ELISA detection of urine DEK to predict and diagnose bladder cancer in humans
The present invention is directed to a method of detecting a DEK protein in a human urine sample using an ELISA assay. Methods and compositions for detection of DEK using mAb 260-6F9F6 (as detection antibody) and mAb 16-2C9C3 (as capture antibody) in human urine are provided herein. Specifically, the ELISA assay utilizes a capture mAb and a detection mAb to yield a high sensitivity of <50 ng/mL. The presence of DEK in urine is useful in predicting or diagnosing the occurrence of bladder cancer in humans.
1. A method of detecting bladder cancer in a human, comprising the steps of:
(a) obtaining a urine sample from a human suspected of suffering from bladder cancer;
(b) concentrating said urine sample;
(c) immobilizing said concentrated urine sample onto a solid surface;
(d) adding an anti-DEK antibody to said immobilized concentrated urine sample so as to allow a complex formation between DEK isoform 2 protein, and said anti-DEK antibody, said DEK Isoform 2 protein being a protein consisting of SEQ ID NO:2
wherein said anti-DEK antibody recognizes DEK isoform 2 protein;
(e) selecting said anti-DEK antibody from the group consisting of a monoclonal antibody 16-2C9C3 or a monoclonal antibody 260-6F9F6;
and
(e) detecting said protein-antibody complex, wherein the presence of said protein-antibody complex is indicative of a bladder cancer in said human.
2. The method of claim 1 , wherein the anti-DEK antibody is a monoclonal antibody 16-2C9C3.
3. The method of claim 2 , wherein the monoclonal antibody 16-2C9C3 comprises: (i) CDR1 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:15; (ii) CDR2 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:17; (iv) CDR1 of the light chain variable region which has an amino acid sequence of SEQ ID NO: 23; (v) CDR2 of the light chain variable region which has the amino acid sequence of SEQ ID NO:24; and (vi) CDR3 of the light chain variable region which has the amino acid sequence of SEQ ID NO:25.
4. The method of claim 1 , wherein the anti-DEK antibody is a monoclonal antibody 260-6F9F6.
5. The method of claim 4 , wherein the monoclonal antibody 260-6F9F6 comprises: (i) CDR1 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:31; (ii) CDR2 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:32; (iii) CDR3 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:33; (iv) CDR1 of the light chain variable region which has an amino acid sequence of SEQ ID NO: 39; (v) CDR2 of the light chain variable region which has the amino acid sequence of SEQ ID NO:40; and (vi) CDR3 of the light chain variable region which has the amino acid sequence of SEQ ID NO:41.
6. A method of detecting bladder cancer in a human, comprising the steps of:
(a) obtaining a urine sample from a human suspected of suffering from bladder cancer;
(b) concentrating said urine sample;
(c) immobilizing a first anti-DEK antibody onto a solid surface; said first anti-DEK antibody is a monoclonal antibody 16-2C9C3;
(d) adding said concentrated urine sample onto said solid surface having said immobilized first anti-DEK antibody and allowing formation of DEK isoform 2 protein and first anti-DEK antibody complex;
(e) removing unbound DEK isoform 2 protein; said DEK Isoform 2 is a protein consisting of SEQ ID NO: 2
(f) adding a second anti-DEK antibody so as to allow formation of a complex between said bound DEK isoform 2 protein with said second anti-DEK antibody said second anti-DEK antibody is a monoclonal antibody 260-6F9F; and
(g) detecting said bound DEK isoform 2 protein with said second anti-DEK antibody complex, wherein said first anti-DEK antibody and said second anti-DEK antibody recognize a different region of DEK isoform 2 protein, and wherein the presence of said protein-antibody complex is indicative of a bladder cancer in said human.
7. The method of claim 6 , wherein the monoclonal antibody 16-2C9C3 comprises: (i) CDR1 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:15; (ii) CDR2 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:16; (iii) CDR3 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:17; (iv) CDR1 of the light chain variable region which has an amino acid sequence of SEQ ID NO: 23; (v) CDR2 of the light chain variable region which has the amino acid sequence of SEQ ID NO:24; and (vi) CDR3 of the light chain variable region which has the amino acid sequence of SEQ ID NO:25.
8. The method of claim 6 , wherein the monoclonal antibody 260-6F9F6 comprises: (i) CDR1 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:31; (ii) CDR2 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:32; (iii) CDR3 of the heavy chain variable region which has the amino acid sequence of SEQ ID NO:33; (iv) CDR1 of the light chain variable region which has an amino acid sequence of SEQ ID NO: 39; (v) CDR2 of the light chain variable region which has the amino acid sequence of SEQ ID NO:40; and (vi) CDR3 of the light chain variable region which has the amino acid sequence of SEQ ID NO:41.
9. A method of detecting bladder cancer in a human, comprising the steps of:
(a) obtaining a urine sample from a human suspected of suffering from bladder cancer;
(b) concentrating said urine sample;
(c) immobilizing a first anti-DEK antibody onto a solid surface;
(d) adding said concentrated urine sample onto said solid surface having said immobilized first anti-DEK antibody and allowing formation of DEK isoform 2 protein and first anti-DEK antibody complex;
(e) removing unbound DEK isoform 2 protein;
(f) adding a second anti-DEK antibody so as to allow formation of a complex between said bound DEK isoform 2 protein with said second anti-DEK antibody; and
(g) detecting said bound DEK isoform 2 protein with said second anti-DEK antibody complex, wherein said first anti-DEK antibody and said second anti-DEK antibody recognize a different region of DEK isoform 2 protein, and wherein the presence of said protein-antibody complex is indicative of a bladder cancer in said human;
wherein the first anti-DEK antibody is a monoclonal antibody 16-2C9C3 and the second anti-DeK antibody is a monoclonal antibody 260-6F9F6.