Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin d metabolite

Abstract

The invention concerns a process for the production of a hybridoma, and of a monoclonal antibody or fragments thereof able to recognize 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 .

1 . Process for the production of a hybridoma, and of a monoclonal antibody or fragments thereof, able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  comprising the following steps:
 a) immunisation of an animal with a hapten, rendered immunogenic, of formula (I), or a salt thereof, or a derivative thereof in which the carboxylic acid function is protected to form an ester, amide or oxazoline,   
       
         
           
           
               
               
           
         
         in which 
         n is an integer between 0 and 3; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are independently hydrogen or a C 1-4  alkyl, 
         R 12  and R 13  are a C 1-4  alkyl; 
         R is a hydrogen or a substituent selected from the group consisting of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; 
         b) sampling of B cells produced by the animal and fusion of the same with myeloma cells to form the hybridoma; 
         c) production, from at least part of the obtained hybridoma, of a monoclonal antibody, or fragments thereof, able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3 . 
       
     
     
         2 . Process according to  claim 1 , wherein the recognition, by said monoclonal antibody, of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  is simultaneous when both antigens are in the same sample. 
     
     
         3 . Process according to  claim 1 , wherein said monoclonal antibody has a recognition percentage of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  ranging from 70 and 110%. 
     
     
         4 . Process according to  claim 1 , wherein the hapten is the derivative of formula (I) in which n is equal to 0; R, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are respectively hydrogen; R 12  and R 13  are respectively methyl group. 
     
     
         5 . Process according to  claim 1 , wherein the hybridoma is selected from the group consisting of the hybridomas deposited in the BCCM/LMBP under deposit numbers LMBP 7011CB, LMBP 7012CB and LMBP 7013CB, LMBP 7204CB and LMBP 7205CB. 
     
     
         6 . Process according to  claim 1 , wherein the hapten is rendered immunogenic by the covalent coupling between the hapten and a carrier protein, said binding enabled via the carboxylic acid function of said hapten. 
     
     
         7 . Hybridoma suitable for the production of a monoclonal antibody or fragments thereof able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3 . 
     
     
         8 . Hybridoma according to  claim 7 , wherein it is selected from the group consisting of the hybridomas deposited in the BCCM/LMBP under deposit numbers LMBP 7011CB, LMBP 7012CB, LMBP 7013CB, LMBP 7204CB and LMBP 7205CB. 
     
     
         9 . Monoclonal antibody or fragments thereof able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  in a sample. 
     
     
         10 . Monoclonal antibody according to  claim 9 , wherein the recognition of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  is simultaneous when both antigens are in the same sample. 
     
     
         11 . Monoclonal antibody according to  claim 9 , wherein it has a recognition percentage of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  of between 70 and 110%. 
     
     
         12 . Monoclonal antibody according to  claim 9 , wherein it is produced from a hybridoma selected from the group consisting of the hybridomas deposited in the BCCM/LMBP under deposit numbers LMBP 7011CB, LMBP 7012CB, LMBP 7013CB, LMBP 7204CB and LMBP 7205CB. 
     
     
         13 . Method of recognition of and/or quantitation of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  in a sample comprising the steps of:
 providing a biological sample to be tested;   testing the sample on a diagnostic device, wherein the diagnostic device uses a hapten consisting of a compound of formula (I), or a salt thereof, or a derivative thereof in which the carboxylic acid function is protected to form an ester, amide or oxazoline,   
       
         
           
           
               
               
           
         
         in which 
         n is an integer between 0 and 3; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are independently hydrogen or a C 1-4  alkyl; 
         R 12  and R 13  are a C 1-4  alkyl; 
         R is a hydrogen or a substituent selected from the group consisting of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; said hapten rendered immunogenic; 
       
       for the production of a hybridoma, and of a monoclonal antibody, or fragments or recombinant fragments thereof, able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3 . 
     
     
         14 . Method according to  claim 13 , wherein said hapten is rendered immunogenic by the binding between the hapten and a carrier protein, said binding enabled via the carboxylic acid function of said hapten. 
     
     
         15 . Method according to  claim 13 , wherein said hapten of formula (I) is the compound of formula (I) in which n is equal to 0; R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are respectively hydrogen; R 12  and R 13  are respectively methyl group. 
     
     
         16 . Method according to  claim 13 , wherein said monoclonal antibody, or the fragments thereof, produced is a monoclonal antibody capable of the simultaneous recognition of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  when both antigens are in the same sample. 
     
     
         17 . Method of recognition of and/or quantitation of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  in a sample comprising the steps of:
 providing a biological sample to be tested;   testing the sample on a diagnostic device, wherein the diagnostic device uses a hapten consisting of a compound of formula (I), or a salt thereof, or a derivative thereof in which the carboxylic acid function is protected to form an ester, amide or oxazoline,   
       
         
           
           
               
               
           
         
         in which 
         n is an integer between 0 and 3; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are independently hydrogen or a C 1-4  alkyl; 
         R 12  and R 13  are C 1-4  alkyl; 
         R is a hydrogen or substituent selected from the group consisting of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; said hapten rendered immunogenic. 
       
     
     
         18 . A diagnostic device able to allow the recognition and/or quantification of 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3  in a sample to be tested comprising a monoclonal antibody able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3 . 
     
     
         19 . A diagnostic device according to  claim 18 , wherein it further comprises a sample of the tracer of formula (II), 
       
         
           
           
               
               
           
         
         in which 
         n is an integer between 0 and 3; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are independently hydrogen or a C 1-4  alkyl; 
         R 12  and R 13  are a C 1-4  alkyl; 
         R is a hydrogen or a substituent selected from the group consisting of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; 
       
       R 16  is the HRP protein (horseradish peroxidase), the alkaline phosphatase protein, the POD protein (peroxidase), a group of formula (III) or (IV), 125I-labeled histamine,  125 I-labeled histidine,  125 I-labeled tyrosine,  125 I-labeled methyl tyrosinate, a fluorescent group, a chemiluminescent group or a group of formula (V) wherein Z is a linker and W is a functional group able to bind with a carbonyl group. 
       
         
           
           
               
               
           
         
       
     
     
         20 . A diagnostic device according to  claim 18 , wherein it is selected from the group consisting of enzyme immunoassays (EIA) device, enzyme-linked immunosorbent assays (ELISA) device, an immunofluorescence assay (IFA) device, a radioimmunoassay device (RIA), a “magnetic separation assays” device (MSA), a “lateral flow assays” device, a “diffusion immunoassays” device, an immuno-precipitation device, an “immunosorbent” or “antigen-down assays” device an immuno-agglutination device, a “chemilunescence immuno assay (CLIA) device, or a device using a biosensor. 
     
     
         21 . A method for the production of a hybridoma comprising the steps of:
 immunisation of an animal or human with a hapten, rendered immunogenic, of general formula (I), or a salt thereof, or a derivative thereof in which the carboxylic acid function is protected to form an ester, amide or oxazoline,   
       
         
           
           
               
               
           
         
         in which 
         n is an integer between 0 and 3; 
         R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 14 , R 15  are independently hydrogen or a C 1-4  alkyl, 
         R 12  and R 13  are a C 1-4  alkyl; 
         R is a hydrogen or a substituent selected from the group consisting of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; and 
         sampling of B cells produced by the animal or human and fusion of the same with myeloma cells to form the hybridoma.