Reagents and methods for detecting a polymorphic protein
View Patent ↗The present invention provides antibodies that differentially react with allelic variants of a polymorphic protein, methods of identifying same, an antigen binding fragment comprised therein, proteins, cells, viral particles, compositions, and kits comprising same. The invention also provides methods for determining a haptoglobin type of a subject and methods for testing a subject for susceptibility to diabetic complications.
1. A method of determining a haptoglobin (Hp) phenotype of a subject, comprising:
contacting a biological sample of said subject with an isolated anti-haptoglobin monoclonal antibody,
wherein the isolated anti-haptoglobin monoclonal antibody, comprises at least one light chain variable region and at least one heavy chain variable region, said light chain variable region comprising: the complementarity determining region light chain (CDRL) 1 consisting of SEQ ID NO:3; a CDRL2 consisting of SEQ ID NO:4; and a CDRL3 consisting of SEQ ID NO:5, and said heavy chain variable region comprising: a complementarity determining region heavy chain (CDRH) 1 consisting of SEQ ID NO:8; a CDRH2 consisting of SEQ ID NO:9; and a CDRH3 consisting of SEQ ID NO:10;
forming a bound complex between the isolated anti-haptoglobin monoclonal antibody and haptoglobin in the biological sample;
quantitatively determining a binding affinity between said haptoglobin and said isolated anti-haptoglobin monoclonal antibody; and
comparing said quantitatively determined binding affinity with a value obtained from quantitatively determining the binding affinity of said isolated anti-haptoglobin monoclonal antibody to an isolated Hp 1-1, Hp 2-1, or Hp 2-2 isoform, wherein the binding affinity determined is indicative of the Hp allele type, thereby determining the phenotype of Hp in said subject.
2. The method of claim 1 , wherein the biological sample is serum or plasma.
3. The method of claim 2 , wherein the plasma has been anticoagulated with citrate, heparin or EDTA.
4. The method of claim 1 , further comprising immobilizing the bound anti-haptoglobin antibody-haptoglobin complex on a substrate resulting in an immobilized complex.
5. The method of claim 4 , wherein the immobilizing comprises binding the anti-haptoglobin monoclonal antibody to the substrate.
6. The method of claim 4 , further comprising:
contacting said immobilized complex with an additional quantity of said monoclonal anti-haptoglobin antibody; and determining a binding affinity between said haptoglobin and said additional quantity of said monoclonal anti-haptoglobin antibody.
7. The method of claim 1 , wherein the monoclonal anti-haptoglobin antibody is a murine hybridoma deposited with the American Type Culture Collection (ATCC) having a patent deposit designation PTA-9815.
8. The method of claim 1 , wherein the isolated anti-haptoglobin monoclonal antibody comprises a light chain variable region consisting of SEQ ID NO:2.
9. The method of claim 1 , wherein the isolated anti-haptoglobin monoclonal antibody comprises a heavy chain variable region consisting of SEQ ID NO:7.
10. The method of claim 1 , wherein the isolated anti-haptoglobin monoclonal antibody comprises a light chain variable region consisting of SEQ ID NO:2 and a heavy chain variable region consisting of SEQ ID NO:7.
11. The method of claim 1 , wherein said antibody light or heavy chain variable region of the isolated anti-haptoglobin monoclonal antibody is at least one of chimerzed, humanized, or CDR-grafted.
12. The method of claim 11 , wherein the isolated anti-haptoglobin monoclonal antibody further comprises at least one compound or polypeptide selected from a detectable label or reporter.
13. The method of claim 12 , wherein the detectable label is an enzyme.
14. The method of claim 13 , wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
15. The method of claim 1 , wherein the isolated anti-haptoglobin monoclonal antibody has a binding affinity for Hp2-2, a binding affinity for Hp2-1, and a binding affinity for Hp1-1, wherein the binding affinity of the isolated anti-haptoglobin monoclonal antibody for Hp2-2 is greater than the binding affinity of the isolated anti-haptoglobin monoclonal antibody for Hp2-1 and Hp-1-1, and the binding affinity of the isolated anti-haptoglobin monoclonal antibody for Hp2-1 is greater than the binding affinity of the isolated anti-haptoglobin monoclonal antibody for Hp-1-1.