G - Physics – 01 – N
Patent
G - Physics
01
N
G01N 33/542 (2006.01) C12Q 1/34 (2006.01) G01N 33/53 (2006.01) G01N 33/58 (2006.01)
Patent
CA 2252931
The invention discloses a method for detecting and/or quantifying a hapten in a homogeneous phase, comprising the following steps: adding a known quantity of a hapten inhibitor complex to the solution containing the hapten to be detected and/or quantified; adding to the solution a quantity of antibodies corresponding to the quantiy of the hapten/inhibitor complex; adding to the solution a type C beta -lactamase having an active site for two substrates in antigenic competition in the said active site, the first substrate being a reporter substrate capable of being transformed into a detectable and/or quantifiable product, preferably by visible UV radiation measurement, the second substrate being the hapten/inhibitor complex acting on the hydrolysis rate of the reporter substrate; detecting and/or quantifying the concentration of the product resulting from the transformation of the reporter substrate, the Km constant of the reporter substrate being at least a hundred times higher than the Km constant of the hapten/inhibitor complex, and the kcat constant being at least ten times higher than the kcat constant of the hapten/inhibitor complex.
La présente invention concerne un procédé de détection et/ou de quantification d'un haptène dans une phase homogène, dans lequel: on additionne une quantité connue d'un complexe inhibiteur - haptène à la solution contenant l'haptène à détecter et/ou à quantifier; on additionne à la solution une quantité d'anticorps correspondant à la quantité du complexe inhibiteur - haptène; on additionne à la solution une .beta.-lactamase de type C possédant un site actif pour deux substrats entrant en compétition sur ledit site actif, le premier substrat étant reporter transformable en un produit détectable et/ou quantifiable, de préférence par mesure de rayonnement UV - visible, le second substrat étant le complexe inhibiteur - haptène, agissant sur la vitesse d'hydrolyse du substrat reporter; on détecte et/ou quantifie la concentration du produit issu de la transformation du substrat reporter, la constante K m, du substrat reporter est au moins 100 fois plus élevée que la constante K m du complexe inhibiteur - haptène, en ce que la constante k cat, est au moins 10 fois plus élevée que la constante k cat du complexe inhibiteur - haptène.
Ghitti Gianangelo
Kohl Michel
Lejeune Robert
Renotte Roger
Sarlet Guy
Gowling Lafleur Henderson Llp
La Region Wallonne
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