Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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Cette étude a été financée par le ministère tunisien de la Santé et le ministère tunisien de l’Enseignement supérieur et de la Recherche scientifique.

La présente étude a été menée conformément aux aspects éthiques établis par le comité d’éthique de l’Institut Pasteur de Tunis.
1. Étapes pré-ELISA : Prérequis et prétraitement
<ol><li>Souches bactériennes et milieux de croissance REMARQUE : Les espèces de mollicutes et les bactéries à paroi utilisées dans la présente étude ainsi que leurs milieux de croissance sont énumérés dans le tableau 1.<ol><li>Croissance des espèces de ...

<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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I., Leinikki, P. <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+infection+by+enzyme+immunoassay.">Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.</a> Journal of Clinical Pathology. 33 (9), 836-840 (1980).</li><li>Sasaki, T., Bonissol, C., Stoilikovic, B., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+cross-reactive+antibodies+to+mycoplasmas+in+human+sera+by+ELISA+and+immunoblotting.">Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting.</a> Microbiology and Immunology. 31 (7), 639-648 (1987).</li><li>Brunner, H., 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=Unexpectedly+high+frequency+of+antibody+to+Mycoplasma+pneumoniae+in+human+sera+as+measured+by+sensitive+techniques.">Unexpectedly high frequency of antibody to Mycoplasma pneumoniae in human sera as measured by sensitive techniques.</a> Journal of Infectious Diseases. 135 (4), 524-530 (1977).</li><li>Plackett, P., Marmion, B. 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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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Cet article présente une description générale d’un test ELISA interne principalement développé pour assurer un dépistage spécifique des M. pneumoniae Infection. Les détails sur le protocole du test ELISA lui-même ainsi que certaines étapes de pré et de post-traitement sont fournis. La spécificité de ce dosage est assurée par la technique d’adsorption. Cette procédure a déjà été décrite dans des tests ELISA développés pour le diagnostic des humains et des oiseaux. Mycoplasmas<su...

Les mycoplasmes sont parmi les procaryotes connus les plus petits et les plus simples. Elles se distinguent principalement des autres bactéries par l’absence de structure de paroi cellulaire. Ainsi, les mycoplasmes ont été classés dans une classe distincte appelée Mollicutes1. La déficience de la paroi cellulaire confère une résistance intrinsèque à ces micro-organismes contre certains agents antimicrobiens et est en grande partie responsable de leur polymorphisme. Les mycoplasmes ont un génome restreint et une taille réduite, ce qui limite leurs capacités métaboliques et biosynthétiques et explique leur nature parasitaire et saprophyte1.
Mycoplasma pneumoniae est l’un des mycoplasmes qui infectent l’homme et est considéré comme le plus virulent2. M. pneumoniae colonise les voies respiratoires supérieures, entraînant une pneumonie atypique chez les enfants et les jeunes adultes. Les signes cliniques engendrés par l’infection à M. pneumoniae sont pseudo-grippaux, avec maux de tête, fièvre et toux3. La cytadhésion de M. pneumoniae aux cellules hôtes est médiée par un organite de fixation comprenant une adhérence majeure P1 et plusieurs protéines accessoires 4,5. D’autres manifestations cliniques peuvent survenir en raison d’une inflammation locale et d’une stimulation du système immunitaire de l’hôte résultant de l’adhérence intime de M. pneumoniae à la muqueuse des voies respiratoires6. Bien que la pneumonie soit une caractéristique de l’infection à M. pneumoniae, il a été révélé que l’infection par cette bactérie peut également être responsable d’un large éventail de manifestations non pulmonaires dans différents sites anatomiques tels que le système nerveux central, le cœur, la peau et les articulations7.
Comme pour toutes les espèces de Mycoplasma, le diagnostic de M. pneumoniae est difficile. Les signes cliniques évoquant la mycoplasmose sont pour la plupart inapparents et non caractéristiques8. Comme il est très difficile de diagnostiquer l’infection à M. pneumoniae en se fiant uniquement aux manifestations cliniques et aux symptômes, le dépistage en laboratoire présente un intérêt particulier9. La détection des colonies de M. pneumoniae par culture est la méthode de référence pour un diagnostic approprié. Cependant, les exigences de croissance fastidieuses et le long temps nécessaire pour obtenir des résultats définitifs (1-2 semaines) compliquent la culture, et signifie donc qu’elle est rarement utilisée pour le diagnostic de routine10. Les technologies d’amplification des acides nucléiques ont été validées en termes de rapidité et d’efficacité, bien qu’en raison de leur coût relativement élevé et de leur indisponibilité dans certains établissements de soins de santé, ces techniques moléculaires ne soient pas considérées comme des tests diagnostiques de première intention. Il est vrai que les tests PCR commerciaux sont largement utilisés pour diagnostiquer les infections à M. pneumoniae, mais ils ne peuvent toujours pas remplacer la sérologie. En outre, l’occurrence fréquente de résultats faussement négatifs et faux positifs a limité l’utilisation de la PCR9. Systématiquement, la sérologie reste la plus pratiquée dans les laboratoires pour le diagnostic de l’infection à M. pneumoniae. Plusieurs approches sérologiques ont été rapportées depuis des décennies, telles que les hémagglutinines froides, le test de fixation du complément 11, le test d’hémagglutination indirecte12, l’immunofluorescence 13 et la technologie ELISA, qui a été appliquée pour la première fois à la sérologie des mycoplasmes au début des années1980 14,15,16. L’un des principaux problèmes rencontrés lors de la réalisation du sérodiagnostic ELISA de l’infection à M. pneumoniae est les réactions croisées, ce qui réduit considérablement la spécificité de la technique. L’adsorption non spécifique de sérums humains avec des antigènes de M. pneumoniae a déjà été signalée; en fait, bon nombre des anticorps détectés par ELISA dans les sérums humains ne sont pas toujours liés aux antigènes mycoplasmiques 17, en raison de la similitude de M. pneumoniae avec certaines bactéries18,19 et certains tissus animaux et humains20.
En raison des lectures de fond élevées observées dans le test ELISA conventionnel pratiqué en laboratoire, l’interprétation des résultats était souvent compliquée et, par conséquent, la délivrance d’un diagnostic correct de M. pneumoniae était une tâche difficile. Face à ce problème, nous avons choisi d’améliorer le test ELISA de M. pneumoniae en supprimant les réactions non spécifiques des antigènes de M. pneumoniae avec des anticorps à tester. À cette fin, nous avons travaillé sur la déplétion sélective des antigènes non spécifiques de M. pneumoniae en utilisant la technique d’adsorption. En fait, l’objectif principal du test ELISA de capture d’antigènes est de détecter spécifiquement l’immunoglobuline (Ig) G de M. pneumoniae dans des échantillons de sérum humain. Le concept de ce test ELISA consiste principalement en la capture sélective d’antigènes spécifiques de M. pneumoniae, avant l’ajout des échantillons de sérum humain. Cette sélectivité est assurée par l’incubation de l’antigène brut M. pneumoniae avec un antisérum polyclonal M. pneumoniae, produit chez le lapin en laboratoire et en le rendant spécifique à l’espèce par adsorption contre un panel de bactéries hétérologues, appartenant ou non à la classe des Mollicutes, partageant des antigènes avec M. pneumoniae espèces et/ou connues pour coloniser les voies respiratoires. La procédure d’adsorption a été répétée trois fois et son efficacité à éliminer la réactivité croisée a été testée par immunobuvardage. Le test ELISA développé est une combinaison d’ELISA sandwich et indirect. Brièvement, les puits de la plaque ELISA sont d’abord recouverts d’un antisérum polyclonal spécifique à M. pneumoniae. Ensuite, l’antigène M. pneumoniae est ajouté et piégé entre l’antisérum et les anticorps présents dans l’échantillon de sérum à tester. Le complexe immunologique formé est détecté par un anticorps secondaire conjugué à l’enzyme (IgG conjuguées à la peroxydase). Les réactions sont visualisées par l’ajout de substrat chromogène et l’absorbance est mesurée par spectrophotométrique. Ce test ELISA interne est présenté schématiquement à la figure 1. Le test ELISA maison s’est avéré efficace pour détecter spécifiquement l’infection à M. pneumoniae et est actuellement l’un des tests les plus pratiqués dans l’activité diagnostique de routine.

<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 est un procaryote déficient de la paroi cellulaire, principalement connu pour coloniser les voies respiratoires humaines et être endémique, avec des pics épidémiques tous les 6 ans, chez les enfants plus âgés et les jeunes adultes. Le diagnostic de M. pneumoniae est difficile en raison de la nature fastidieuse de l’agent pathogène et de la possibilité d’un portage asymptomatique. Le diagnostic en laboratoire de l’infection à M. pneumoniae basé sur le titrage des anticorps dans les échantillons de sérum des patients reste la méthode la plus pratiquée. En raison du problème potentiel de réactivité immunologique croisée avec l’utilisation de sérum polyclonal pour M. pneumoniae, un test immuno-enzymatique de capture d’antigènes (ELISA) a été développé pour améliorer la spécificité du diagnostic sérologique. Les plaques ELISA sont recouvertes d’anticorps polyclonaux contre M. pneumoniae, élevés chez le lapin et rendus spécifiques après adsorption contre un panel de bactéries hétérologues qui partagent des antigènes avec des espèces de M. pneumoniae et/ou sont connues pour coloniser les voies respiratoires . Les antigènes homologues de M. pneumoniae ayant réagi sont alors spécifiquement reconnus par leurs anticorps correspondants dans les échantillons de sérum. L’optimisation ultérieure des paramètres physico-chimiques auxquels l’ELISA de capture d’antigènes est soumis a conduit à un ELISA hautement spécifique, sensible et reproductible.

L’activité immunoblotting de l’antisérum polyclonal non adsorbé Mycoplasma pneumoniae contre les bactéries hétérologues La réactivité croisée existe bel et bien comme le montrent les résultats de l’immunoblot (Figure 2) et par rapport au témoin positif (Lane 1), certains des antigènes de M. pneumoniae sont partagés avec les bactéries dépistées. L’intensité de ces réactions croisées était varia...

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

Dans l’infection à Mycoplasma pneumoniae, les tests sérologiques peuvent générer de bons résultats, mais avec une faible spécificité en raison de la réaction immunologique croisée. Le test ELISA interne de capture d’antigènes, décrit dans cet article, garantit une spécificité élevée de l’espèce et s’est avéré être un test de dépistage fiable pour un diagnostic précis de M. pneumoniae.

DOSAGE IMMUNO-ENZYMATIQUE DE CAPTURE D’ANTIGÈNE POUR LA DÉTECTION SPÉCIFIQUE DE MYCOPLASMA PNEUMONIAE

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

Comme pour tous les patients atteints de mycoplasmes, leur diagnostic de Mycoplasm pneumoniae est difficile en raison de réactions croisées. Notre ELISA proposé est basé sur la déplétion sélective des antigènes non spécifiques de Mycoplasm pneumoniae à l’aide d’une technique d’adsorption. Le test ELISA interne de capture d’antigènes décrit dans cette vidéo garantit une grande spécificité spacieuse.Et en fait, il s’est avéré être un test de dépistage fiable pour un diagnostic précis de l’infection à Mycoplasm pneumoniae. Imen Chniba, doctorante, présentera les étapes pré-ELISA. Et les étapes ELISA seront Nadine Khadraoui, une technicienne de notre laboratoire.Pour commencer la procédure d’adsorption de l’antigène, tirez les 12 antigènes bactériens hétérologues dans un tube de microcentrifugation et ajustez la concentration en protéines correspondant à la moitié de l’antisérum à adsorber. Ensuite, centrifuger le mélange d’antigènes à 14 000 g pendant 10 minutes à quatre degrés Celsius pour recueillir la pastille. Ensuite, remettez la pastille en suspension avec les IgG polyclonales anti-Mycoplasma pneumoniae purifiées et incuber pendant deux heures à 37 degrés Celsius avec une agitation lente.A la fin de l’incubation, centrifuger à nouveau la suspension à 14 000 G pendant 10 minutes à quatre degrés Celsius et récupérer le surnageant correspondant à l’antisérum polyclonal spécifique Mycoplasma pneumoniae. Pour enduire une plaque ELISA de 96 puits d’un anticorps de capture, diluez l’anticorps à 10 microgrammes par millilitre et 0,1 tampon de bicarbonate molaire à pH 9,6. Ensuite, ajoutez 100 microlitres de l’anticorps dilué à chaque puits de la microplaque et incuber pendant une nuit à quatre degrés Celsius.Le lendemain, retirez la solution de revêtement et lavez la plaque cinq fois avec le tampon de lavage. Ensuite, bloquez les surfaces non liées des puits revêtus en ajoutant 100 microlitres de la solution de blocage à chaque puits. Après avoir incubé la plaque pendant une heure à température ambiante, retirez la solution de blocage à l’aide d’une pipette et lavez la plaque comme indiqué précédemment.Ajouter une quantité égale de protéines entières de Mycoplasma pneumoniae à une concentration de 10 nanogrammes par puits et laisser la réaction antigène-anticorps se produire pendant deux heures à température ambiante. Après l’incubation, retirer l’excès d’antigène et laver la plaque comme indiqué. Ensuite, diluez les échantillons de test de sérum humain et faites référence aux témoins séropositifs et négatifs.Ensuite, ajoutez 100 microlitres des échantillons d’essai dilués et des témoins dans les puits et les doublons appropriés. Marquez les puits ne contenant ni antigène ni sérum comme étant vides. Après avoir incubé la plaque pendant 90 minutes à température ambiante, retirez la solution de sérum et lavez la plaque cinq fois avec le tampon de lavage.Pipeter 100 microlitres des anticorps de détection conjugués enzymatiques dilués appropriés à chaque puits et incuber pendant une heure à 37 degrés Celsius dans l’obscurité. Après avoir retiré les corps de détection non liés et lavé la plaque, ajoutez 100 microlitres de solution de chromogène TMB pour visualiser la liaison antigène-anticorps. Laissez la plaque se développer pendant 30 minutes à température ambiante.Ensuite, arrêtez la réaction en ajoutant 100 microlitres d’acide sulfurique à 7,5%. Enfin, lisez l’absorbance à 450 nanomètres à l’aide d’un lecteur de microplaques et triez les résultats en fonction du calcul de l’indice de positivité. L’analyse par immunotransfert entre l’antisérum polyclonal non adsorbé de Mycoplasma pneumoniae et les antigènes bactériens hétérologues a révélé une réactivité croisée mais d’intensité variable.Par exemple, les antigènes de Mycoplasma gallisepticum et de Mycoplasma imitans ont montré la plus forte réactivité bien qu’il s’agisse de mycoplasmes aviaires. En revanche, aucun des deux isolats cliniques humains d’Ureaplasma urealyticum n’a montré de réaction. Cependant, les antigènes des espèces de mycoplasmes génitaux humains restants et d’autres antigènes bactériens ont produit des réactions croisées considérables avec l’antisérum.L’analyse par immunotransfert a confirmé l’efficacité de l’adsorption polyclonale de l’antisérum Mycoplasma pneumoniae contre les antigènes bactériens hétérologues. La procédure d’adsorption a éliminé toutes les réactions croisées, rendant ainsi l’antisérum Mycoplasma pneumoniae spécifique pour tous les tests sérologiques et immunoblotting ultérieurs. De plus, l’ensemble de sérums humains testés à l’aide du test ELISA de capture d’antigènes développé dans cette étude s’est avéré positif pour les IgG de Mycoplasma pneumoniae avec une bonne spécificité.La spécificité de cet ELISA est principalement assurée par la technique d’adsorption, car il est primordial d’effectuer cette étape critique avec soin et de la répéter au moins deux ou trois fois. Le principe de cette technique peut être appliqué pour diagnostiquer d’autres espèces de mycoplasmes ou d’autres bactéries en général. Cependant, la sélection des antigènes bactériens pour l’adsorption ne doit pas être pertinente et adéquate.

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

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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.

La détection des protéines ubiquitination

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

Recherche

Biologie

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.

ELIME (Enzyme Linked Immuno Magnetic électrochimique) Méthode de détection des mycotoxines

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.

Un allèle spécifique Assay expression du gène pour l&#39;épreuve la base fonctionnelle des associations génétiques

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.

Assay immunoprécipitation de la chromatine des gènes spécifiques de tissus en utilisant des embryons de souris en début de

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.

Optimisé basé sur la PCR Détection de Mycoplasma

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.

Une microplaque de dosage rapide et spécifique pour la détermination de l&#39;ascorbate intra-et extracellulaire dans les cellules cultivées

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.

Un dosage multi-détection pour moustiques vecteurs du paludisme

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.

Détection de intracellulaire Gene Expression dans des cellules vivantes de murin, humain et porcin Origine utilisant des nanoparticules marquées par fluorescence

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.

Essai cinétique à molécule unique à haut débit parallèle pour le clivage d’ADN spécifique au site

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.