Test de confirmation : Western Blot

Lors d'un dépistage du VIH, si le test ELISA est négatif, un second test est réalisé par sécurité. Si au moins un de ces deux tests ELISA est positif, un test Western Blot (WB) est alors effectué.  En effet, le test ELISA effectue une recherche générique de présence du virus, ce qui peut entraîner des faux-positifs. Le test Western Blot, plus cher et compliqué est donc utilisé pour confirmer la validité de ces tests positifs en vérifiant la présence d'anticorps anti-VIH (recherche d'anticorps spécifiques à certaines protéines virales).
 
Logo astuce

Le saviez-vous ?
Son nom vient d'un jeu de mot, issu d'un autre test ( le Southern Blot, du nom de son inventeur Edwin Southern, qui concerne l'ADN). En anglais, blot signifie tache ; en effet cette technique utilise une migration de protéines sur une membrane, puis une révélation dont les résultats ressemblent à une empreinte sur buvard.
 
En résumé...
 
Le test ELISA est utilisé pour le dépistage. Le test WB - plus long et spécifique - n'est utilisé qu'en confirmation du premier dans les cas positifs.

Déroulement du test

Le test de confirmation Western Blot se déroule en trois temps:
  1. Séparation des protéines virales
  2. Recherche des anticorps spécifiques
  3. Diagnostic sur la séropostivité du patient

Isolement des protéines virales (Ag)

Préparation de l'échantillon

  1. Spécificité oblige, les agents viraux doivent être séparés. Par différentes méthodes (agent chimique, ultrasons, etc...) le virus est en quelque sorte broyé et tous ses constituants sont mélangés.
  2. L'échantillon est porté à ébullition : cela brise toutes les liaisons responsables de l'enroulement de la protéine. Celle ci se déroule complètement.
Sont ajoutés dans la solution :
 
- du TRIS: appelé tampon, il maintient un pH stable (utile quand on travaille sur des protéines).
 
- du DTT : composant sulfhydril, il empêche la protéine de reformer des liens (ponts disulfures).
 
- du SDS : il « entoure » complètement la protéine et la charge négativement tout en l'empêchant de se replier. Toutes les protéines sont donc à égalité au niveau des charges.
 
- du glycérol : augmente le poids. Il est plus facile de déposer le mélange dans les petits puis : celui-ci coule directement au fond.
 
L'intérêt de tout ces ajouts est la préparation de la séparation des protéines par électrophorèse.

L'électrophorèse

Principe : Il s'agit d'une migration des protéines sur un gel de polyacrylamide soumis à un courant électrique. Pour bien l'imaginer, il est possible d'assimiler ce gel à un réseau de pores plus ou moins serrées selon la composition du mélange choisi. L'échantillon de protéines est déposé dans des petits puits au sommet du bloc de gel. Ces petits puits sont creusés lors de la préparation du gel, ils sont distancés suffisamment pour que les échantillons ne se mélangent pas.
Gel d'électrophorèse
 
Désormais, la préparation détaillée plus haut prend tout son sens : le mélange a été homogénéisé au niveau des charges et de la forme des protéines et celles-ci, soumises au courant, vont migrer vers le bas du gel en fonction de leur poids moléculaire ! Plus une protéine est grande, plus elle mettra de temps à se faufiler entre les pores du gel.
Après un certain temps défini, le courant est arrêté. Les protéines ont migré et se sont réparties en bandes. A ce stade, nous possédons donc un gel avec des bandes (encore incolores !), chacune étant représentée par une certaine protéine.
Maintenant que les protéines virales sont isolées, nous allons faire entrer en jeu le sérum du patient. Mais avant il nous faut transférer les protéines virales sur un support adéquat : il n'est pas possible de manipuler sur le gel.

Transfert sur nitrocellulose

Des bandes de buvard en nitrocellulose sont utilisées. Le gel est simplement appliqué sur le gel d'électrophorèse pour récupérer les protéines.
Bande de notrocellulose

Recherche des Anticorps spécifiques chez le patient

Le buvard est à présent incubé dans le sérum de notre patient. Si celui-ci présente des anticorps spécifiques aux diverses protéines virales, ceux-ci vont se répartir sur les bandes du buvard, selon les protéines reconnues.
 
Immersion de la bande
Le buvard est retiré. En théorie, si le patient est infecté par le VIH, à la surface du buvard nous avons les protéines virales par dessus lesquelles se sont fixés les anticorps. Des anticorps secondaires peuvent être ajoutés. Comme auparavant pour le test ELISA, ceux-ci sont couplés à un révélateur enzymatique. Les anticorps du patient sont alors révélés !
 
Le test est déclaré positif si il présente des anticorps contre au moins deux des trois glycoprotéines virales (gp160, gp120 ou gp41) ou s'il présente la protéine p24 et la glycoprotéine gp160 (début d'infection)
 
Il ne faut pas oublier les témoins essentiels pour chaque manipulation, ce sont des références.
Un témoin positif = le sérum témoin contient tous les anticorps. Toutes les bandes virales sont révélées : cela permet de les localiser et de comparer avec celles du témoin.
Un témoin négatif = aucun anticorps n'est présent dans le sérum témoin. La révélation ne doit présenter aucune coloration pour que le test soit jugé fiable.

Evolution particulière et symptomes de la maladie

évolution de la maladie
Evolution typique après infection par VIH-1 (d'après l'Université Libre de Bruxelles)
courbe verte : état du système immunitaire (évolution de du taux des lymphocytes T CD4+ de la personne infectée)
courbe rouge : progression du virus (évolution du taux de virus VIH-1 (mesure de son ARN) circulant dans le sang)
 
Primo-infection : le virus pénètre dans les cellules du système immunitaire.
Virémie aiguë : le virus infecte les cellules du système immunitaire et les détruit. Beaucoup de nouveaux virus sont crées et parallèlement, un nombre important de lymphocytes T CD4+ sont détruits. A ce stade une fatigue physique et des symptomes gripaux peuvent être visibles. Cependant le système immunitaire remonte la pente et arrive à maîtriser l'avancée de l'infection.
S'ensuit alors une phase de latence, pouvant durer une vingtaine d'année, qui est sans symptome. Le taux de lymphocytes se maintient ; le virus se multiplie mais reste sous contrôle du système immunitaire. Au fil du temps, le taux de lymphocytes baisse de plus en plus et le virus, entamant une multiplication rapide, finit par prendre le dessus.
Commence alors la phase SIDA proprement dite : le patient ne possède plus de système immunitaire efficace l'organisme ne parvient plus à assurer sa défense contre les maladies opportunistes. En effet, on ne meurt pas "directement" de l'infection du VIH, mais par des maladies que l'on développe suite à l'anéantissemnt de notre système immunitaire.
 
Pour être efficace, les dépistages ne peuvent être fait n'importe quand. Ils se font en fonction de l'état présumé d'avancement de la maladie et de la quantité d'Ag potentiellement présents :
- dès le 10ème jour:  détection de l'ARN proviral (obtenu par amplification PCR)
- dès le 15ème jour : détection de l'Ag P24
- dès le 22ème jour : dépistage par test ELISA 
 
En résumé...
 
  • Si Elisa 1 négatif = on effectue un Elisa 2 de confirmation sur même échantillon
  • Si Elisa 1 ou 2 positif = confirmation par WB sur nouveau prélèvement (au cas où la séropositivité serait due à un problème dans le premier échantillon, une contamination par exemple)
Dans certains cas le test n'est pas performant :
  • Test effectué trop tôt : pas encore assez d'Ac dans le sang.
  • Test effectué très tard : peu d'Ac ce qui témoigne de l'effondrement du système immunitaire.

Références

- virus de l'immunodefiscience humaine (cours du CHU de besançon)
- cours de L1 et L2 en biologie
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