High-sensitivity proteolysis assay
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.