Competition based-detection assays
View Patent ↗Disclosed herein are methods and kits which are useful for detecting presence of an enzyme and the relative amount of glycan associated with the enzyme in a test sample based upon the enzyme's ability to competitively inhibit the binding of a ligand in 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 of detecting a relative amount of glycan associated with an enzyme in a test sample, the method comprising: (a) contacting the test sample with at least one capture agent under conditions appropriate for binding of glycosylated enzyme in the test sample to the capture agent, wherein if the glycosylated enzyme is present in the test sample a bound enzyme is formed; (b) separating the bound enzyme from the test sample; c) contacting the bound enzyme with a ligand for the capture agent and (d) detecting the extent to which the bound enzyme inhibits binding of the ligand to the capture agent, wherein the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of the relative amount of glycan associated with the enzyme in the test sample; wherein the capture agent comprises a biotinylated lectin.
2. The method of claim 1 , wherein the lectin is 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, or ricinus communis agglutinin I.
3. The method of claim 1 , wherein the lectin is affixed on a solid support.
4. The method of claim 3 , wherein the solid support comprises avidin or streptavidin.
5. The method of claim 3 , wherein the solid support comprises a multiple well microtiter plate.
6. The method of claim 4 , wherein the solid support comprises at least one bead.
7. The method of claim 1 , wherein the ligand is a competitive inhibitor which competes with an enzyme suspected of being present in the test sample for binding to the capture agent.
8. The method of claim 7 , wherein the enzyme is idursulfase and the competitive inhibitor is agalsidase alfa.
9. The method of claim 7 , wherein the enzyme is heparan N-sulfatase and the competitive inhibitor is agalsidase alfa.
10. The method of claim 7 , wherein the enzyme is aryl sulfatase A and the competitive inhibitor is agalsidase alfa.
11. The method of claim 1 , wherein the greater the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of a greater relative amount of glycan associated with the enzyme in the test sample, and wherein the lesser the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of a lesser relative amount of glycan associated with the enzyme in the test sample.
12. The method of claim 1 , wherein the step of detecting the extent to which the bound enzyme inhibits binding of the ligand to the capture agent comprises determining the amount of the ligand bound to the capture agent.
13. The method of claim 12 , wherein the step of determining the amount of the ligand bound to the capture agent comprises detecting the intrinsic enzymatic activity of the ligand bound to the capture agent.
14. The method of claim 13 , wherein the step of detecting intrinsic enzymatic activity of the ligand bound to the capture agent is performed by contacting the ligand bound to the capture agent with a substrate.
15. The method of claim 14 , wherein the substrate is reactive with the ligand bound to the capture agent.
16. The method of claim 14 , wherein the conversion of the substrate to product is indicative of intrinsic enzymatic activity.
17. The method of claim 13 , wherein the step of detecting intrinsic enzymatic activity comprises quantitatively determining the presence of a product formed after contacting the ligand bound to the capture agent with the substrate.
18. The method of claim 17 , wherein the presence of the product is indicative of intrinsic enzymatic activity.
19. The method of claim 14 , wherein the ligand is agalsidase alfa and the substrate is 4-nitrophenyl-α-D-galactopyranoside.
20. The method of claim 1 , wherein the enzyme is idursulfase and the glycan is sialic acid.
21. The method of claim 1 , wherein the enzyme is idursulfase and the glycan is mannose-6-phosphate.
22. The method of claim 1 , wherein the enzyme is heparan N-sulfatase and the glycan is sialic acid.
23. The method of claim 1 , wherein the enzyme is heparan N-sulfatase and the glycan is mannose-6-phosphate.
24. The method of claim 1 , wherein the enzyme is aryl sulfatase A and the glycan is sialic acid.
25. The method of claim 1 , wherein the enzyme is aryl sulfatase A and the glycan is mannose-6-phosphate.
26. The method of claim 1 , wherein the step of separating the bound enzyme from the test sample is performed by washing.
27. A method of detecting a relative amount of glycan associated with an enzyme in a test sample, the method comprising: (a) contacting a test sample with at least one capture agent under conditions appropriate for binding of glycosylated enzyme, wherein if the glycosylated enzyme is present in the test sample a bound enzyme is formed; (b) separating the bound enzyme from the test sample; (c) contacting the bound enzyme with at least one ligand for the capture agent; and (d) detecting the extent to which the bound enzyme inhibits binding of the ligand to the capture agent, wherein the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of the relative amount of glycan associated with the enzyme in the test sample; wherein the capture agent comprises a biotinylated lectin.
28. The method of claim 27 , wherein the lectin is 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, or ricinus communis agglutinin I.
29. The method of claim 27 , wherein the lectin is affixed on a solid support.
30. The method of claim 29 , wherein the solid support comprises avidin or streptavidin.
31. The method of claim 29 , wherein the solid support comprises a multiple well microtiter plate.
32. The method of claim 30 , wherein the solid support comprises at least one bead.
33. The method of claim 27 , wherein the ligand is a competitive inhibitor which competes with an enzyme suspected of being present in the test sample for binding to the capture agent.
34. The method of claim 33 , wherein the enzyme is idursulfase and the competitive inhibitor is agalsidase alfa.
35. The method of claim 33 , wherein the enzyme is heparan N-sulfatase and the competitive inhibitor is agalsidase alfa.
36. The method of claim 33 , wherein the enzyme is aryl sulfatase A and the competitive inhibitor is agalsidase alfa.
37. The method of claim 27 , wherein the greater the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of a greater relative amount of the glycan associated with the enzyme glycoform in the test sample, and wherein the lesser the extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of a lesser relative amount of glycan associated with the enzyme glycoform in the test sample.
38. The method of claim 27 , wherein the step of detecting the extent to which the bound enzyme inhibits binding of the ligand to the capture agent comprises determining the amount of the ligand bound to the capture agent.
39. The method of claim 38 , wherein the step of determining the amount of the ligand bound to the capture agent comprises detecting the intrinsic enzymatic activity of the ligand bound to the capture agent.
40. The method of claim 39 , wherein the step of detecting intrinsic enzymatic activity of the ligand bound to the capture agent is performed by contacting the ligand bound to the capture agent with a substrate.
41. The method of claim 40 , wherein the substrate is reactive with the ligand bound to the capture agent.
42. The method of claim 40 , wherein the conversion of the substrate to product is indicative of intrinsic enzymatic activity.
43. The method of claim 39 , wherein the step of detecting intrinsic enzymatic activity comprises quantitatively determining the presence of a product formed after contacting the ligand bound to the capture agent with the substrate.
44. The method of claim 43 , wherein the presence of the product is indicative of intrinsic enzymatic activity.
45. The method of claim 40 , wherein the ligand is agalsidase alfa and the substrate is 4-nitrophenyl-α-D-galactopyranoside.
46. The method of claim 39 , wherein the enzyme is idursulfase and the glycan is sialic acid.
47. The method of claim 39 , wherein the enzyme is idursulfase and the glycan is mannose-6-phosphate.
48. The method of claim 39 , wherein the enzyme is heparan N-sulfatase and the glycan is sialic acid.
49. The method of claim 39 , wherein the enzyme is heparan N-sulfatase and the glycan is mannose-6-phosphate.
50. The method of claim 39 , wherein the enzyme is aryl sulfatase A and the glycan is sialic acid.
51. The method of claim 39 , wherein the enzyme is aryl sulfatase A and the glycan is mannose-6-phosphate.
52. The method of claim 39 , wherein the bound enzyme is separated from the test sample by washing.