Method for measuring the protease activity of c3 and c5 convertase of the alternative complement pathway
AssigneeAlexion Pharmaceuticals, Inc.
InventorsKrista K. JOHNSON, Christen D. FORBES
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Abstract
A method for measuring the protease activity of a convertase of the alternative complement pathway is provided. The method typically comprises immobilizing a biotinylated C3b on a solid phase coated with a biotin binding protein. Substantially homogeneous components of the alternative complement pathway may be incubated with the immobilized C3b in a serum free and a gelatin free buffer, to form a convertase. The activity of the convertase is generally measured with an immunoassay.
Claims23
1-46. (canceled)
A method for measuring the protease activity of C3 convertase comprising the steps of:
- a. binding C3b covalently attached to a tag comprising biotin or an analog or derivative thereof to a biotin binding protein immobilized on a solid phase;
- b. incubating the immobilized tagged-C3b, in the presence of Factor D, and Factor B in a buffer to form C3 convertase (C3b,Bb);
- c. adding C3 to the incubation of (b) and incubating the mixture under conditions sufficient to cleave C3 with the C3 convertase (C3b,Bd) to form a C3a and C3b;
- and
- d. measuring the amount of the C5a with an immunoassay,
- wherein each of the individual components tagged-C3b, Factor B, Factor D, and C3 are substantially homogeneous.
A method for measuring the protease activity of C5 convertase comprising the steps of:
- a. binding C3b covalently attached to a tag comprising biotin or an analog or derivative thereof to a biotin binding protein immobilized on a solid phase;
- b. incubating the immobilized tagged-C3b, in the presence of Factor D, and Factor B in a buffer to form C5 convertase;
- c. adding C5 to the incubation of (b) and incubating the mixture under conditions sufficient to cleave C5 with the C5 convertase to form a C5a and CSb;
- and
- d. measuring the amount of the C5a with an immunoassay,
- wherein each of the individual components bio tagged-C3b, Factor B, Factor D, and C5 are substantially homogeneous.
A kit for measuring the protease activity of C3 convertase of the alternative pathway using substantially homogeneous components of the alternative complement pathway, the kit comprising:
- a. substantially homogeneous C3b covalently attached to a tag comprising biotin or an analog or derivative thereof;
- b. a solid phase coated with a biotin binding protein;
- c. substantially homogeneous Factor B, Factor D and C3;
- and,
- d. an anti-C3a antibody.
A kit for measuring the protease activity of C5 convertase of the alternative pathway using substantially homogeneous components of the alternative complement pathway, the kit comprising:
- a. substantially homogeneous C3b covalently attached to a tag comprising biotin or an analog or derivative thereof;
- b. a solid phase coated with a biotin binding protein;
- c. substantially homogeneous Factor B, Factor D and C5;
- and,
- d. an anti-C5a antibody.
Dependencies are inferred from the claim text.