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I. -1-; and K. La, Conjugaison de la MC-LR aux protéines porteuses : l'ovalbumine (OVA)

. , Dosage protéique à l'Acide Bicinchoninique (BCA)

I. ?. ,

I. .. -5.-western-blot????????????????????????????????, , vol.4

. , Préparation d'anticorps monoclonaux par la technologie des hybridomes

. , Sélection par Phage Display de scFv dirigés contre la MC-LR

. , Préparation de la banque de phages

. , Sélection des phages spécifiques à la MC-LR

. , Séquençage des 11 clones sélectionnés

. , 11 I-10.a-Le vecteur d'expression pET9c, Clonage et Production des scFv sous forme soluble

. , Séquences optimisées

. , Caractérisation biochimique et validation de la fonctionnalité

. , b-Etude des interactions des anticorps commerciaux avec la MC-LR par SPR

. , Etude des interactions du scFv 3A8 avec la MC

, Conjugaison de la MC-LR aux protéines porteuses : l'ovalbumine (OVA) et la KLH

, Celui-ci réagit avec les groupements carboxyliques et induit la formation d'un intermédiaire (l'ester actif O-acyliso-urée) réagissant à son tour rapidement avec un groupement amine pour former une liaison amide et libérer un produit secondaire, l'iso-urée. Une liaison entre le groupement carboxylique de l'un

, Réactifs Protéines porteuses 0.5 mg (OVA ou KLH Thermo Scientific, # 77120 et 77600 respectivement)

. ?-tampon-de,

, ? EDC 10 mg

?. Mc,

, Protocole La protéine porteuse et la MC-LR sont dissoutes dans respectivement 50 µL et 125 µL de tampon de conjugaison puis mélangées ensemble (volume final 175 µL). 100 µL d'EDC 10 mg/mL préparés extemporanément sont ajoutés au mélange

, Dosage protéique à l'Acide Bicinchoninique (BCA)

. Réactifs-?-ampoule and . Bsa,

?. Kit and B. , B9643-1L) et solution B de sulfate de cuivre (Sigma, # C2284-25mL) utilisées en ratio A/B de 50/1, solution A d'acide bicinchoninique

, Protocole Une gamme étalon d'Albumine de Sérum Bovin (BSA) est préparée en balayant les concentrations de 0

, Le réactif de détection BCA est préparé extemporanément à partir d'une solution A d'acide bicinchoninique et d'une solution B de sulfate de cuivre en

, ?L de chaque point de gamme et de chaque échantillon sont déposés en duplicats dans une plaque 96

, L'absorbance est mesurée à 560 nm après incubation de la plaque pendant 30 minutes à 37°C. L'intensité de la coloration est mesurée par spectrophotométrie sur un dosage immuno-enzymatique qui permet l'étude d'interaction entre la MC-LR et les anticorps commerciaux AD4G2 et MC10E7. L'utilisation d'anticorps conjugués à un marqueur enzymatique adaptés permet la révélation, puits, auxquels sont ajoutés 200 µl du réactif BCA

, Réactifs ? Tampon Carbonate d'immobilisation : solution A : Na2CO3 0,1 M, solution B : NaHCO3 0,1 M (30 mL de solution A + 70 mL de solution B), vol.9, p.6

?. Pbs, NaCl 137 mM, KCl 2,7 mM, NaHPO4 10 mM et KH2PO4 1,8 mM ? Tampon de saturation : PBS 1x et gélatine en poudre 1%. ? Tampon de lavage PBST : PBS 1x et Tween-20 0,05%. ? Anticorps

?. Anticorps-secondaire, Anti-IgG de souris couplé HRP (Abliance # BI2413C)

, Substrat de l'enzyme HRP : ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (Merck Millipore, 194434.

L. Protocole and . Est, L'immobilisation de l'OVA-MC s'effectue après dilution dans le tampon carbonate à 2,5 µg/mL à raison de 100 µl/puits en duplicats (les conditions de la KLH-MC sont similaires dans le cas d'un ELISA visant à tester le couplage de cette protéine porteuse). La plaque est incubée 2h à 37°C puis lavée 3 fois avec 300 µL de PBST. Entre chaque étape, réalisé dans des plaques 96 puits Maxisorb (Nunc)

, µL de la solution d'ABTS sont déposés par puits, puis l'A405 nm est lue après incubation de la plaque à l'obscurité, pendant 30 minutes

, Préparation d'anticorps monoclonaux par la technologie des hybridomes I-6.a-Immunisation des souris Deux souris (SWISS) ont été immunisées par voie sous-cutanée en utilisant la KLH-MC à 5 ?g/mL homogénéisée dans l'adjuvant complet de Freund. Après deux semaines, chaque souris a reçu une nouvelle injection sous-cutanée, comme décrit précédemment, mais en présence d'adjuvant incomplet de Freund. Après dix jours

, Une semaine après la dernière injection, du sang a été prélevé et le titre en anticorps spécifiques a été évalué par ELISA sur des dilutions successives du sérum, Trois injections sous-cutanées supplémentaires ont été effectuées à 20 jours d'intervalle avec 20 µg de chaque antigène émulsionné dans l'adjuvant incomplet de Freund

, L'animal ayant le meilleur titre en anticorps a reçu une dernière injection de 5 ?g d'antigène dilué dans 100 ?l de PBS, par voie intraveineuse. Trois jours plus tard l'animal a été sacrifié et sa rate prélevée

, I-6.b-Production d'anticorps monoclonaux

, Après centrifugation, les cellules sont mises en culture dans un milieu sélectif HAT (hypoxanthine, aminoptérine et thymidine), réparties dans des plaques 96 puits à raison d'une cellule par puits, et enfin incubées à 37°C sous atmosphère de 5% de CO2. Quinze jours plus tard, le surnageant prélevé des puits est testé par ELISA. Les lignées cellulaires d'hybridomes positives sélectionnées sont ensuite sous-clonées par la méthode de dilutions limites à raison d'une cellule tous les 3 puits. Vingt jours après le clonage, le test ELISA est répété pour confirmer la présence d'anticorps spécifiques dans le surnageant des cellules hybrides ainsi sous-clonées, Les cellules spléniques ont été fusionnées aux cellules de myélome SP2/0 préalablement mises en culture dans du milieu DMEM 10 (Dulbecco's Modified Eagle Medium mélangé avec du sérum bovin foetal à 10%)

, I-6.c-Caractérisation immunologique-ELISA

, mg/mL de KLH/MC dilués avec du tampon carbonate ont été immobilisés sur les puits des plaques NUNC 96 puits pendant 12 à 16 heures à 4°C. Après l'étape d'immobilisation

, L'anticorps secondaire utilisé est l'anti-Ig G de souris conjugué à la peroxydase (dilué à 1 :4000 dans du tampon d'incubation). La révélation de l'activité enzymatique est assurée grâce à une solution d'orthophénylènediamine (OPD). De l'acide sulfurique est ajoutée15 minutes plus tard pour arrêter la réaction, Ensuite, la plaque est lavée de nouveau et incubée avec les sérums d'animaux immunisés dilués dans un tampon d'incubation

L. , ELISA sandwich est la même que l'ELISA effectué dans le cadre de la vérification du couplage (Annexe I-3) mais ici, l'ajout des scFv remplace celui des anticorps AD4G2/MC10E7. Les scFv purs sont en principe à 10 µM et peuvent être dilués d'un facteur

, L'anticorps secondaire est l'anti-His couplé à l'HRP (Clinisciences, p.8036

, Etude des interactions des anticorps commerciaux avec la MC-LR par SPR Réactifs ? Tampon d'immobilisation des anticorps ou fragments d'anticorps : Acétate de Sodium 10 mM ? Tampon de course : HBS-P pH

, une dilution a été préalablement effectuée à 100 µg/mL dans le tampon d'acétate de Sodium (10 mM à pH 5,0) avant d'être injectés à un débit de 3 µL/min pendant 8 minutes jusqu'à atteindre la RL visée (ici 20 000 RU), Protocole Pour immobiliser les anticorps commerciaux sur la puce CM5

, Les constantes d'affinité sont déterminées avec le logiciel BIAeval permettant un ajustement des données expérimentales à un modèle mathématique. L'alignement des résultats a été réalisé avec le modèle Steady State Affinity

, Pour l'analyse des interactions avec la MC-LR, la méthode Single Cycle Kinetics permettant l'injection de concentrations croissantes d'analyte sans régénération a été utilisée. Cinq concentrations croissantes successives de MCLR ont ensuite été injectées à 10 µL/min pendant 500 secondes, Etude des interactions du scFv 3A8 avec la MC-LR par SPR 50µg/mL de scFv 3A8 dilués en acétate de sodium 10 mM pH, vol.3

, d-ELISA compétitif Pour effectuer l'ELISA compétitif, un protocole semblable à celui de l'ELISA Sandwich a été utilisé

. Toutefois, une étape entre les lavages effectués après incubation avec les scFv et l'ajout des anticorps secondaires l'anti His /HRP a été ajoutée

, Cette même démarche a été utilisée avec un extrait brut d'un échantillon prélevé sur le terrain. Dans ce cas, l'anticorps commercial AD4G2 a été utilisé à la place des scFv. L'anticorps secondaire était alors l'anti IgG/HRP (Abliance, # BI2413C)