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) polymer 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 an antibody that specifically binds the PEG polymer of the polymer:protein conjugate, the antibody detectable when bound to the polymer:protein conjugate,
detecting the level of the 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 antibody detected bound to the polymer:protein conjugate when compared to a known control, and wherein antibody binding is dose dependent and linear with respect to an amount of bound PEG.
2. The method of claim 1 , wherein the PEG is from 3 to 100 kDa.
3. The method of claim 2 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 60 kDa.
4. The method of claim 2 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 40 kDa.
5. The method of claim 2 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 15 kDa.
6. The method of claim 2 , wherein the PEG has a molecular weight in a range of about 5 kDa to about 10 kDa.
7. The method of claim 1 , wherein the antibody comprises a detectable label.
8. The method of claim 1 , wherein the polymer:protein conjugate is bound to a carrier matrix prior to binding with the antibody.
9. The method of claim 8 , wherein the level of antibody detected is measured as absorbance of the detectable label.
10. 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.
11. The method of claim 10 , 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.
12. The method of claim 10 , wherein the molecular weight of the polymer-protein complex correlates with the protein subunit comprising the polymer molecule.
13. The method of claim 1 , wherein the protein or protein complex is a blood clotting factor or a blood clotting factor complex.
14. The method of claim 13 wherein the blood clotting factor or blood clotting factor complex is human.