Immunoassay for detection of very low density lipoprotein and antibodies useful therefor

Abstract

The present invention provides a method for directly measuring apolipoprotein B-100 (apoB) or cholesterol associated with very low density lipoprotein (VLDL) in a fluid sample. In one embodiment the method involves the specific capture of intact VLDL particles from a fluid sample with a specific VLDL binding agent. The quantity of VLDL-apoB present in the sample is then measured by detecting the amount of VLDL-apoB bound to the binding agent-VLDL complexes formed in the reaction. In an alternative embodiment of the method, intact VLDL particles from a fluid sample are also captured with a specific VLDL binding agent and thereafter the cholesterol associated with the bound VLDL is determined. The cholesterol contained in the binding-agent-VLDL complexes can be detected by reacting the complexes with labeled cholesterol specific binding agents and measuring the amount of label bound therto, or by releasing the cholesterol in the complexes and measuring the amount of cholesterol released. VLDL specific binding reagents are also provided.

What is claimed is:  
     
         1 . A method for determining the amount of apoB associated with VLDL in a sample comprising: 
 (a) mixing a sample and a VLDL-specific binding agent for a time and under conditions to form binding-agent-VLDL complexes; and    (b) determining the amount of apoB associated with VLDL bound to said binding-agent-VLDL complexes.    
     
     
         2 . The method of  claim 1  wherein said VLDL-specific binding agent is coupled to a solid support.  
     
     
         3 . The method of  claim 2  further comprising the step of separating the solid support from the sample before determining the amount of apoB bound to said binding-agent-VLDL complexes.  
     
     
         4 . The method of  claim 2  wherein the solid support is selected from the group consisting of nitrocellulose, latex, nylon, polystyrene.  
     
     
         5 . The method of  claim 2  wherein the solid support is selected from the group consisting of beads, particles, magnetic particles, and glass fiber.  
     
     
         6 . The method of  claim 1  further comprising the step of separating said binding-agent-VLDL complexes prior to step (b).  
     
     
         7 . The method of  claim 6  wherein said VLDL-specific binding agent is conjugated to a first charged substance and said separation comprises: 
 (a) contacting said binding-agent-VLDL complexes with an insoluble solid phase material which is oppositely charged with respect to said first charged substance, such that said solid phase material attracts and attaches to said first charged substance; and  
 (b) separating said solid phase material and said sample.  
 
     
     
         8 . The method of  claim 7  wherein said charged substances are anionic and cationic monomers or polymers.  
     
     
         9 . The method of  claim 1  wherein the VLDL-specific binding agent is an antibody or fragment thereof that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding.  
     
     
         10 . The method of  claim 9  wherein said antibody is a monoclonal antibody.  
     
     
         11 . The method of  claim 10  wherein said monoclonal antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211.  
     
     
         12 . The method of  claim 10  wherein said monoclonal antibody is 18-358-211.  
     
     
         13 . A method for determining the amount of apoB associated with VLDL in a sample comprising the steps of: 
 (a) contacting said sample with an indicator reagent wherein said indicator reagent is a monoclonal antibody or fragment thereof that specifically binds to said apoB associated with VLDL and with a solid support coated with VLDL for a time and under conditions to permit binding of said indicator reagent with said VLDL in said sample and with said bound VLDL; and    (b) determining said amount of apoB associated with VLDL in said test sample by detecting the reduction in binding of said indicator reagent to said solid support as compared to the signal generated from a negative sample to indicate the presence of VLDL in said test sample.    
     
     
         14 . The method of  claim 13  wherein said indicator reagent is Mab 18-358-211.  
     
     
         15 . A method for determining the amount of cholesterol associated with VLDL in a sample comprising: 
 a. mixing a sample and a VLDL-specific binding agent for a time and under conditions to form binding-agent-VLDL complexes; and    b. determining the amount of cholesterol bound to said binding-agent-VLDL complexes.    
     
     
         16 . The method of  claim 15  wherein said VLDL-specific binding agent is coupled to a solid support.  
     
     
         17 . The method of  claim 15  further comprising the step of separating said binding-agent-VLDL complexes prior to step (b).  
     
     
         18 . The method of  claim 15  wherein the VLDL-specific binding agent is a monoclonal antibody, polyclonal antibody or fragment thereof that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding.  
     
     
         19 . The method of  claim 18  wherein said antibody is a monoclonal antibody.  
     
     
         20 . The method of  claim 19  wherein said monoclonal antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211.  
     
     
         21 . The method of  claim 16  further comprising the step of separating the solid support from the sample before determining the amount of cholesterol bound to said binding-agent-VLDL complexes.  
     
     
         22 . The method of  claim 16  wherein the solid support is selected from the group consisting of nitrocellulose, latex, nylon and polystyrene.  
     
     
         23 . The method of  claim 16  wherein the solid support is selected from the group consisting of beads, particles, magnetic particles, and glass fiber.  
     
     
         24 . The method of  claim 15  wherein said determination comprises releasing said cholesterol bound to said binding agent-VLDL complexes and measuring the amount of cholesterol released.  
     
     
         25 . The method of  claim 15  wherein said determination comprises mixing said binding-agent-VLDL complexes with a cholesterol specific binding agent coupled to a detectable label for a time and under conditions suitable to form binding-agent-VLDL-cholesterol specific binding agent complexes and determining the amount of label bound to said binding-agent-VLDL-cholesterol specific binding agent complex.  
     
     
         26 . The method of  claim 17  wherein said VLDL-specific binding agent is conjugated to a first charged substance; and said separation comprises: 
 (a) contacting said binding-agent-VLDL complexes with an insoluble solid phase material which is oppositely charged with respect to said first charged substance, such that said solid phase material attracts and attaches to said first charged substance; and  
 (b) separating said solid phase material and said sample.  
 
     
     
         27 . The method of  claim 26  wherein said charged substances are anionic and cationic monomers or polymers.  
     
     
         28 . The method of  claim 17  wherein said determination comprises releasing said cholesterol bound to said binding-agent-VLDL complexes and measuring the amount of cholesterol released.  
     
     
         29 . The method of  claim 17  wherein said determination comprises mixing said binding-agent-VLDL complexes with a cholesterol specific binding agent coupled to a detectable label such that a second complex is formed and determining the amount of label bound to said second complex.  
     
     
         30 . An antibody or fragment thereof specific for VLDL wherein said antibody binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding.  
     
     
         31 . The antibody of  claim 30  selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211.  
     
     
         32 . The antibody of  claim 31  which is produced by a hybridoma cell line having ATCC Accession No. HB-12392.  
     
     
         33 . A hybridoma cell line that produces a monoclonal antibody which binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding.  
     
     
         34 . The hybridoma cell line of  claim 33  wherein said monoclonal antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211.  
     
     
         35 . The hybridoma cell line of  claim 33  having ATCC Accession No. HB-12392.  
     
     
         36 . A monoclonal antibody specific for VLDL prepared by the method comprising the steps of: 
 (a) immunizing a mouse or a rat with Apo CIII;    (b) making a suspension of the mouse or rat spleen cells;    (c) fusing the spleen cells with mouse or rat myeloma cells in the presence of a fusion promoter;    (d) culturing the fused cells;    (e) determining the presence of anti-VLDL antibody in the culture media;    (f) cloning a hybridoma producing antibody that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of LDL binding; and    (g) obtaining the antibody from said hybridoma.