High-sensitivity proteolysis assay

Abstract

The present invention includes a highly sensitive method for detecting the presence of proteases in a sample which are present at very low levels.

1 . A method for determining the presence of proteolytic activity in a sample comprising incubating a mixture comprising the sample with a peptide substrate for said protease for at least about 8 hours and determining proteolysis of said substrate in the incubated mixture. 
     
     
         2 . The method of  claim 1  wherein the incubated mixture comprises a reducing agent. 
     
     
         3 . The method of  claim 1  wherein proteolysis of the substrate is determined by heating an aliquot of the incubated mixture to at least 90° C. for at least 5 minutes, electrophoresing the heated aliquot on a SDS-polyacrylamide gel staining the gel with a protein indicator stain; observing protein in the gel; and, based on said observation, determining if proteolytic degradation of the substrate occurred. 
     
     
         4 . The method of  claim 3  wherein the gel is a 4-12% or 4-20% discontinuous SDS-polyacrylamide gel. 
     
     
         5 . The method of  claim 3  wherein about 12 μg of the peptide substrate is electrophoresed on the gel. 
     
     
         6 . The method of  claim 1  wherein the pH of the incubated mixture is about 8.8. 
     
     
         7 . The method of  claim 6  wherein the incubated mixture comprises Tris pH 8.8 buffer. 
     
     
         8 . The method of  claim 1  wherein the incubated mixture is incubated for about 18 hours. 
     
     
         9 . The method of  claim 1  wherein the peptide substrate is an immunoglobulin polypeptide. 
     
     
         10 . The method of  claim 9  wherein the concentration of the immunoglobulin in the incubated mixture is about 1 mg/ml. 
     
     
         11 . The method of  claim 1  wherein proteolysis is determined by SDS-polyacrylamide gel electrophoresis analysis. 
     
     
         12 . The method of  claim 1  wherein the incubation is carried out at room temperature. 
     
     
         13 . The method of  claim 1  wherein the peptide substrate is an anti-IGF1R antibody or an anti-IL10 antibody. 
     
     
         14 . The method of  claim 1  wherein the sample is cellular growth media. 
     
     
         15 . The method of  claim 14  wherein the growth media comprises a hydrolysate. 
     
     
         16 . The method of  claim 15  wherein the hydrolysate is a plant-derived hydrolysate. 
     
     
         17 . A method for producing an antibody or antigen-binding fragment thereof comprising determining the presence of proteolytic activity in culture medium by the method of  claim 1  and, if no proteolytic activity is detected, culturing one or more host cells expressing the antibody or fragment in said medium. 
     
     
         18 . The method of  claim 17  further comprising isolating the antibody or fragment from the medium. 
     
     
         19 . The method of  claim 17  wherein the antibody is an anti-IGF1R antibody.