Antibody production methods related to disruption of peripheral tolerance in B lymphocytes

Abstract

The subject invention relates a method for the production of monoclonal antibodies. The method utilizes an immunized animal having antibody-producing cells with disrupted peripheral tolerance. The invention also provides a method for the use of such monoclonal antibodies, and polyclonal antibodies derived from an immunized animal having antibody-producing cells with disrupted peripheral tolerance, for in vitro and in vivo clinical diagnostics and therapeutics.

1 . A method for production of a monoclonal antibody to an antigen comprising: 
 (a) immunizing an animal, the animal having antibody-producing cells with a manipulated characteristic that facilitates the antibody-producing cell's ability to produce antibodies, with said antigen to permit said antibody-producing cells to produce antibodies to said antigen;    (b) removing at least a portion of said antibody-producing cells from said animal,    (c) forming a hybridoma by fusing one of said antibody-producing cells with an immortalizing cell wherein said hybridoma is capable of producing a monoclonal antibody to said antigen,    (d) propagating said hybridoma, and    (e) harvesting the monoclonal antibodies produced by said hybridoma.    
     
     
         2 . The method of  claim 1 , wherein said manipulated characterisitic comprises disrupted peripheral tolerance.  
     
     
         3 . The method of  claim 1 , wherein the animal is selected from the group consisting of a mouse, rat, pig, guinea pig, poultry, a goat, a sheep, primate and a rabbit.  
     
     
         4 . The method of  claim 3 , wherein said animal is a mouse.  
     
     
         5 . The method of  claim 4 , wherein said mouse is a transgenic mouse overexpressing CD19.  
     
     
         6 . The method of  claim 1 , wherein said antibody-producing cells comprise B lymphocytes.  
     
     
         7 . The method of  claim 1 , wherein said monoclonal antibodies produced comprise antibodies having a high affinity for said antigen.  
     
     
         8 . A method for production of a monoclonal antibody to an antigen comprising: 
 (a) immunizing an animal, the animal having antibody-producing cells with disrupted peripheral tolerance, with said antigen to permit said antibody-producing cells to produce antibodies to said antigen;    (b) removing at least a portion of said antibody-producing cells from said animal,    (c) forming a hybridoma by fusing one of said antibody-producing cells with an immortalizing cell wherein said hybridoma is capable of producing a monoclonal antibody to said antigen,    (d) propagating said hybridoma, and    (e) harvesting the monoclonal antibodies produced by said hybridoma.    
     
     
         9 . The method of  claim 8 , wherein said animal is selected from the group consisting of a mouse, rat, pig, guinea pig, poultry, a goat, a sheep, primate and a rabbit.  
     
     
         10 . The method of  claim 9 , wherein said animal is a mouse.  
     
     
         11 . The method of  claim 10 , wherein said mouse is a transgenic mouse overexpressing CD19.  
     
     
         12 . The method of  claim 8 , wherein said antibody-producing cells comprise B lymphocytes.  
     
     
         13 . The method of  claim 8 , wherein said monoclonal antibodies produced comprise antibodies having a high affinity for said antigen.  
     
     
         14 . A method for production of polyclonal antibodies to an antigen comprising immunizing an animal having antibody-producing cells with disrupted peripheral tolerance with said antigen to permit said antibody-producing cells to produce antibodies to said antigen and separating serum, which contains said polyclonal antibodies, from said animal.  
     
     
         15 . The method of  claim 14 , wherein said animal is selected from the group consisting of a mouse, rat, pig, guinea pig, poultry, a goat, a sheep, primate and a rabbit.  
     
     
         16 . The method of  claim 15 , wherein said animal is a mouse.  
     
     
         17 . The method of  claim 16 , wherein said mouse is a transgenic mouse overexpressing CD19.  
     
     
         18 . The method of  claim 14 , wherein said antibody-producing cells comprise B lymphocytes.  
     
     
         19 . A diagnostic assay kit for detecting the presence of an antigen in a biological sample, the kit comprising a first container containing a first antibody capable of immunoreacting with the antigen, wherein the first antibody is produced from an animal having antibody-producing cells with disrupted peripheral tolerance and the first antibody is present in an amount sufficient to perform at least one assay.  
     
     
         20 . The assay kit of  claim 19 , further comprising a second container containing a second antibody that immunoreacts with the first antibody, wherein second antibody is produced from an animal having antibody-producing cells with disrupted peripheral tolerance.  
     
     
         21 . The assay kit of  claim 20 , wherein the first antibody and the second antibody comprise monoclonal antibodies.  
     
     
         22 . The assay kit of  claim 21 , wherein said first antibody comprises an antibody having a high affinity for said antigen.  
     
     
         23 . The assay kit of  claim 20 , wherein the first antibody is affixed to a solid support.  
     
     
         24 . The assay kit of  claim 20 , wherein the first and second antibodies each further comprise an indicator.  
     
     
         25 . An assay kit of  claim 24 , wherein the indicator is a radioactive label or an enzyme.  
     
     
         26 . A method of producing a non-human animal with an immune system having cells with a predetermined characteristic, the method comprising the steps of: 
 (a) obtaining an animal having immune system cells with a particular characteristic;    (b) obtaining another animal having immune system cells with either a same or a different characteristic from the animal of step (a); and    (c) breeding the animal of step (a) with the animal of step (b) to produce an animal with an immune system having cells with a predetermined characteristic.    
     
     
         27 . The method of  claim 26 , wherein said animals are selected from the group consisting of a mouse, rat, pig, guinea pig, poultry, a goat, a sheep, primate and a rabbit.  
     
     
         28 . The method of  claim 27 , wherein said animals are transgenic animals.