Binding agent and assay for PIVKA
The present disclosure relates to specific binding agents binding to different PIVKA-II forms as compared to antibodies known so far in the art. The present disclosure also relates to methods of using the specific binding agents to detect the presence of PIVKA-II.
1. A composition comprising an antibody containing CDRs that comprise the following amino acid sequences:
(i) a CDR1 comprising the amino acid sequence of SEQ ID NO:10, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:12 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:13, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain;
(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:21, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain;
(iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:21, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain; or
(iv) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain.
2. The composition of claim 1 , wherein the antibody is a monoclonal antibody.
3. The composition of claim 1 , wherein the antibody binds to a synthetic peptide of SEQ ID NO:1, has an at least 10-fold binding preference for the peptide of SEQ ID NO:1 as compared to a synthetic peptide of SEQ ID NO:2 and binds to a synthetic peptide of SEQ ID NO:3 at least as good as compared to the peptide of SEQ ID NO:1.
4. The composition of claim 3 , wherein the antibody also has a binding preference for a peptide of SEQ ID NO:1 as compared to a peptide of SEQ ID NO:4.
5. The composition of claim 1 , wherein the antibody binds to a synthetic peptide of SEQ ID NO:1 has an at least 15-fold binding preference for the peptide of SEQ ID NO:1 as compared to a synthetic peptide of SEQ ID NO: 2.
6. The composition of claim 5 , wherein the antibody also has a binding preference for a peptide of SEQ ID NO:1 as compared to a peptide of SEQ ID NO:4.
7. The composition of claim 1 , wherein the antibody is a specific binding agent binding to PIVKA-II.
8. A method of detecting PIVKA-II in a sample, the method comprising the steps of:
a) contacting the sample with the antibody according to claim 1 for a time and under conditions sufficient for the formation of antibody-PIVKA-II complex; and
b) detecting the presence of the antibody-PIVKA-II complex, wherein the presence of the antibody-PIVKA-II complex indicates the presence of PIVKA-II in the sample.
9. The method of claim 8 wherein
step a) further comprises contacting the sample with a second specific binding agent to PIVKA-II, wherein the second specific binding agent is detectably labeled, for a time and under conditions sufficient to form an antibody-PIVKA-II-second specific binding agent complex; and wherein
step b) comprises measuring the complex formed in (a), thereby detecting PIVKA-II in the sample.
10. The method according to claim 9 , wherein the second specific binding agent is a polyclonal or a monoclonal antibody.
11. The method according to claim 9 , wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within F1/F2.
12. The method according to claim 9 , wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within F1.
13. The method according to claim 9 , wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within the Gla-domain of PIVKA-II.
14. A method of diagnosing HCC in a patient suspected of having HCC, comprising the steps of:
a) obtaining a sample from the patient;
b) contacting the sample with a first specific binding agent to PIVKA-II according to claim 1 and a second specific binding agent to PIVKA-II, wherein the second specific binding agent is detectably labeled, for a time and under conditions sufficient to form a first specific binding agent-PIVKA-II-second specific binding agent complex;
c) measuring the complex formed in (b), thereby measuring the amount of PIVKA-II present in the sample, wherein an amount of PIVKA-II greater than a reference level is indicative of the presence of HCC in the patient.