Method and compositions for specifically detecting physiologically acceptable polymer molecules
View Patent ↗The present invention relates to a method for determining the amount of a physiologically acceptable polymer molecule bound to a protein, an antibody or other composition being capable of specifically binding to a physiologically acceptable polymer molecule, and a kit containing said antibody or composition.
1. A method for determining the number of polyethylene glycol (PEG) molecules in a polymer:protein conjugate having one or more PEG polymer molecules bound to the protein, the method comprising,
contacting the polymer:protein conjugate with a polyclonal antibody that specifically binds the PEG polymer of the polymer:protein conjugate, said polyclonal antibody detectable when bound to the polymer:protein conjugate, and
detecting the level of the polyclonal antibody bound to the polymer:protein conjugate,
wherein the number of PEG polymer molecules in the polymer:protein conjugate correlates with the level of the polyclonal antibody detected bound to the polymer:protein conjugate when compared to a known control, and wherein polyclonal antibody binding is dose dependent and linear with respect to an amount of bound PEG, and wherein the protein is a blood clotting factor or a blood clotting factor complex.
2. The method of claim 1 , wherein the antibody comprises a detectable label.
3. The method of claim 2 , wherein the detectable label is selected from the group consisting of an enzyme, a radioactive label, a fluorophore, an electron dense reagent, biotin, digoxigenin, haptens, and proteins which are made detectable by addition of any of these labels.
4. The method of claim 1 , wherein the polymer:protein conjugate is bound to a carrier matrix prior to binding with the antibody.
5. The method of claim 4 , wherein the carrier matrix is selected from the group consisting of a microcarrier, a particle, a membrane, a strip, paper, a film, a bead or a plate.
6. The method of claim 4 , wherein the level of antibody detected is measured as absorbance of the detectable label.
7. The method of claim 1 , wherein the polymer:protein conjugate is isolated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane prior to the detecting.
8. The method of claim 7 , wherein the number of polymers in the polymer:protein conjugate is calculated based on the molecular weight of the protein-polymer conjugate compared to a known control.
9. The method of claim 7 , wherein the molecular weight of the polymer-protein complex correlates with the protein subunit comprising the polymer molecule.
10. The method of claim 1 wherein the blood clotting factor or blood clotting factor complex is human.
11. The method of claim 1 wherein the blood clotting factor is selected from the group consisting of Factor II, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand Factor, protein C and antithrombin III.
12. The method of claim 10 wherein the blood clotting factor complex is FactorVIII:VWF.
13. The method of claim 1 , wherein the PEG polymer is releasable.
14. The method of claim 1 , wherein the PEG polymer is hydrolyzable.
15. The method of claim 1 wherein the blood clotting factor is Factor VIII.
16. The method of claim 1 , wherein the PEG is from 3 to 100 kDa.
17. The method of claim 16 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 60 kDa.
18. The method of claim 16 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 40 kDa.
19. The method of claim 16 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 15 kDa.
20. The method of claim 16 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 10 kDa.