Enzymatic activity-based detection
View Patent ↗Disclosed herein are methods and kits which are useful for detecting presence of an enzyme in a test sample based upon the intrinsic enzymatic activity of such test sample. The present invention provides the ability to evaluate cell culture conditions and optimize the desired glycoform content of recombinantly prepared enzymes.
1. A method for detecting the presence of an enzyme in a test sample, wherein said method comprises the steps of: (a) contacting said test sample with at least one capture agent under conditions appropriate for binding of glycosylated enzyme in said test sample to said capture agent, wherein if said glycosylated enzyme is present in said test sample a bound enzyme is formed, and wherein said capture agent comprises a biotinylated lectin; (b) separating said bound enzyme from said test sample; and (c) detecting intrinsic enzymatic activity of said bound enzyme, wherein the presence of intrinsic enzymatic activity is indicative of the presence of enzyme in said test sample.
2. The method of claim 1 , wherein said lectin is affixed on a solid support.
3. The method of claim 1 , wherein said step of detecting intrinsic enzymatic activity of said bound enzyme is performed by contacting said bound enzyme with a substrate.
4. The method of claim 3 , wherein said substrate is reactive with an enzyme suspected of being present in said test sample.
5. The method of claim 4 , wherein said enzyme is agalsidase alfa and said substrate is 4-nitrophenyl-α-D-galactopyranoside.
6. The method of claim 4 , wherein said step of detecting intrinsic enzymatic activity comprises quantitatively determining the depletion of said substrate.
7. The method of claim 4 , wherein said step of detecting intrinsic enzymatic activity comprises quantitatively determining the presence of a product formed after contacting said bound enzyme with said substrate.
8. The method of claim 1 , wherein said lectin is selected from the group consisting of concanavalin A, wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lens culinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II, Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigra lectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotus tetragonolobus lectin, galanthus nivalis lectin, euonymus europaeus lectin, and ricinus communis agglutinin I.
9. A method for detecting the presence of an enzyme in a test sample, wherein said method comprises the steps of: (a) contacting a test sample with at least one capture agent under conditions appropriate for binding of glycosylated enzyme, wherein if said glycosylated enzyme is present in said test sample a bound enzyme is formed; (b) separating said bound enzyme from said test sample; (c) contacting said bound enzyme with at least one substrate; and (d) detecting the presence or absence of intrinsic enzymatic activity of said bound enzyme, wherein the presence of intrinsic enzymatic activity is indicative of the presence of enzyme in said test sample, wherein said enzyme is agalsidase alfa, and wherein said substrate is 4-nitrophenyl-α-D-galactopyranoside.
10. The method of claim 9 , wherein said capture agent comprises a lectin.
11. The method of claim 9 , wherein said step of detecting intrinsic enzymatic activity comprises quantitatively determining the depletion of said substrate.
12. The method of claim 9 , wherein said step of detecting intrinsic enzymatic activity comprises quantitatively determining the presence of a product formed after contacting said bound enzyme with said substrate.
13. The method of claim 12 , wherein the presence of said product is indicative of intrinsic enzymatic activity.
14. The method of claim 10 , wherein said lectin is selected from the group consisting of concanavalin A, wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lens culinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II, Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigra lectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotus tetragonolobus lectin, galanthus nivalis lectin, euonymus europaeus lectin, and ricinus communis agglutinin I.