Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
Uploaded: 2023-02-24T12:40:00
Description: Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

이 연구는 튀니지 보건부와 튀니지 고등 교육 및 과학 연구부의 자금 지원을 받았습니다.

본 연구는 튀니스 파스퇴르 연구소의 윤리위원회가 설립 한 윤리적 측면에 따라 수행되었습니다.
1. 사전 ELISA 단계: 전제 조건 및 전처리
<ol><li>박테리아 균주 및 성장 배지 참고: 본 연구에 사용된 Mollicutes 종과 벽으로 둘러싸인 박테리아 및 성장 배지는 표 1에 나열되어 있습니다.<ol><li>연체 동물의 성장<ol><li>각 종의 글리세롤 스톡으로부터 200 μL...

<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. D., Schmid, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae:+Usual+suspect+and+unsecured+diagnosis+in+the+acute+setting.">Mycoplasma pneumoniae: Usual suspect and unsecured diagnosis in the acute setting.</a> The Journal of Emergency Medicine. 30 (4), 371-375 (2006).</li><li>Baseman, J. B., Cole, R. M., Krause, D. C., Leith, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+cytadhesion+of+Mycoplasma+pneumoniae.">Molecular basis for cytadhesion of Mycoplasma pneumoniae.</a> Journal of Bacteriology. 151, 1514-1522 (1982).</li><li>Waldo, R. H., Krause, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+stability,+and+function+of+cytadhesin+P1+and+accessory+protein+B/C+complex+of+Mycoplasma+pneumoniae.">Synthesis, stability, and function of cytadhesin P1 and accessory protein B/C complex of Mycoplasma pneumoniae.</a> Journal of Bacteriology. 188 (2), 569-575 (2006).</li><li>Waites, K. B., Balish, M. F., Atkinson, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+and+detection+of+Mycoplasma+pneumoniae+infections.">New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.</a> Future Microbiology. 3 (6), 635-648 (2008).</li><li>Narita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+extrapulmonary+manifestations+of+Mycoplasma+pneumoniae+infection+with+special+reference+to+pneumonia.">Pathogenesis of extrapulmonary manifestations of Mycoplasma pneumoniae infection with special reference to pneumonia.</a> Journal of Infection and Chemotherapy. 16 (3), 162-169 (2010).</li><li>Kashyap, S., Sarkar, 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+pneumonia:+Clinical+features+and+management.">Mycoplasma pneumonia: Clinical features and management.</a> Lung India. 27 (2), 75-85 (2010).</li><li>Zhang, L., Zong, Z. Y., Liu, Y. B., Ye, H., Lv, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+versus+serology+for+diagnosing+Mycoplasma+pneumoniae+infection:+A+systematic+review+&amp;+meta-analysis.">PCR versus serology for diagnosing Mycoplasma pneumoniae infection: A systematic review &amp; meta-analysis.</a> Indian Journal of Medical Research. 134 (3), 270-280 (2011).</li><li>Saraya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae+infection:+Basics.">Mycoplasma pneumoniae infection: Basics.</a> Journal of General and Family Medicine. 18 (3), 118-125 (2017).</li><li>Jacobs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+diagnosis+of+Mycoplasma+pneumoniae+infections:+a+critical+review+of+current+procedures.">Serological diagnosis of Mycoplasma pneumoniae infections: a critical review of current procedures.</a> Clinical Infectious Diseases. 17, 79-82 (1993).</li><li>Kok, T. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+pneumoniae+infections:+diagnosis+based+on+immunofluorescence+titer+of+IgG+and+IgM+antibodies.">Mycoplasma pneumoniae infections: diagnosis based on immunofluorescence titer of IgG and IgM antibodies.</a> Mayo Clinic Proceedings. 61 (10), 830-831 (1986).</li><li>Busolo, F., Tonin, E., Conventi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay+for+detection+of+Mycoplasma+pneumoniae+antibodies.">Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antibodies.</a> Journal of Clinical Microbiology. 12 (1), 69-73 (1980).</li><li>Van Griethuysen, A. 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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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이 백서는 주로 특정 스크리닝을 보장하기 위해 개발된 사내 ELISA에 대한 일반적인 설명을 제공합니다. M. pneumoniae 감염. ELISA 분석 자체의 프로토콜과 일부 전처리 및 후처리 단계에 대한 세부 정보가 제공됩니다. 이 분석의 특이성은 흡착 기술에 의해 보장됩니다. 이 절차는 이전에 인간 및 조류 진단을 위해 개발 된 ELISA 검사에서 설명되었습니다. Mycoplasmas17,</sup...

마이코플라스마 는 알려진 가장 작고 단순한 원핵생물 중 하나입니다. 그들은 주로 세포벽 구조가 없기 때문에 다른 박테리아와 구별됩니다. 따라서 마이코 플라스마는 Mollicutes1이라는 별도의 클래스로 분류되었습니다. 세포벽 결핍은 일부 항균제에 대해 이러한 미생물에 대한 내재적 내성을 부여하며 다형성에 크게 책임이 있습니다. 마이코플라스마는 게놈이 작고 크기가 작아 대사 및 생합성 능력을 제한하고 기생 및 부생균 특성을 설명합니다1.
마이코플라스마 폐렴은 사람을 감염시키는 마이코플라스마 중 하나이며 가장 독성이 강한 것으로 생각됩니다2. M. pneumoniae는 상부 호흡기를 식민지화하여 어린이와 젊은 성인의 비정형 성 폐렴을 유발합니다. M. pneumoniae 감염으로 인한 임상 징후는 두통, 발열 및 기침과 함께 독감과 유사합니다3. 숙주 세포에 대한 M. pneumoniae의 세포 부착은 P1 주요 접착 및 여러 보조 단백질 4,5를 포함하는 부착 소기관에 의해 매개됩니다. 기도 점막에 대한 M. pneumoniae의 친밀한 부착으로 인한 숙주 면역계의 국소 염증 및 자극으로 인해 더 많은 임상 증상이 발생할 수 있습니다6. 폐렴은 M. pneumoniae 감염의 특징이지만,이 박테리아에 의한 감염은 또한 중추 신경계, 심장, 피부 및 관절과 같은 다양한 해부학 적 부위에서 광범위한 비 폐 증상을 유발할 수 있음이 밝혀졌습니다7.
모든 마이코플라스마 종에 관해서는 M. pneumoniae의 진단이 어렵습니다. 마이코 플라스마 증을 유발하는 임상 징후는 대부분 명백하지 않고 특징이 없습니다8. 임상 증상과 증상에만 의존하여 M. pneumoniae 감염을 진단하는 것은 매우 어렵 기 때문에 실험실 스크리닝이 특히 중요합니다9. 배양으로 M. pneumoniae 콜로니를 검출하는 것은 적절한 진단을위한 황금 표준 방법입니다. 그러나 까다로운 성장 요구 사항과 최종 결과 (1-2 주)를 제공하는 데 필요한 긴 시간은 문화를 복잡하게 만들어 일상적인 진단에 거의 사용되지 않음을 의미합니다10. 핵산 증폭 기술은 속도와 효율성 측면에서 검증되었지만 상대적으로 높은 비용과 일부 의료 시설에서 사용할 수 없기 때문에 이러한 분자 기술은 1 차 진단 테스트로 간주되지 않습니다. 상용 PCR 검사가 M. pneumoniae 감염을 진단하는 데 널리 사용되는 것은 사실이지만 여전히 혈청학을 대체 할 수는 없습니다. 또한 위음성 및 위양성 결과가 모두 자주 발생하여 PCR9의 사용이 제한되었습니다. 일상적으로, 혈청학은 M. pneumoniae 감염의 진단을 위해 실험실에서 가장 많이 시행되고 있습니다. 차가운 혈구 응집소, 보체 고정 검사 11, 간접 혈구 응집 검사 12, 면역 형광검사 13 및 1980 년대 초 마이코 플라스마 혈청학에 처음 적용된 ELISA 기술과 같은 여러 혈청 학적 접근법이 수십 년 동안보고되었습니다14,15,16. M. pneumoniae 감염의 ELISA 혈청 진단을 수행 할 때 발생하는 주요 문제 중 하나는 교차 반응이며, 이는 기술의 특이성을 상당히 낮 춥니 다. M. pneumoniae 항원을 사용한 인간 혈청의 비특이적 흡착은 이전에 보고되었습니다. 사실, 인간 혈청에서 ELISA에 의해 검출된 많은 항체는 일부 박테리아(18,19) 및 일부 동물 및 인간 조직(20)과의 M. pneumoniae의 공통성으로 인해 마이코플라스마 항원(17)에 항상 결합되지 않을 수 있다.
실험실에서 시행 된 기존의 ELISA 검사에서 관찰 된 높은 배경 판독 값으로 인해 결과 해석이 종종 복잡했기 때문에 적절한 M. pneumoniae 진단을 전달하는 것은 어려운 과제였습니다. 이 문제에 직면하는 동안 우리는 테스트 할 항체와 M. pneumoniae 항원의 비특이적 반응을 제거하여 M. pneumoniae ELISA를 개선하기로 결정했습니다. 이를 위해 흡착 기술을 사용하여 비특이적 M. pneumoniae 항원의 선택적 고갈에 대해 연구했습니다. 사실, 항원 포획 ELISA의 주요 목표는 인간 혈청 샘플에서 M. pneumoniae 면역 글로불린 (Ig) G를 특이 적으로 검출하는 것입니다. 이 ELISA의 개념은 주로 인간 혈청 샘플을 추가하기 전에 M. pneumoniae 특이 항원의 선택적 포획으로 구성됩니다. 이 선택성은 실험실에서 토끼에서 생산되는 M. pneumoniae polyclonal antiserum과 함께 M. pneumoniae 원유 항원을 배양하고 Mollicutes 클래스에 속하거나 속하지 않는 이종 박테리아 패널에 대한 흡착에 의해 종 특이 적으로 만들어 M. pneumoniae 와 항원을 공유함으로써 보장됩니다. 종 및/또는 호흡기를 식민지화하는 것으로 알려져 있습니다. 흡착 절차를 세 번 반복하고, 교차 반응을 제거하는 그의 효율을 면역 블로팅에 의해 시험하였다. 개발된 ELISA 분석은 샌드위치와 간접 ELISA의 조합입니다. 간단히 말해서, ELISA 플레이트의 웰은 먼저 M. pneumoniae에 특이적인 다클론 항혈청으로 코팅됩니다. 이어서, M. pneumoniae 항원을 첨가하고, 항혈청과 시험될 혈청 샘플에 존재하는 항체 사이에 포획한다. 형성된 면역 복합체는 2 차 효소 - 접합 된 항체 (퍼 옥시다제 - 접합 된 IgG)에 의해 검출된다. 반응은 발색 기질의 첨가에 의해 시각화되고, 흡광도는 분광 광도계로 측정된다. 이 사내 ELISA는 그림 1에 개략적으로 나와 있습니다. 수제 ELISA는 M. pneumoniae 감염을 특이적으로 감지하는 데 효율적인 것으로 입증되었으며 현재 일상적인 진단 활동에서 가장 많이 시행되는 테스트 중 하나입니다.

<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 는 세포벽 결핍 원핵생물로, 주로 인간의 호흡기를 식민지화하고 풍토병으로 알려져 있으며 6년마다 전염병이 최고조에 달하는 것으로 알려져 있습니다. M. pneumoniae 의 진단은 병원균의 까다로운 특성과 무증상 운송의 가능성 때문에 어렵습니다. 환자의 혈청 샘플에서 항체 적정에 기초한 M. pneumoniae 감염의 실험실 진단은 가장 많이 시행되는 방법으로 남아 있습니다. M. pneumoniae에 대한 다클론 혈청의 사용으로 인한 면역 학적 교차 반응의 잠재적 인 문제 때문에 혈청 학적 진단의 특이성을 향상시키기 위해 항원 포획 효소 결합 면역 흡착 분석 (ELISA)이 개발되었습니다. ELISA 플레이트는 M. pneumoniae polyclonal 항체로 코팅되고, 토끼에서 자라며, M. pneumoniae 종과 항원을 공유하거나 호흡기를 식민지화하는 것으로 알려진 이종 박테리아 패널에 대해 흡착 후 특이적으로 렌더링됩니다 . 반응된 M. pneumoniae 상동 항원은 혈청 샘플에서 그의 상응하는 항체에 의해 특이적으로 인식된다. 항원-포획 ELISA가 적용되는 물리화학적 파라미터의 추가 최적화는 매우 특이적이고 민감하며 재현 가능한 ELISA를 유도했습니다.

이종 박테리아에 대한 흡착되지 않은 다클론 마이코플라스마 폐렴 항혈청의 면역블로팅 활성 교차 반응은 면역블로팅 결과(그림 2)에서 입증된 바와 같이 실제로 존재하며 양성 대조군(레인 1)과 비교하여 일부 M. pneumoniae 항원은 선별된 박테리아와 공유됩니다. 이러한 교차 반응의 강도는 다양했습니다. 예를 들어, M. ...

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

마이코플라스마 폐렴 감염에서 혈청 검사는 좋은 결과를 생성할 수 있지만 면역학적 교차 반응으로 인해 특이도가 낮습니다. 이 논문에 설명된 사내 항원 포획 ELISA는 높은 종 특이성을 보장하며 M. pneumoniae의 정확한 진단을 위한 신뢰할 수 있는 선별 검사로 나타났습니다.

마이코플라스마 폐렴의 특이적 검출을 위한 항원-포획 효소 결합 면역흡착 분석

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

모든 마이코플라스마의 환자와 마찬가지로 마이코플라스마 폐렴 진단은 교차 반응으로 인해 어렵습니다. 우리가 제안한 ELISA는 흡착 기술을 사용하여 비특이적 마이코플라스마 폐렴 항원의 선택적 고갈을 기반으로 합니다. 이 비디오에 설명된 사내 항원 캡처 ELISA는 높은 넓은 특이성을 보장합니다.그리고 실제로, 그것은 마이코 플라스마 폐렴 감염의 정확한 진단을위한 신뢰할 수있는 선별 검사로 입증되었습니다. ELISA 이전 단계를 시연하는 것은 박사 과정 학생 인 Imen Chniba입니다. 그리고 ELISA 단계는 우리 실험실의 기술자 인 Nadine Khadraoui가 될 것입니다.항원 흡착 절차를 시작하려면 12개의 이종 박테리아 항원을 미세 원심분리 튜브로 당기고 흡착할 항혈청의 절반에 해당하는 단백질 농도를 조정합니다. 다음으로, 항원 혼합물을 섭씨 4도에서 10분 동안 14, 000 G에서 원심분리하여 펠렛을 수집하였다. 이어서, 정제된 폴리클로날 항-마이코플라스마 폐렴 IgG와 함께 펠렛을 재현탁시키고, 느린 교반과 함께 섭씨 37도에서 2시간 동안 인큐베이션한다.배양이 끝날 때, 현탁액을 섭씨 4도에서 10 분 동안 14, 000 G에서 다시 원심 분리하고 특정 다클론 마이코 플라스마 폐렴 항혈청에 상응하는 상청액을 회수한다. 96웰 ELISA 플레이트를 포획 항체로 코팅하려면 항체를 밀리리터당 10마이크로그램 및 pH 9.6에서 0.1몰 탄산염 중탄산염 완충액으로 희석합니다. 이어서, 희석된 항체 100 마이크로리터를 마이크로플레이트의 각 웰에 첨가하고, 섭씨 4도에서 밤새 인큐베이션한다.다음날, 코팅 용액을 제거하고 세척 완충액으로 플레이트를 5회 세척한다. 다음으로, 각 웰에 100 마이크로 리터의 블로킹 용액을 첨가하여 코팅 된 웰의 결합되지 않은 표면을 차단한다. 플레이트를 실온에서 1시간 동안 인큐베이션한 후, 피펫으로 블로킹 용액을 제거하고 이전에 입증된 바와 같이 플레이트를 세척한다.동일한 양의 Mycoplasma pneumoniae 전체 단백질을 웰당 10나노그램의 농도로 추가하고 항원-항체 반응이 실온에서 2시간 동안 일어나도록 합니다. 배양 후, 과량의 항원을 제거하고 입증된 바와 같이 플레이트를 세척한다. 다음으로, 인간 혈청 시험 샘플을 희석하고 양성 및 음성 혈청 대조군을 참조한다.그런 다음 100 마이크로리터의 희석된 테스트 샘플과 대조군을 적절한 웰과 복제본에 추가합니다. 항원이나 혈청이 들어 있지 않은 우물을 공백으로 표시하십시오. 플레이트를 실온에서 90분 동안 배양한 후, 혈청 용액을 제거하고 세척 완충액으로 플레이트를 5회 세척하였다.각 웰에 적절한 희석된 효소 접합 검출 항체를 100 마이크로리터의 피펫으로 넣고 암실에서 섭씨 37도에서 1시간 동안 배양한다. 결합되지 않은 검출체를 제거하고 플레이트를 세척한 후 100마이크로리터의 TMB Chromogen 용액을 추가하여 항원-항체 결합을 시각화합니다. 플레이트를 실온에서 30 분 동안 현상시킵니다.그런 다음 100 마이크로 리터의 7.5 % 황산을 첨가하여 반응을 중지하십시오. 마지막으로, 마이크로플레이트 리더를 사용하여 450나노미터에서 흡광도를 판독하고 양성 지수의 계산을 기반으로 결과를 정렬합니다. Mycoplasma pneumoniae 비 흡착 된 폴리 클로날 항 혈청과 이종 박테리아 항원 사이의 면역 블롯 분석은 교차 반응성을 나타내지 만 강도는 다양했습니다.예를 들어, 마이코플라스마 갈리셉티쿰과 마이코플라스마 모방 항원은 조류 마이코플라스마임에도 불구하고 가장 강한 반응성을 나타냈습니다. 대조적으로, 우레아플라스마 우레알리티쿰의 두 인간 임상 분리 물 중 어느 것도 반응을 보이지 않았습니다. 그러나 나머지 인간 생식기 마이코플라스마 종의 항원 및 기타 박테리아 항원은 항혈청과 상당한 교차 반응을 일으켰습니다.면역블롯 분석은 이종성 박테리아 항원에 대한 마이코플라스마 폐렴 다클론 항혈청 흡착의 효율을 확인하였다. 흡착 절차는 모든 교차 반응을 제거하여 Mycoplasma pneumoniae 항혈청을 이후의 모든 혈청학적 및 면역블로팅 검사에 특이적으로 만들었습니다. 또한, 본 연구에서 개발된 항원 포획 ELISA를 사용하여 시험된 인간 혈청 세트는 마이코플라스마 폐렴 IgG에 대해 양호한 특이성으로 양성으로 입증되었다.이 ELISA의 특이성은이 중요한 단계를 신중하게 수행하고 적어도 두 번 또는 세 번 반복하는 것이 기본이기 때문에 주로 흡착 기술에 의해 보장됩니다. 이 기술의 원리는 일반적으로 다른 마이코 플라스마 종 또는 다른 박테리아를 진단하는 데 적용될 수 있습니다. 그러나, 흡착을위한 박테리아 항원의 선택은 부적절하고 적절해야한다.

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.

단백질 Ubiquitination의 감지

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

연구

생물학

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.

진균 독소의 검출을위한 ELIME (효소가 단백질 자기 전기를 연결) 방법

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.

유전자 협회의 기능 기초를 테스트하는 Allele 특정 유전자 발현 분석

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.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

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.

Mycoplasma의 최적화된 PCR 기반의 감지

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.

배양 된 세포의 내부와 세포 외 아스 코르 빈산의 결정에 대한 신속하고 특정 마이크로 분석

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.

말라리아 모기 전송하기위한 다중 탐지 분석

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.

형광 표지 된 나노 입자를 사용하여 쥐, 인간과 돼지 유래의 살아있는 세포에서 세포 내 유전자 발현의 검출

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.

현장 별 DNA 분열을 위한 병렬 높은 처리량 단 분자 운동 분석

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.