Method for determining biological agents in living target cells

Abstract

Method for determining the titer (concentration) of biological agents, such as viral vectors for gene transfer, in real time, in living target cells and uses thereof in the field of gene therapy and diagnosis.

1 °) Method for determining the titer of a biologic agent interacting with living target cells, characterized in that it comprises at least the following steps: 
 (a 1 ) the incubation of said biological agent at unknown initial concentration C, with said target cells at constant concentration D,  
 (b 1 ) the measurement at various successive times t of the intensity i of the same signal, which results from the reaction biological agent+living target cells,  
 (c 1 ) the determination of the time t β  corresponding to the i=β value, chosen in the interval β min <β<β max , such as β min  and β max  correspond to the values of i at the inflexion point of the i=f(t) curve for respectively the minimal and maximal concentrations of a standard biological agent, for which the t β =f(C) curve is pre-established, and  
 (d 1 ) the determination of the initial concentration C of the biologic agent, with the help of said t β =f(C) standard curve.  
 
     
     
         2 °) Method according to  claim 1 , characterized in that, simultaneously or before the step (a 1 ), said standard curve is established according to the following steps: 
 (a 0 ) the preparation of n serial dilutions of a standard biological agent at a known initial concentration C 0 , corresponding to said biological agent final concentrations C 1 , C 2 , . . . C n , respectively,  
 (b 0 ) the incubation of each standard biological agent dilution obtained in (a 0 ), with said target cells at a constant concentration D,  
 (c 0 ) the determination of the signal intensity i which results from the reaction biological agent+living target cells, for each standard biological agent concentration C 1 , C 2 , . . . C n ′ at various successive times t 1  to t n ′ 
 (d 0 ) the drawing of the i=f(t) curve for each value of C 1 , C 2 , . . . C n ′ 
 (e 0 ) the determination of the B value of i, such that β min <β<B max  and B min  and B max  correspond to the values of i at the inflexion point of the i=f(t) curve for C n  and C 1 , respectively, and  
 (f 0 ) the drawing of the t β =f (C) standard curve for the i=β value.  
 
     
     
         3 °) Method according to  claim 1  or  claim 2 , characterized in that said biological agent is selected from the group consisting of viruses, viral and non-viral vectors for genes transfer, vaccines, antibodies and recombinant proteins.  
     
     
         4 °) Method according to anyone of  claims 1  to  3 , characterized in that said signal is selected from the group consisting of fluorescence, luminescence, absorbance and cell numeration.  
     
     
         5 °) Kit for the quantification or the detection of a biological agent, characterized in that it comprises: 
 living target cells at a constant concentration D,  
 a standard biological agent at known concentration C, and  
 the t β =f(C)standard curve of said standard biological agent.