bildirilmiştir; Aslında, insan serumlarında ELISA tarafından tespit edilen antikorların çoğu, M. pneumoniae'nin bazı bakteriler 18,19 ve bazı hayvan ve insan dokuları20 ile ortaklığından dolayı her zaman mikoplazmal antijenlere 17 bağlı olmayabilir.
Laboratuvarda uygulanan geleneksel ELISA testinde gözlenen yüksek arka plan okumaları nedeniyle, sonuçların yorumlanması genellikle karmaşıktı ve bu nedenle uygun bir M. pneumoniae tanısının verilmesi zor bir görevdi. Bu sorunla karşı karşıya kalırken, M. pneumoniae antijenlerinin spesifik olmayan reaksiyonlarını test edilecek antikorlarla ortadan kaldırarak M. pneumoniae ELISA'yı iyileştirmeyi seçtik. Bu amaçla, adsorpsiyon tekniğini kullanarak spesifik olmayan M. pneumoniae antijenlerinin seçici tükenmesi üzerinde çalıştık. Aslında, antijen yakalama ELISA'nın temel amacı, insan serum örneklerinde M. pneumoniae immunoglobulin (Ig) G'yi spesifik olarak tespit etmektir. Bu ELISA kavramı, esas olarak, insan serum örneklerini eklemeden önce, M. pneumoniae-spesifik antijenlerin seçici olarak yakalanmasından oluşur. Bu seçicilik, laboratuvarda tavşanlarda üretilen M. pneumoniae ham antijeninin M. pneumoniae poliklonal antiserum ile inkübe edilmesi ve Mollicutes sınıfına ait olan veya olmayan heterolog bakterilerden oluşan bir panele karşı adsorpsiyon yoluyla türe özgü hale getirilmesi, antijenlerin M. pneumoniae ile paylaşılmasıyla sigortalanır. türler ve / veya solunum yollarını kolonize ettiği bilinmektedir. Adsorpsiyon prosedürü üç kez tekrarlandı ve çapraz reaktiviteyi ortadan kaldırma etkinliği immünoblotlama ile test edildi. Geliştirilen ELISA testi, sandviç ve dolaylı ELISA'nın bir kombinasyonudur. Kısaca, ELISA plakasının kuyucukları ilk önce M. pneumoniae'ye özgü bir poliklonal antiserum ile kaplanır. Daha sonra, M. pneumoniae antijeni eklenir ve antiserum ile test edilecek serum örneğinde bulunan antikorlar arasında sıkışır. Oluşan immünolojik kompleks, ikincil bir enzim konjuge antikoru (peroksidaz konjuge IgG) ile tespit edilir. Reaksiyonlar kromojenik substratın eklenmesiyle görselleştirilir ve absorbans spektrofotometrik olarak ölçülür. Bu kurum içi ELISA, şematik olarak Şekil 1'de sunulmuştur. Ev yapımı ELISA, M. pneumoniae enfeksiyonunu spesifik olarak tespit etmede etkili olduğunu kanıtladı ve şu anda rutin tanı aktivitesinde en çok uygulanan testlerden biridir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-chloro-1-naphtol</td>
			<td>Sigma-Aldrich</td>
			<td>C6788-50 TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50 Cap</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL1066&#160;</td>
			<td>VWR</td>
			<td>P0058-N1L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco PPLO Broth</td>
			<td>BD</td>
			<td>255420</td>
			<td>Frey media</td>
		</tr>
		<tr>
			<td>ELISA plate-Reader</td>
			<td>MULTISKAN GO, Thermo Scientific</td>
			<td>Ref: 51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Capricorn Scientific</td>
			<td>FBS-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-human IgG</td>
			<td>Abcam</td>
			<td>ab6759</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>656120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 37%</td>
			<td>Prolab</td>
			<td>2025.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), solution 30%</td>
			<td>Scharlau</td>
			<td>HI01361000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Prepared in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Mycoplasma pneumoniae polyclonal antiserum produced in rabbit</td>
			<td>Produced in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Serum was produced by rabbit immunization at the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Nicotinamide adenine dinucleotide</td>
			<td>Sigma-Aldrich</td>
			<td>N7004-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Maxisorp flat-bottom 96-well microtiter plate&#160;</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium (1 MIU)</td>
			<td>PANPHARMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>fluka chemika</td>
			<td>77660</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>MP Biomedicals</td>
			<td>902887</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Novachim</td>
			<td>PS02805</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4) 95-97%</td>
			<td>Merck</td>
			<td>1007311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>USB</td>
			<td>22215</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate (3,3&#8217;, 5,5&#8217; TetraMethyl-Benzidine solution)</td>
			<td>Abcam</td>
			<td>ab142042</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6791-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>1379-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, powder, Ultrapure</td>
			<td>Thermo Scientific&#160;</td>
			<td>J23547&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae

Mycoplasma pneumoniae, esas olarak insan solunum yollarını kolonize ettiği ve endemik olduğu bilinen, daha büyük çocuklarda ve genç erişkinlerde her 6 yılda bir epidemik zirvelere ulaşan, hücre duvarı eksikliği olan bir prokaryottur. M. pneumoniae'nin teşhisi, patojenin titiz doğası ve asemptomatik taşıma olasılığı nedeniyle zordur. M. pneumoniae enfeksiyonunun hastaların serum örneklerinde antikor titrasyonuna dayalı laboratuvar tanısı en çok uygulanan yöntem olmaya devam etmektedir. M. pneumoniae için poliklonal serum kullanımı ile immünolojik çapraz reaktivitenin potansiyel problemi nedeniyle, serolojik tanının özgüllüğünü arttırmak için antijen yakalama enzimine bağlı immünosorbent testi (ELISA) geliştirilmiştir. ELISA plakaları M. pneumoniae poliklonal antikorları ile kaplanır, tavşanlarda yetiştirilir ve M. pneumoniae türleri ile antijenleri paylaşan ve / veya solunum yollarını kolonize ettiği bilinen heterolog bakterilerden oluşan bir panele karşı adsorpsiyondan sonra spesifik hale getirilir. Reaksiyona giren M. pneumoniae homolog antijenleri daha sonra serum örneklerinde karşılık gelen antikorları tarafından özel olarak tanınır. Antijen yakalama ELISA'sının maruz kaldığı fizikokimyasal parametrelerin daha fazla optimizasyonu, oldukça spesifik, hassas ve tekrarlanabilir bir ELISA'ya yol açmıştır.

Adsorbe edilmemiş poliklonal Mycoplasma pneumoniae antiserumunun heterolog bakterilere karşı immünoblotlama aktivitesi Çapraz reaktivite, immünoblotlama sonuçlarında (Şekil 2) görüldüğü gibi gerçekten de mevcuttur ve pozitif kontrol (Şerit 1) ile karşılaştırıldığında, M. pneumoniae antijenlerinin bir kısmı taranan bakterilerle paylaşılmaktadır. Bu çapraz reaksiyonların yoğunluğu değişke...

Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Mycoplasma pneumoniae enfeksiyonunda, seroloji testleri immünolojik çapraz reaksiyon nedeniyle düşük özgüllükle iyi sonuçlar verebilir. Bu yazıda açıklanan kurum içi antijen yakalama ELISA, yüksek tür özgüllüğünü garanti eder ve M. pneumoniae'nin doğru teşhisi için güvenilir bir tarama testi olduğu gösterilmiştir.

Mycoplasma pneumoniae'nin Spesifik Tespiti için Antijen Yakalama Enzimine Bağlı İmmünosorbent Testi

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks ...

Tüm mikoplazma hastalarına gelince, çapraz reaksiyonlar nedeniyle Mikoplazma pnömonisi tanısı zordur. Önerdiğimiz ELISA, bir adsorpsiyon tekniği kullanılarak spesifik olmayan Mikoplazma pnömoni antijenlerinin seçici tükenmesine dayanmaktadır. Bu videoda açıklanan şirket içi antijen yakalama ELISA, yüksek ferah özgüllüğü garanti eder.Ve aslında, Mikoplazma pnömoni enfeksiyonunun doğru teşhisi için güvenilir bir taramalı test olduğu kanıtlanmıştır. ELISA öncesi adımları gösteren, doktora öğrencisi Imen Chniba olacak. Ve ELISA adımları, laboratuvarımızdan bir teknisyen olan Nadine Khadraoui olacak.Antijen adsorpsiyon prosedürüne başlamak için, 12 heterolog bakteriyel antijeni bir mikrosantrifüj tüpüne çekin ve adsorbe edilecek antiserumun yarısına karşılık gelen protein konsantrasyonunu ayarlayın. Daha sonra, peleti toplamak için antijen karışımını 14.000 G'de dört santigrat derecede 10 dakika boyunca santrifüj edin. Daha sonra, peleti saflaştırılmış poliklonal anti-Mycoplasma pneumoniae IgG ile tekrar askıya alın ve yavaş ajitasyonla 37 santigrat derecede iki saat inkübe edin.Kuluçka sonunda, süspansiyonu dört santigrat derecede 10 dakika boyunca 14.000 G'de tekrar santrifüj edin ve spesifik poliklonal Mycoplasma pneumoniae antiserumuna karşılık gelen süpernatantı geri kazanın. 96 kuyucuklu bir ELISA plakasını yakalama antikoru ile kaplamak için, antikoru mililitre başına 10 mikrograma ve pH 9.6'da 0.1 molar karbonat bikarbonat tamponuna seyreltin. Daha sonra, mikroplakanın her bir kuyucuğuna seyreltilmiş antikorun 100 mikrolitresini ekleyin ve gece boyunca dört santigrat derecede inkübe edin.Ertesi gün, kaplama çözeltisini çıkarın ve plakayı yıkama tamponu ile beş kez yıkayın. Daha sonra, kaplanmış kuyucukların bağlanmamış yüzeylerini, her bir kuyucuğa 100 mikrolitre blokaj çözeltisi ekleyerek bloke edin. Plakayı oda sıcaklığında bir saatten inkübe ettikten sonra, bloke edici çözeltiyi bir pipetle çıkarın ve plakayı daha önce gösterildiği gibi yıkayın.Kuyucuk başına 10 nanogram konsantrasyonda eşit miktarda Mycoplasma pneumoniae bütün proteinleri ekleyin ve antijen-antikor reaksiyonunun oda sıcaklığında iki saat boyunca gerçekleşmesine izin verin. Kuluçkayı takiben, fazla antijeni çıkarın ve plakayı gösterildiği gibi yıkayın. Daha sonra, insan serum test örneklerini seyreltin ve pozitif ve negatif serum kontrollerine referans verin.Daha sonra, seyreltilmiş test numunelerinden 100 mikrolitre ekleyin ve uygun kuyucuklara ve kopyalara kontroller yapın. Ne antijen ne de serum içeren kuyucukları boş olarak işaretleyin. Plakayı oda sıcaklığında 90 dakika inkübe ettikten sonra, serum çözeltisini çıkarın ve plakayı yıkama tamponu ile beş kez yıkayın.Pipet, uygun seyreltilmiş enzimin 100 mikrolitresi, her bir kuyucuğa konjuge tespit antikorlarını konjuge eder ve karanlıkta 37 santigrat derecede bir saat boyunca inkübe eder. Bağlanmamış algılama gövdelerini çıkardıktan ve plakayı yıkadıktan sonra, antijen-antikor bağlanmasını görselleştirmek için 100 mikrolitre TMB Kromojen çözeltisi ekleyin. Plakanın oda sıcaklığında 30 dakika boyunca gelişmesine izin verin.Ardından, 100 mikrolitre% 7.5 sülfürik asit ekleyerek reaksiyonu durdurun. Son olarak, bir mikroplaka okuyucu kullanarak 450 nanometredeki absorbansı okuyun ve sonuçları pozitiflik indeksinin hesaplanmasına göre sıralayın. Mycoplasma pneumoniae non adsorbe poliklonal antiserum ve heterolog bakteriyel antijenler arasındaki immünoblot analizinde çapraz reaktivite saptandı, ancak değişen yoğunlukta.Örneğin, Mycoplasma gallisepticum ve Mycoplasma imitans antijenleri, kuş mikoplazmaları olmasına rağmen en güçlü reaktiviteyi göstermiştir. Buna karşılık, Ureaplasma urealyticum'un iki insan klinik izolatından hiçbiri herhangi bir reaksiyon göstermedi. Bununla birlikte, kalan insan genital mikoplazma türlerinin ve diğer bakteriyel antijenlerin antijenleri, antiserum ile önemli çapraz reaksiyonlar sağlamıştır.İmmünoblot analizi, Mycoplasma pneumoniae poliklonal antiserum adsorpsiyonunun heterolog bakteriyel antijenlere karşı etkinliğini doğruladı. Adsorpsiyon prosedürü tüm çapraz reaksiyonları ortadan kaldırdı, böylece Mycoplasma pneumoniae antiserumunu sonraki tüm serolojik ve immünoblotlama testleri için spesifik hale getirdi. Ayrıca, bu çalışmada geliştirilen antijen yakalama ELISA kullanılarak test edilen insan serumları seti, iyi bir özgüllüğe sahip Mycoplasma pneumoniae IgG için pozitif olduğunu kanıtladı.Bu ELISA'nın özgüllüğü esas olarak adsorpsiyon tekniği ile sigortalanır, çünkü bu kritik adımı dikkatli bir şekilde gerçekleştirmek ve en az iki veya üç kez tekrarlamak ilkeldir. Bu tekniğin prensibi, diğer mikoplazma türlerini veya genel olarak diğer bakterileri teşhis etmek için uygulanabilir. Bununla birlikte, adsorpsiyon için bakteriyel antijenlerin seçimi alakasız ve yeterli olmalıdır.

antigen-capture-enzyme-linked-immunosorbent-assay-for-specific

Research

JoVE Journal

Biology

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Protein Ubikitinasyon Tespiti

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biyoloji

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Mikotoksin Algılama ELIME (Enzim İmmüno Manyetik Elektrokimyasal Bağlantılı) Yöntem

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Genetik Dernekleri Fonksiyonel Dayanak Test bir Allel özel Gen İfadesi Testi

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Erken evre Fare Embriyolar kullanarak Doku özel Genler Kromatin Immunoprecipitation Testi

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma optimize edilmiş PCR tabanlı Algılama

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Kültür Hücrelerinin Intra-ve Ekstrasellüler Askorbat Belirlenmesi için hızlı ve spesifik bir Mikroplate Deneyi

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Sıtma verici Sivrisinekler için bir çok algılama Deneyi

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Floresan etiketli Nanopartiküller kullanma Fare, İnsan ve domuz Menşe Canlı hücreler hücre içi Gen İfade Algılama

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Siteye Özgü DNA Bölünmesi için Paralel Yüksek İş Kinetik Test

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.