Method of in-vitro immunoassay

Abstract

The present invention relates to a novel method of in-vitro assay of the molecules of the extracellular matrix synthesized by cells in culture and uses thereof in the form of a kit for in-vitro measurement and/or for a method of screening of cosmetic and/or pharmaceutical ingredients.

1 - 22 . (canceled) 
     
     
         23 . In-vitro immunoassay method for at least one molecule of the extracellular matrix synthesized by cells in culture comprising at least the steps:
 a) a step of cell culture the cells   b) a preferred step of collecting the culture medium   c) a step of cell lysis with a solution of quaternary amine   d) a step of collecting the cell lysate   e) a step of assaying said molecule in the extracellular matrix by an immunological technique.   
     
     
         24 . Assay method according to  claim 23  wherein step b) of collecting the culture medium is carried out. 
     
     
         25 . Assay method according to  claim 23  wherein the molecule of the ECM is selected from the proteins constituting the ECM, the glycoproteins constituting the ECM, the glycosaminoglycans constituting the ECM, the proteoglycans constituting the ECM and the growth factors contained in the ECM. 
     
     
         26 . Assay method according to  claim 25  wherein the molecule of the ECM is selected from type I, III, V, VI, XII, XIV, XVI, IV and VII collagens, elastin, tropoelastin, fibrillin 1, LOX, LOXL, EBP (Elastin Binding Protein), fibulins 3 and 5, Emilin 1 and 2, perlecan, versican, decorin, biglycan, lumican, fibronectin, laminin, hyaluronic acid, heparan sulphate, nidogen-1. 
     
     
         27 . Assay method according to  claim 25  wherein the molecule of the ECM is selected from VEGF, PDGF, FGF-2, FGF-7, and HGF. 
     
     
         28 . Assay method according to  claim 23  wherein the cells in culture are selected from stromal cells, preferably fibroblasts, osteoblasts and/or adipoblasts, epithelial cells, preferably keratinocytes or endothelial cells. 
     
     
         29 . Assay method according to  claim 28  wherein the cells in culture are selected from fibroblasts and/or keratinocytes and/or adipocytes. 
     
     
         30 . Assay method according to  claim 23  wherein the cells in culture are maintained in culture at confluency for a time ranging from 24 to 96 hours before step c) is carried out. 
     
     
         31 . Assay method according to  claim 30  wherein an active ingredient of cosmetic and/or pharmaceutical interest is added to the culture medium. 
     
     
         32 . Assay method according to  claim 31  wherein the ingredient is selected from vitamin C, preferably at a dose in the range from 5 μM to 100 μM and TGF beta preferably at a dose in the range from 1 ng/ml to 100 ng/ml. 
     
     
         33 . Assay method according to  claim 24  wherein the molecule of the ECM and/or a precursor of the molecule of the ECM is assayed by an immunological method in the sample of culture medium taken in step b), preferably by ELISA type assay. 
     
     
         34 . Assay method according to  claim 23  wherein the solution of quaternary amine is a solution of ammonium, optionally in the form of salts. 
     
     
         35 . Assay method according to  claim 34  wherein the solution of quaternary amine is selected from an ammonium chloride solution, an ammonium hydroxide solution and mixtures thereof. 
     
     
         36 . Assay method according to  claim 23  wherein the solution of quaternary amine is at a concentration in the range from 10 mM to 200 mM. 
     
     
         37 . Assay method according to  claim 23  wherein the solution of quaternary amine is applied on the cells for a time in the range from 5 to 60 minutes. 
     
     
         38 . Assay method according to  claim 23  wherein an assay of DNA and/or of molecule of the ECM is carried out in the cell lysate collected in step d). 
     
     
         39 . Assay method according to  claim 23  wherein step e) of immunoassay of said molecule contained in the extracellular matrix is carried out by the TRF fluorescence method. 
     
     
         40 . Assay method according to  claim 23  comprising at least the steps:
 a) a step of cell culture the cells 
 b) a step of collecting the culture medium and of assaying the molecule of the ECM and/or a precursor of the molecule of the ECM 
 c) a step of cell lysis with a solution of quaternary amine 
 d) a step of collecting the cell lysate and of assaying the DNA and/or of assaying the molecule of the ECM and/or a precursor of the molecule of the ECM 
 e) a step of assaying said molecule of the ECM contained in the extracellular matrix by an immunological technique. 
 
     
     
         41 . Method of screening and/or of investigation in vitro of at least one ingredient of cosmetic and/or pharmaceutical interest for its properties of increasing or decreasing the quantity of a molecule in the extracellular matrix comprising at least the steps:
 a) a step of cell culture the cells in the presence of the ingredient   b) preferably a step of collecting the culture medium and more preferably of assaying the molecule of the ECM and/or a precursor of the molecule of the ECM in said culture medium thus taken,   c) a step of cell lysis with a solution of quaternary amine   d) a step of collecting the cell lysate and preferably of assaying the DNA and/or of assaying the molecule of the ECM and/or a precursor of the molecule of the ECM   e) a step of assaying said molecule of the ECM contained in the extracellular matrix by an immunological technique.   
     
     
         42 . Kit for assay by the immunological method useful according to  claim 23  comprising a solution of quaternary amine and an antibody directed against the molecule of the ECM, optionally bound to a secondary antibody. 
     
     
         43 . Method of selection of a lysis solution useful in the assay method according to  claim 23 , wherein the assay method according to the invention is applied with a lysis solution to be tested for at least 10 min, preferably 10 min, and the ratio of the result of the assay of the molecule of the ECM obtained in step e) to the result of the assay of the DNA in the cell lysate in step d) is compared with that obtained with an ammonium hydroxide lysis solution at 20 mM applied for the same time, preferably 10 minutes.