Search

US-12624090-B2 - Method and antibody for detection of HBcAg

US12624090B2US 12624090 B2US12624090 B2US 12624090B2US-12624090-B2

Abstract

In the field of Hepatitis B virus (HBV) detection, disclosed are a method for detecting HBcAg by means of using a double antibody sandwich method, and an antibody and kit for detecting HBcAg; also included is a monoclonal antibody that can be used in the immunological detection of HBcAg in a tissue or cell sample.

Inventors

  • Zimin Chen
  • Junhui Xiong
  • Jiaqi Liu
  • Shaojuan Wang
  • Shengxiang Ge
  • Quan Yuan
  • Liuwei Song
  • XuDong Sun

Assignees

  • XIAMEN INNODX BIOTECH CO., LTD
  • XIAMEN UNIVERSITY

Dates

Publication Date
20260512
Application Date
20210118
Priority Date
20200119

Claims (19)

  1. 1 . A method for detecting the presence or level of HBcAg protein in a sample, comprising the steps of: (1) contacting the sample with a first antibody to form an antibody-antigen complex, wherein the first antibody is selected from an antibody or antigen-binding fragment thereof that specifically binds to positions 150-183 of HBcAg protein; (2) contacting the antibody-antigen complex with a second antibody to form an antibody-antigen-antibody complex, wherein the second antibody is selected from an antibody or antigen-binding fragment thereof that specifically binds to positions 141-154 of HBcAg protein; and (3) determining an amount of the antibody-antigen-antibody complex; wherein the first antibody is selected from the following antibodies or antigen-binding fragments thereof: (i) an antibody or an antigen-binding fragment thereof, which comprises: a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): HCDR1 having a sequence set forth in SEO ID NO: 3, HCDR2 having a sequence set forth in SEO ID NO: 4, and HCDR3 having a sequence set forth in SEO ID NO: 5; and, a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): LCDR1 having a sequence set forth in SEO ID NO: 6, LCDR2 having a sequence set forth in SEO ID NO: 7, and LCDR3 having a sequence set forth in SEO ID NO: 8; or, (ii) an antibody or an antigen-binding fragment thereof, which comprises: a heavy chain variable region (VH) comprising 3 CDRs contained in the VH set forth in SEO ID NO: 1; and a light chain variable region (VL) comprising 3 CDRs contained in the VL set forth in SEO ID NO: 2; wherein the 3 CDRs contained in the VH and the 3 CDRs contained in the VL are defined by the Kabat, Chothia or IMGT numbering system; or, (iii) an antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line 18B2-2 which is deposited in the China Center for Type Culture Collection (CCTCC) and has the deposit number of CCTCC NO. C2019303; and wherein the second antibody is selected from the following antibody or antigen-binding fragment thereof: (i) an antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): HCDR1 having a sequence set forth in SEO ID NO: 11, HCDR2 having a sequence set forth in SEO ID NO: 12, and HCDR3 having a sequence set forth in SEO ID NO: 13; and a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): LCDR1 having a sequence set forth in SEO ID NO: 14, LCDR2 having a sequence set forth in SEO ID NO: 15, and LCDR3 having a sequence set forth in SEO ID NO: 16; or, (ii) an antibody or antigen-binding fragment thereof, which comprises: a heavy chain variable region (VH) comprising 3 CDRs contained in the VH set forth in SEO ID NO: 9; and a light chain variable region (VL) comprising 3 CDRs contained in the VL set forth in SEO ID NO: 10; wherein the 3 CDRs contained in the VH and the 3 CDRs contained in the VL are defined by the Kabat, Chothia or IMGT numbering system; or, (iii) an antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line 2A7 which is deposited in the China Center for Type Culture Collection (CCTCC) and has the deposit number of CCTCC NO. C2019302.
  2. 2 . The method according to claim 1 , wherein the second antibody bears a detectable label; or, the determining as described in step (3) comprises using a third antibody with a detectable label.
  3. 3 . The method according to claim 1 , wherein, in step (3), the determining is selected from enzyme immunoassay or chemiluminescence immunoassay.
  4. 4 . The method according to claim 1 , wherein the first antibody is coated on the surface of a solid carrier.
  5. 5 . The method according to claim 1 , wherein the sample is selected from the group consisting of whole blood, plasma and serum.
  6. 6 . The method according to claim 1 , wherein, prior to step (1), the method further comprises a step of treating the sample, wherein the treating comprises: mixing a lysing agent with the sample so as to lyse virus; and/or prior to step (2) and/or step (3), the method further comprises a washing step.
  7. 7 . A monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to HBcAg, wherein, (i) the monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): HCDR1 having a sequence set forth in SEQ ID NO: 11, HCDR2 having a sequence set forth in SEQ ID NO: 12, and HCDR3 having a sequence set forth in SEQ ID NO: 13; and, a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): LCDR1 having a sequence set forth in SEQ ID NO: 14, LCDR2 having a sequence set forth in SEQ ID NO: 15, and LCDR3 having a sequence set forth in SEQ ID NO: 16; or, (ii) an antibody or antigen-binding fragment thereof, which comprises: a heavy chain variable region (VH) comprising 3 CDRs contained in a heavy chain variable region set forth in SEQ ID NO: 9; and, a light chain variable region (VL) comprising 3 CDRs contained in a light chain variable region set forth in SEQ ID NO: 10; wherein the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system; or, (iii) an antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line 2A7 which is deposited in the China Center for Type Culture Collection (CCTCC) and has the deposit number of CCTCC NO. C2019302.
  8. 8 . The monoclonal antibody or antigen-binding fragment thereof according to claim 7 , wherein the monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO: 9 or a sequence having a sequence identity of at least 80% compared thereto and the VH comprises HCDR1-HCDR3 set forth in SEQ ID NO: 11-13, respectively; and a light chain variable region (VL) having the sequence set forth in SEQ ID NO: 10 or a sequence having a sequence identity of at least 80% compared thereto and the VL comprises LCDR1-LCDR3 set forth in SEQ ID NO: 14-16, respectively.
  9. 9 . The monoclonal antibody or antigen-binding fragment thereof according to claim 7 , wherein the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).
  10. 10 . A method for detection of HBcAg in a sample, wherein the detection is an immunological detection using the monoclonal antibody or antigen-binding fragment thereof according to claim 7 as the detection reagent.
  11. 11 . The monoclonal antibody or antigen-binding fragment thereof according to claim 7 , which is characterized by one or more of the following: (i) the monoclonal antibody is an IgG, IgM, IgE, IgD or IgA antibody; (ii) the antigen-binding fragment is selected from the group consisting of Fab, Fab′, (Fab′) 2 , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); (iii) the monoclonal antibody is a murine antibody, a chimeric antibody or a humanized antibody.
  12. 12 . The method according to claim 10 , which is characterized by one or more of the following: (i) the sample is a tissue sample or a cell sample; (ii) the immunological detection is selected from immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF) and Western Blot; (iii) the detection reagent is the monoclonal antibody or the antigen-binding fragment thereof bearing a detectable label, or is the monoclonal antibody or the antigen-binding fragment thereof and a secondary antibody bearing a detectable label.
  13. 13 . The method according to claim 1 , wherein the first antibody is selected from an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1-HCDR3 set forth in SEQ ID NO: 1-3, respectively and has the sequence set forth in SEQ ID NO: 1 or a sequence having a sequence identity of at least 80% compared thereto; and the VL comprises LCDR1-LCDR3 set forth in SEQ ID NO: 6-8, respectively and has the sequence set forth in SEQ ID NO: 2 or a sequence having a sequence identity of at least 80% compared thereto.
  14. 14 . The method according to claim 1 , wherein the second antibody is selected from an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1-HCDR3 set forth in SEQ ID NO: 11-13, respectively and has the sequence set forth in SEQ ID NO: 9 or a sequence having a sequence identity of at least 80% compared thereto; and the VL comprises LCDR1-LCDR3 set forth in SEQ ID NO: 14-16, respectively and has the sequence set forth in SEQ ID NO: 10 or a sequence having a sequence identity of at least 80% compared thereto.
  15. 15 . The method according to claim 1 , wherein the first antibody and/or the second antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).
  16. 16 . The method according to claim 1 , wherein the first antibody and/or the second antibody is an IgG, IgM, IgE, IgD or IgA antibody.
  17. 17 . The method according to claim 1 , wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab′, (Fab′) 2 , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); and/or, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.
  18. 18 . The method according to claim 2 , wherein the detectable label is selected from enzyme, chemiluminescent reagent, fluorescent dye or biotin.
  19. 19 . The method according to claim 4 , wherein the solid carrier is selected from magnetic bead or microtiter plate.

Description

CROSS-REFERENCE TO RELATED APPLICATION The application is a § 371 national phase of International Application No. PCT/CN2021/072483, filed on Jan. 18, 2021, which claims priority to Chinese Application No. 202010059190.X, filed Jan. 19, 2020, the entire contents of both applications are hereby incorporated by reference. Incorporation by Reference of Sequence Listing Provided as a Text File A Sequence Listing is provided herewith in a text file entitled IEC200405PUS_ST25.txt, created on Aug. 26, 2025, and having a size of 17,678 bytes. The contents of this text file are incorporated herein by reference in its entirety. TECHNICAL FIELD The present invention relates to the field of hepatitis B virus (HBV) detection. Specifically, the present invention provides a method for the detection of HBcAg by using a double-antibody sandwich method, as well as an antibody and kit used for the detection. The present invention also provides a monoclonal antibody that can be used for HBcAg immunological detection of tissue or cell samples. BACKGROUND ART Hepatitis B virus infection, especially chronic HBV infection, is one of the most important public health problems worldwide (Dienstag J L. Hepatitis B virus infection. N Engl J Med 2008 Oct. 2; 359(14):1486-1500). At present, HBV serum markers (such as the Hepatitis B serologic test of HBsAg, HBsAb, HBeAg, HBeAb, HBcAb) are widely used as the routine detection standard for current HBV infection and past HBV infection. However, the high mutation rate combined with the huge number of HBV carriers leads to a high incidence of false negatives in the conventional Hepatitis B serologic test of HBsAg, HBsAb, HBeAg, HBeAb and HBcAb. In addition, the Hepatitis B serologic test of HBsAg, HBsAb, HBeAg, HBeAb and HBcAb cannot quantitatively reflect the degree of virus replication and infectivity, often have results that are suspicious and difficult to explain, and also cannot directly determine whether the tested individuals are not infected with HBV. HBV DNA is a direct indicator of HBV replication, and its dot blot test (or PCR test) is the gold standard for judging infection and infectivity in hepatitis B patients and HBV carriers. Both PCR test and dot blot test can be used as direct indicators of HBV infection and infectivity, but they are not suitable for large-scale screening and routine use. Among all serum marker antigens (HBV PreS1, HBcAg, HBxAg, DNAP, HBV PreS2, etc.) that may be highly related to HBV DNA, HBcAg has always been considered to be an antigen directly related to HBV DNA, and the detection of HBcAg has unique significance in quantification of replicative viruses and diagnosis of HBsAg-negative HBV-infected patients and HBV patients. At present, there is no specific HBcAg detection reagent developed in the market. The HBcAg immunodiagnostic reagents that have been reported or have been developed usually adopt the pre-treatment of sample before detection (lysing virus, rupturing membrane and inactivating HBcAb) or detection of HBcAg-HBcAb immune complex; however, the former method has complex procedures and is not easy to be accepted by clinical customers, and is not suitable for large-scale screening of blood donors and epidemiological investigations, while the latter method is difficult to achieve the ideal specificity and sensitivity due to the particularity of the detection method. In 2006, it was reported in the patent “Method and diagnostic kit for combined detection of hepatitis B virus pre-S1 antigen and core antigen” that virus particles were captured by using sAg antibody followed by membrane rupture, lysis of virus and detection of cAg in core particle for HBcAg detection, but its sensitivity was unsatisfactory. It has urgent practical significance in evaluation of antiviral efficacy and prognosis of HBV patients to develop a simple, accurate and highly sensitive HBcAg luminescent detection reagent. Contents of the Present Invention After extensive experimental research, the inventors unexpectedly found a pair of antibodies that bind to specific epitopes, which is particularly suitable for the detection of HBcAg by double-antibody sandwich method. On this basis, the inventors developed a new HBcAg quantitative detection kit and detection method. The detection method reaches a level of sensitivity comparable to that of DNA method and realizes rapid and high-throughput detection, and thus has great clinical application value. Kit Therefore, in a first aspect, the present invention provides a kit, which comprises: (i) a first antibody, an isolated nucleic acid molecule encoding the first antibody, a vector comprising the isolated nucleic acid molecule, or a recombinant cell expressing the first antibody; wherein the first antibody is selected from an antibody or antigen-binding fragment thereof that is capable of specifically binding to an epitope contained in positions 150-183 of HBcAg protein; and,(ii) a second antibody, an isolated nucleic acid molecule