METHOD AND ANTIBODY FOR DETECTION OF HBCAG
20230069418 · 2023-03-02
Inventors
- Zimin CHEN (Xiamen, CN)
- Junhui XIONG (Xiamen, CN)
- Jiaqi LIU (Xiamen, CN)
- Shaojuan WANG (Xiamen, CN)
- Shengxiang Ge (Xiamen, CN)
- Quan Yuan (Xiamen, CN)
- Liuwei Song (Xiamen, CN)
- Xudong Sun (Xiamen, CN)
Cpc classification
G01N2469/10
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
International classification
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.
Claims
1. A kit, which comprises: (i) a first antibody, which is selected from an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope contained in positions 150-183 of HBcAg protein; and, (ii) a second antibody, which is selected from an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope contained in positions 141-154 of HBcAg protein.
2. The kit according to claim 1, wherein the second antibody is selected from an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope contained in positions 141-152 of HBcAg protein.
3. The kit according to claim 1 or 2, wherein the first antibody is selected from the following antibody or antigen-binding fragment 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 SEQ ID NO: 3, HCDR2 having a sequence set forth in SEQ ID NO: 4, and HCDR3 having a sequence set forth in SEQ ID NO: 5; and/or, a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): LCDR1 having a sequence set forth in SEQ ID NO: 6, LCDR2 having a sequence set forth in SEQ ID NO: 7, and LCDR3 having a sequence set forth in SEQ 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 SEQ ID NO: 1; and/or, a light chain variable region (VL) comprising 3 CDRs contained in the VL set forth in SEQ ID NO: 2; preferably, the 3 CDRs contained in the VH, and/or 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.
4. The kit according to claim 3, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO: 1; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 1; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 1; and/or, (b) a light chain variable region (VL), which comprises an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 2; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 2; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 2; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 1 and a VL having the sequence set forth in SEQ ID NO: 2.
5. The kit according to any one of claims 1 to 4, 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 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/or, 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 the VH set forth in SEQ ID NO: 9; and/or, a light chain variable region (VL) comprising 3 CDRs contained in the VL set forth in SEQ ID NO: 10; preferably, the 3 CDRs contained in the VH, and/or 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.
6. The kit according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO: 9; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 9; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 9; and/or, (b) a light chain variable region (VL), which comprises an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 10; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 10; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 10; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or antigen-binding fragment thereof comprises a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:10.
7. The kit according to any one of claims 1 to 6, wherein the first antibody and/or the second antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the first antibody and/or the second antibody comprises a murine heavy chain constant region and a murine light chain constant region; preferably, the first antibody and/or the second antibody is an IgG, IgM, IgE, IgD or IgA antibody.
8. The kit according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab′, (Fab′).sub.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.
9. The kit according to any one of claims 1 to 8, wherein the second antibody bears a detectable label, or the kit further comprises a third antibody capable of specifically binding to the second antibody, the third antibody bears a detectable label; preferably, the detectable label is selected from enzyme (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagent (e.g., acridinium ester compound), fluorescent dye or biotin.
10. The kit according to any one of claims 1 to 9, wherein the kit further comprises a solid carrier; preferably, the solid carrier is selected from magnetic bead or microtiter plate (e.g., microwell plate or ELISA plate); preferably, the first antibody is coated on the surface of the solid carrier.
11. The kit according to any one of claims 1 to 10, wherein the kit further comprises one or more reagents or devices selected from the group consisting of: a standard (e.g., a series of samples containing different known amounts of HBcAg); a positive control sample (e.g., a sample containing a known amount of HBcAg); a negative control sample (e.g., a sample not containing HBcAg); a lysing agent used to lyse HBV virus; and, a device for collection or storage of a sample to be tested (e.g., a blood collection device).
12. 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 defined as in any one of claims 1 to 8; and, (ii) a second antibody, an isolated nucleic acid molecule encoding the second antibody, a vector comprising the isolated nucleic acid molecule, or a recombinant cell expressing the second antibody; wherein the second antibody is defined as in any one of claims 1 to 8.
13. The kit according to claim 12, wherein, the recombinant cell expressing the first antibody is a host cell comprising an isolated nucleic acid molecule encoding the first antibody or a vector comprising the isolated nucleic acid molecule, the recombinant cell expressing the second antibody is a host cell comprising an isolated nucleic acid molecule encoding the second antibody or a vector comprising the isolated nucleic acid molecule.
14. The kit according to claim 12, wherein, the recombinant cell expressing the first antibody is 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 the recombinant cell expressing the second antibody is 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.
15. 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 defined as in any one of claims 1 to 8; (2) contacting the antibody-antigen complex with a second antibody to form an antibody-antigen-antibody complex, wherein the second antibody is defined as in any one of claims 1 to 8; and (3) determining an amount of the antibody-antigen-antibody complex.
16. The method according to claim 15, 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; preferably, the detectable label is selected from enzyme (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagent (such as acridinium ester compound), fluorescent dye or biotin.
17. The method according to claim 15 or 16, wherein, in step (3), the determining is selected from enzyme immunoassay or chemiluminescence immunoassay.
18. The method according to any one of claims 15 to 17, wherein the first antibody is coated on the surface of a solid carrier; preferably, the solid carrier is selected from magnetic bead or microtiter plate (e.g., microwell plate or ELISA plate).
19. The method according to any one of claims 15 to 18, wherein the sample is selected from the group consisting of whole blood, plasma and serum.
20. The method according to any one of claims 15-19, 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.
21. Use of the kit according to any one of claims 1 to 14 in the manufacture of a detection kit for detecting the presence or level of HBcAg protein in a sample.
22. 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/or, 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/or, a light chain variable region (VL) comprising 3 CDRs contained in a light chain variable region set forth in SEQ ID NO: 10; preferably, 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.
23. The monoclonal antibody or antigen-binding fragment thereof according to claim 22, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO: 9; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 9; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 9; and/or, (b) a light chain variable region (VL), which comprises an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 10; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence set forth in SEQ ID NO: 10; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence set forth in SEQ ID NO: 10; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the monoclonal antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 9 and a VL having the sequence set forth in SEQ ID NO: 10.
24. The monoclonal antibody or antigen-binding fragment thereof according to claim 22 or 23, wherein the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the monoclonal antibody is an IgG, IgM, IgE, IgD or IgA antibody.
25. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 22 to 24, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab′, (Fab′).sub.2, Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); and/or, the monoclonal antibody is a murine antibody, a chimeric antibody or a humanized antibody.
26. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 22 to 25 in the manufacture of a reagent for detecting HBcAg in a sample; preferably, the sample is a tissue sample (e.g., tissue section) or a cell sample; preferably, the detection is an immunological detection; preferably, the immunological detection is selected from immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF) and Western Blot; preferably, the monoclonal antibody or the antigen-binding fragment thereof bears a detectable label, or the reagent further comprises a secondary antibody bears a detectable label; preferably, the detectable label is selected from enzyme (e.g., horseradish peroxidase or alkaline phosphatase), fluorescent dye or biotin; preferably, the secondary antibody is an anti-immunoglobulin antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0141]
[0142]
[0143]
[0144]
[0145]
[0146]
[0147] Sequence Information
[0148] Information of some sequences referred to in the present application is described in the table below.
TABLE-US-00001 SEQ ID NO: Description Sequence information 1 18B2-2 VH QIQLVQSGPELKKPGETVKISCKASGYAFTDYPVHWVKQAPGKGL KWMGWINTETGEPTYADDFKGRFAFSLEASANTAYLQINNLKNED TATYFCDHYSMDYWGQGTSVTVSS 2 18B2-2 VL DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQ SPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC QQLVEYPFTFGSGTKLEIK 3 18B2-2 GYAFTDYP HCDR1 4 18B2-2 INTETGEP HCDR2 5 18B2-2 DHYSMDY HCDR3 6 18B2-2 KSLLYKDGKTY LCDR1 7 18B2-2 LMS LCDR2 8 18B2-2 QQLVEYPFT LCDR3 9 2A7 VH QVQLQQPGAELVKPGASVKLSCKASGYTFTRYWMHWVMQRPGQ DLEWIGEINPINGRTNYNEKFRRKATLTVDKSSSTVYIQFSSLTSED SAVYFCTREGYRNDYYYAMDFWGRGTSVTVSS 10 2A7 VL DIQMTQTSSSLSASPGDRVTISCRASQGINNYLNWYKQKTDGTFKL LIYYTSYLHSGVPSRFSGRGSGTDYSLTISNLEPEDVATYYCQQYG KLPWTFGGGTKLEIK 11 2A7 HCDR1 GYTFTRYW 12 2A7 HCDR2 INPINGRT 13 2A7 HCDR3 TREGYRNDYYYAMDF 14 2A7 LCDR1 QGINNY 15 2A7 LCDR2 YTS 16 2A7 LCDR3 QQYGKLPWT 17 C183 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEH CSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNV NMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNA PILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC
[0149] Deposit of Biological Materials:
[0150] The invention relates to the following biological materials that have been deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China):
[0151] 1) Hybridoma cell line 18B2-2, which has the deposit number of CCTCC NO. C2019303, and the deposit time of Nov. 28, 2019;
[0152] 2) Hybridoma cell line 2A7, which has the deposit number of CCTCC NO. C2019302, and the deposit time of Nov. 28, 2019.
EXAMPLES
[0153] The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit the present invention.
[0154] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Refined Laboratory Manual for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturer. The reagents which source is not indicated in the examples are all conventional reagents in the art or commercially available reagents. Those skilled in the art appreciate that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention as sought to be protected.
Example 1: Preparation of c183 Antigen
[0155] 1.1 C183 clone (its sequence was set forth in SEQ ID NO: 17) was constructed, and the C183B antigen was prepared by using the E. coli expression system.
[0156] 1.2 Purification of C183 antigen
[0157] The bacterial liquid was collected and sonicated, the sonication liquid was centrifuged at 12,000 rpm and 10° C. for 10 min, and the supernatant was collected. Then, it was allowed to stand in water bath at 65° C. for 20 min, and the supernatant was collected.
[0158] Samples were dialyzed against 1×PB7.4, followed by medium-pressure DEAE-FF chromatography (GE medium).
[0159] The breakthrough peak solution (target protein) was collected, followed by purification by Capto Core700. Then the first sample peak was collected.
Example 2: Preparation of Anti-HBcAg Mouse Monoclonal Antibody
[0160] 2.1 Mouse immunization
[0161] 2.1.1 Preparation of immunogen: The immunogen was the HBcAg (C183 antigen) protein recombinantly expressed in E. coli. The recombinant antigen was diluted to 0.4 mg/mL, and mixed with an equal volume of Freund's adjuvant to form a water-in-oil emulsion (method to judge whether the mixture was completely emulsified: a small drop of the mixture was dropped on the surface of water, and if the mixture kept accumulated and did not disperse, it was considered that it had been substantially homogenized). Freund's complete adjuvant was used for the initial immunization, while incomplete Freund's adjuvant was used for the subsequent booster immunization, and no adjuvant was added for the last booster immunization 72 h before the fusion.
[0162] 2.1.2 Basic immunization of mice: 6-8 week old BALB/c female mice were immunized by subcutaneous multi-point injection with the above immunogen, with the injection dose of 500 μL/mice/time, and 200 μL of eye vein blood was collected before each immunization for later titer determination. Booster immunizations were given every 2 weeks. Serum titers were measured by indirect ELISA. When the serum titers of mice reached a plateau, the immunization stopped and the mice rested for two months before the fusion.
[0163] 2.1.3 Booster immunization 72 hours before fusion (Final boost): 72 hours before the fusion of mouse spleen cells and mouse myeloma cells, booster immunization of spleen was performed. The immunogen for this booster did not contain adjuvant, and 100 μl of 0.5 mg/mL recombinant protein was injected. Before the spleen immunization, the mice were anesthetized with ether, and then the abdominal skin was opened to expose the spleen, 100 μL of antigen was injected along the longitudinal direction of spleen, and then the abdominal incision was quickly sutured.
[0164] 2.2 Preparation and screening of fusion hybridoma
[0165] After the booster immunization 72 h before the fusion, the mouse spleen was taken to make a cell suspension and subjected to fusion with mouse myeloma cells Sp2/0 to obtain hybridoma cells. Feeder cells were prepared prior to this. During the culture of hybridoma cells, a large number of myeloma cells and spleen cells after fusion died one after another in the 1640-HAT medium, and single cells or a few scattered cells were not easy to survive, thus other cells have to be added to make them survive. The live cells that were added were called feeder cells. In this laboratory, mouse peritoneal macrophages or 13-day-old mouse thymocytes were used as feeder cells.
[0166] 2.2.1 Preparation of mouse macrophages: It was performed according to the following steps: (i) one 6-week-old BALB/c mouse was killed by stretching neck, rinsed with tap water, and soaked in 75% ethanol solution for 5 min; the mouse was placed on an ultra-clean workbench with the abdomen facing upwards; the abdominal skin of the mouse was lifted with tweezers, a small cut was made, and the skin was teared upward and downward with large tweezers to fully expose the abdomen; (ii) the peritoneum was lifted with sterile ophthalmic forceps, then an appropriate amount of culture medium was injected into the abdominal cavity with a 5 mL syringe, the limbs of the mouse were slightly lifted with another sterile eye forceps, and the culture medium was finally aspirated using a syringe and placed into a centrifuge tube; (iii) the peritoneal cell fluid was dissolved in HAT medium or HT medium to form macrophage feeder cells at a concentration of 2×10.sup.5/mL; (iv) it was added to a 96-well cell culture plate with 0.1 mL per well, and cultured in an incubator; or it could also be directly mixed with fused cells and added to a 96-well cell culture plate.
[0167] 2.2.2 Preparation of mouse thymocytes: It was performed according to the following steps: (i) one 13-day-old BALB/c mouse was sacrificed by stretching neck, rinsed with tap water, and soaked in 75% ethanol solution for 5 min; the mouse was placed on an ultra-clean workbench with the abdomen facing upwards; (ii) the mouse abdominal skin was lifted with forceps, and the outer skin of the abdomen and thorax was cut; (iii) the thoracic cavity was opened with another pair of clean scissors, the milky white thymus was taken out with tweezers, and subjected to homogenization and filtration through a 200-mesh cell sieve to obtain thymic feeder cell fluid.
[0168] 2.2.3 Preparation of mouse myeloma cells: It was performed according to the following steps: (i) the mouse myeloma cell line Sp2/0-Ag14 (Sp2/0) was easy to culture and had a high fusion rate, thus was the most ideal fusion cell at present, but the Sp2/0 hybridoma cell line was more sensitive to culture conditions than NS-1 and had poor growth at the condition of excessive dilution (density below 3×10.sup.5/mL) and alkaline pH (pH above 7.3); (ii) the cells in logarithmic growth phase were selected for the fusion; (iii) before the fusion, the myeloma cells were transferred from the culture flask into a centrifuge tube, and washed three times with RPMI-1640 medium (1000 rpm×5 min); the cells were resuspended in RPMI-1640 medium and counted; (iv) generally, the mouse myeloma cells should be resuscitated 5 days before the fusion, and about 6 flasks of 35 cm.sup.2 Sp2/0 cells were needed for each fusion.
[0169] 2.2.4 Preparation of immune splenocytes: It was performed according to the following steps: (i) the BALB/C mice for the fusion were sacrificed by bloodletting after removing the eyeballs, and the collected blood sample was made into antiserum, which could be used as a positive control for antibody detection. The mice were rinsed with tap water, soaked in 75% ethanol solution for 5 min, then placed on the mouse dissection board in the ultra-clean bench, in the right lateral position. (ii) the abdominal cavity was aseptically opened, the spleen was taken out, cut into small pieces with scissors and placed on a 200-mesh cell screen, then squeezed and ground with a grinding rod (inner core of syringe), and at the same time, added with RPMI-1640 medium dropwise with a pipette. (iii) an appropriate amount of RPMI-1640 culture medium was replenished, allowed to stand for 3-5 min, and 2/3 of the upper suspension was transferred into a 50 mL plastic centrifuge tube; the above process was repeated 2-3 times. (iv) the cells were washed 3 times with RPMI-1640 medium (1000 rpm×10 min). (v) the cells were resuspended in RPMI-1640 medium and counted.
[0170] 2.2.5 Preparation of hybridoma by PEG-medicated fusion: It was performed according to the following steps: (1) before fusion, 1 mL of PEG-1500, 10 mL of RPMI-1640 serum-free medium and 200 mL of complete medium were pre-heated to 37° C.; (ii) the prepared myeloma cells and spleen cells were mixed in a 50 mL centrifuge tube (1×10.sup.8 spleen cells+1×10.sup.7 myeloma cells, about 10:1), centrifuged at 1500 rpm for 8 min; after centrifugation, the bottom of tube was gently flicked to loosen the cells to form a paste; (iii) 0.8 mL (1×10.sup.8 spleen cells+0.8 mL PEG) was added into the centrifuge tube with a 1 mL pipette, stirred gently while adding, and PEG was added within 60 seconds on average, then 10 mL of the complete medium of RPMI-1640 pre-heated at 37° C. was added under gentle stirring; finally, RPMI-1640 medium was replenished to 40 mL, and centrifuged at 1000 rpm for 5 min; (iv) the supernatant was discarded, a small amount of HT medium was added to disperse the cells carefully, the cells were transferred into the prepared HT medium, added to a 96-well cell culture plate with 0.1 mL per well, and cultured in a CO.sub.2 incubator; (v) after 12 hours, an appropriate amount of HAT complete medium was prepared, and added dropwise to each well with 0.1 mL per well; after 5 days, the HT complete medium was used to replace 50% to 100% the cell supernatant in the well; after about 9 to 14 days, the supernatant was collected for detection.
[0171] 2.2.6 Screening of hybridoma: Indirect ELISA was used for screening, coating with 100 ng/mL recombinant antigen was performed with 0.1 mL per well, then 50 uL of cell supernatant was added for detection, and positive clone wells were selected.
[0172] 2.2.7 Cloning of hybridoma cells: By using the limiting dilution method, the cells were firstly diluted in series to according to a certain concentration, and then inoculated into each well of a 96-well cell culture plate, so that only one cell grew in the well as much as possible; the positive clone of hybridoma generally needed to be cloned 2-3 times until it was 100% positive and confirmed as a stable clone.
[0173] 2.3 Production of monoclonal antibody ascites
[0174] 2-3 BALB/c mice were injected with 0.5 mL of liquid paraffin oil into the abdominal cavity. After 1 week, the hybridoma cells in logarithmic growth phase were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The hybridoma cells were suspended with serum-free medium, and the number of cells was adjusted to (1-2)×10.sup.6/mL, and 0.5 mL of the cells was injected intraperitoneally into each mouse. After 7-10 days, the abdomen of the mice was obviously enlarged, and the mice were sacrificed by stretching neck, rinsed with tap water, soaked in 75% ethanol for 5 min, and the limbs of mouse were fixed with injection needles on the mouse dissection table with the abdomen facing up. The abdominal skin of mouse was lifted with tweezers, a small open was made by cutting, then incision was made by cutting from both sides to the back of mouse, and the skin was teared up and down with large tweezers to fully expose the abdomen. The peritoneum was lifted with sterile ophthalmic forceps, a small slit was made in the center of the peritoneum, and then a 1 mL pipette was used to take out all the ascites in the abdominal cavity through the small slit. The collected ascites could be mixed and centrifuged in a centrifuge tube at 3000 rpm for 20 min. The supernatant was collected after centrifugation.
[0175] 2.4 Purification of monoclonal antibody ascites
[0176] The purified monoclonal antibody was obtained by ammonium sulfate precipitation and purification by Protein A affinity chromatography (purchased from GE, USA).
[0177] The following monoclonal antibodies were obtained by the above method: 1B11, 2A7, 6E1, 14C6, 18B2-2, 14C7, 5H4, 1F9. Among them, the obtained hybridoma cell lines 2A7 and 18B2-2 were deposited as described above in the China Center for Type Culture Collection (CCTCC).
Example 3: In Vitro Epitope Identification of Anti-HBcAg Mouse Monoclonal Antibody
[0178] 3.1 Peptide synthesis
[0179] Using the HBV sequence GenBank ID: CAA59669.1 as the reference sequence, 31 polypeptides were synthesized (entrusted to Shanghai Sangon Biotechnology Co., Ltd.). These 31 polypeptides (s1 to s31) together covered the full-length 183 amino acids of HBcAg. The polypeptide information of 51 to S31 was shown in Table 1 below, and the full-length amino acid sequence of HBcAg was set forth in GenBank: GU357842.1.
TABLE-US-00002 TABLE 1 Peptide information of S1 to S31 Amino Amino SEQ ID Name acid position acid sequence NO: S1 HBcAg-aa1-aa15 MDIDPYKEFGATVEL 18 S2 HBcAg-aa6-aa20 YKEFGATVELLSFLP 19 S3 HBcAg-aa11-aa25 ATVELLSFLPSDFFP 20 S4 HBcAg-aa16-aa30 LSFLPSDFFPSIRDL 21 S5 HBcAg-aa21-aa35 SDFFPSVRDLLDTAS 22 S6 HBcAg-aa26-aa40 SVRDLLDTASALYRE 23 S7 HBcAg-aa31-aa45 LDTASALYREALESP 24 S8 HBcAg-aa36-aa50 ALYREALESPEHCSP 25 S9 HBcAg-aa41-aa55 ALESPEHCSPHHTAL 26 S10 HBcAg-aa46-aa60 EHCSPHHTALRQAIL 27 S11 HBcAg-aa51-aa65 HHTALRQAILCWGEL 28 S12 HBcAg-aa56-aa70 RQAILCWGELMNLAT 29 S13 HBcAg-aa61-aa75 CWGELMTLATWVGVN 30 S14 HBcAg-aa66-aa80 MTLATWVGVNLEDPA 31 S15 HBcAg-aa71-aa85 WVGVNLEDPASRDLV 32 S16 HBcAg-aa76-aa90 LEDPASRDLVVSYVN 33 S17 HBcAg-aa81-aa95 SRDLVVSYVNTNMGL 34 S18 HBcAg-aa86-aa100 VSYVNTNMGLKFRQL 35 S19 HBcAg-aa91-aa105 TNMGLKFRQLLWFHI 36 S20 HBcAg-aa96-aa110 KFRQLLWFHISCLTF 37 S21 HBcAg-aa101-aa115 LWFHISCLTFGRETV 38 S22 HBcAg-aa106-aa120 SCLTFGRETVIEYLV 39 S23 HBcAg-aa111-aa125 GRETVIEYLVSFGVW 40 S24 HBcAg-aa116-aa130 IEYLVSFGVWIRTPP 41 S25 HBcAg-aa121-aa135 SFGVWIRTPPAYRPP 42 S26 HBcAg-aa126-aa140 IRTPPAYRPPNAPIL 43 S27 HBcAg-aa131-aa145 AYRPPNAPILSTLPE 44 S28 HBcAg-aa136-aa150 NAPILSTLPETTVVR 45 S29 HBcAg-aa141-aa154 STLPETTVVRRRGR 46 S30 HBcAg-aa141-aa152 STLPETTVVRRR 47 S31 HBcAg-aa150-aa183 RRRGRSPRRRTPSPRRR 48 RSQSPRRRRSQSRESQC
[0180] 3.2 Analysis of reactivity of anti-HBcAg mouse monoclonal antibody with polypeptides S1 to S31
[0181] 3.2.1 Preparation of reaction plate
[0182] The polypeptide was diluted with 50 min CB buffer (NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, final concentration 50 min, pH 9.6) at pH 9.6 to a final concentration of 5 μg/mL; 100 μL of coating solution was added to each well of a 96-well ELISA plate, the coating was performed at 2-8° C. for 16-24 hours and then at 37° C. for 2 hours; washing was performed once with PBST washing solution (20 min PB7.4, 150 min NaCl, 0.1% Tween20); then 200 μL of blocking solution (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer solution containing 20% calf serum and 1% casein with pH 7.4) was added to each well, allowed to stand at 37° C. for blocking for 2 hours; the blocking solution was discarded. After drying, it was stored in aluminum foil bag at 2-8° C. for later use.
[0183] 3.2.2 ELISA detection of anti-HBcAg mouse monoclonal antibody
[0184] The anti-HBcAg mouse monoclonal antibody obtained in 2.1 was diluted to 1 μg/mL with PBS solution containing 20% newborn bovine serum for qualitative ELISA detection.
[0185] Sample reaction: 36 ELISA plates that had been coated with polypeptides were taken, added with 100 μL of diluted sample to each well, and placed in an incubator to react at 37° C. for 30 minutes.
[0186] Enzyme label reaction: After the sample reaction step was completed, the ELISA plate was washed 5 times with PBST wash solution (20 min PB7.4, 150 min NaCl, 0.1% Tween20), and 100 μL reaction solution of HRP-labeled goat-anti-mouse IgG (GAM) was added to each well, and placed in an incubator to react at 37° C. for 30 minutes.
[0187] Color development reaction: After the enzyme label reaction step was completed, the ELISA plate was washed 5 times with PBST wash solution (20 min PB7.4, 150 min NaCl, 0.1% Tween20), and 50 μL of TMB color developer (purchased from Beijing Wantai Bio-pharmaceutical Co., Ltd.) was added to each well, and placed in an incubator to react at 37° C. for 15 minutes.
[0188] Termination of reaction and measurement of reading value: After the color development reaction step was completed, 50 μL of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well of the ELISA plate, and the OD450/630 values of each well were measured by a microplate reader.
[0189] Judgment of the reactivity of anti-HBcAg mouse monoclonal antibody with 36 kinds of polypeptides: The judgment was performed according to the reading values after the reaction. If the ratio of measured value/background value was greater than 5, it was judged as positive.
[0190] 3.2.3 Analysis of recognition properties of anti-HBcAg mouse monoclonal antibody
[0191] The results were shown in Table 2. The recognition types of the obtained anti-HBcAg mouse monoclonal antibodies could be divided into 5 groups (according to their recognition properties), namely: sA, sB, sC, sD, sE, among which the polypeptides recognized by the sA group antibodies were S29/S30, in which 2A7, 14C6, and 14C7 belonged to the sA group; the polypeptides recognized by the sB group antibodies were S31, in which 1F9 and 18B2-2 belonged to the sB group; the polypeptides recognized by the sC group antibodies were S1; the polypeptides recognized by the sD group antibodies were s26, s27; and the polypeptides recognized by the sE group antibodies were s15 and s16.
TABLE-US-00003 TABLE 2 Analysis of recognition properties of monoclonal antibodies Name of monoclonal Isotype of monoclonal polypeptides Group antibody antibody recognized sA HBc-2A7 IgG2B S29/S30 sA HBc-14C6 IgG1 S29/S30 sC HBc-5H4 IgG1 S1 sA HBc-14C7 IgG1 S29/S30 sB HBc-18B2-2 IgG1 S31 sD HBc-1B11 IgG1 S26/S27 sE HBc-6E1 IgG2A S15/S16 sB HBc-1F9 IgG2B S31
[0192] The detection results of antibodies 2A7, 18B2-2 with the corresponding epitopes were shown in Table 3, which showed that the epitope of 2A7 was in 141-152aa, and the epitope of 18B2-2 was in 150-183aa.
TABLE-US-00004 TABLE 3 Reaction of 2A7/18B2-2 with specific epitopes Name of monoclonal polypeptides Reaction, OD450 Group antibody recognized (1 ug/ml) sA 2A7 S30 2.111 sB 18B2-2 s31 2.220
Example 4: Epitope Identification In Vivo
[0193] 4.1 Eukaryotic plasmid construction
[0194] The sequences in the fragment with a total of 222 amino acids consisting of HBcAg sequence of HBV gene and the sequence from −29 to −1 at its N-terminal were constructed into the downstream of CMV promoter in eukaryotic expression vector (EHRP vector, obtained from National Research Center of Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center of Xiamen University), and the schematic diagram of its structure was shown in
[0195] 4.2 Eukaryotic expression and western blot evaluation
[0196] 293β5 cells were placed in a 6-well plate, and when the cell density reached about 80-90% after 12 hours of adherence, transfection was performed, the constructed eukaryotic expression plasmids were transfected into 293β5 cells using lipo3000 transfection reagent. The replacement with medium (DMEM 30 10% Gibco FBS) was performed 12 hours after the transfection, the culture was continued for 48 h. The cell supernatant was discarded, the cells was washed once with PBS, 300 μL of cell lysis solution was added to each well and allowed to stand at 4° C. for 1 h to perform lysis. The lysate was collected in a 1.5 ml EP tube, centrifuged at 12000 rpm at 4° C. for 10 min, the supernatant was collected into a clean 1.5 ml EP tube, and the lysed sample was subjected to western blot analysis (the secondary antibody was goat-anti-mouse-HRP, purchased from proteintec company).
[0197] 4.3 Analysis of results
[0198] The monoclonal antibodies were evaluated using antigens of different lengths constructed by in vivo eukaryotic expression, and the results were shown in
Example 5: Screening of Magnetic Bead-Coated Monoclonal Antibody and Acridinium Ester-Labeled Monoclonal Antibody in Double Antibody-Sandwich Method
[0199] In this example, the experimental conditions of conventional double antibody-sandwich method were adopted to screen the optimal pairing of the monoclonal antibody for coating magnetic bead and the monoclonal antibody for labeling with acridinium ester.
[0200] 5.1 Preparation of HBcAg magnetic bead-coated monoclonal antibody
[0201] The magnetic microparticle solution was prepared, and the magnetic microparticles were magnetic beads coated with hydrophilic polymers and carboxyl groups on the surface with the particle size of 1.5-3 um. The preparation method was as follows: magnetic microparticles, EDC and NHS at the mass ratio of 1:1:1 were added with 50 min MES solution with pH 5.0 to make the concentration of magnetic microparticles to be 4 mg/mL. It was then loaded on a vertical rotator for activation at environmental temperature of 25° C. for 20 minutes. The activated magnetic microparticles and the anti-HBcAg monoclonal antibody at a ratio of 15 μg of anti-HBcAg monoclonal antibody per mg of magnetic microparticles were loaded on a vertical rotator for labeling, and the reaction was performed at environmental temperature of 25° C. for 3 h. The magnetic microparticles after the reaction were washed 3 times with washing solution, then a phosphate buffer at pH 7.4 containing glycine, 0.5% bovine serum albumin and 0.05% Triton X-100 were added to make the magnetic microparticles to have a concentration of 4 mg/mL, and loaded on the vertical rotator for termination at the reaction temperature of 25° C. for 2 h. The magnetic microparticles after the termination were washed 3 times with washing solution, and a phosphate buffer at pH 7.4 containing 0.5% (W/V) bovine serum albumin, 0.5% (W/V) casein, 0.05% (W/V) Triton X-100, and preservative was added to make the magnetic microparticles to have a concentration of 4 mg/mL, and was stored at 2-8° C. for later use.
[0202] The monoclonal antibodies (18B2-2, 1F9) recognizing aa150-183 were coated with MS300 magnetic beads according to the above-mentioned method to prepare magnetic microparticle solutions.
[0203] 5.2 Preparation of HBcAg acridinium ester-labeled monoclonal antibody
[0204] The acridinium ester-labeled antibody solution was prepared, and the preparation method was as follows: 50 ug of the anti-HBcAg monoclonal antibody to be labeled was added with NaCl-containing phosphate buffer to reach a volume of 300 μL, then 5 μL of acridinium ester stock solution was added, shaken and mixed, and the reaction was performed in the dark at room temperature for 30 min; after the reaction, 200 μL of a phosphate buffer containing NaCl and glycine was added, mixed by manually turning upside down for 20 times, and the reaction was performed at room temperature for 30 min in the dark; after the reaction, the product was transferred into a dialysis bag, and dialyzed in the dark at 2-8° C. against a dialysate that was 20 mM PBS buffer at pH 7.4, the PBS buffer was changed every 2 h for a total of 3 times to remove the unlabeled acridinium ester; the labeled product was added with 10% (W/V) bovine serum albumin according to the actual volume so that the bovine serum albumin had a final concentration of 0.1% (V/V, 1:100), then added with an equal volume of glycerol, mixed by manually turning upside down, and stored at −15° C. in the dark for later use.
[0205] Six HBcAg monoclonal antibodies (1B11, 2A7, 6E1, 14C6, 14C7, 5H4) were labeled with acridinium ester by the above-mentioned method.
[0206] 5.3 Experimental method
[0207] 5.3.1 Samples:
[0208] HBV virus positive (PCR detection) clinical serum sample was provided, and diluted with 20% NBS to reach different DNA loads of 1*10.sup.7, 1*10.sup.6, 1*10.sup.5, 1*10.sup.4, 1*10.sup.3 to obtain positive samples for detection; and HBV virus negative (PCR detection) clinical serum sample was also provided.
[0209] 5.3.2 Loading sample:
[0210] 25 ul sample was added with 12.5 ul of 20% LDS, mixed and then incubated at 37° C. for 30 min 30 ul of 10% CHAPS was added and mixed to neutralize, then 50 ul of magnetic bead-coated monoclonal antibody was added and incubated at 37° C. for 15 min After the incubation was completed, washing was performed with a phosphate buffer containing 0.05-0.08% Tween 20, then 50 ul of acridinium ester-labeled monoclonal antibody was added, shaken and mixed, incubated at 37° C. for 10 min. After incubation, washing was performed with a phosphate buffer containing 0.05-0.08% Tween 20, and 100-200 ul of pre-trigger solution was added to perform pre-trigger. After the pre-trigger solution was removed, 100-200 ul of trigger solution was added to perform trigger and detection.
[0211] The orthogonal detection of each magnetic bead-coated monoclonal antibody paired with each acridinium ester-labeled monoclonal antibody was performed by the above-mentioned method, and the P/N (ratio of the mean value of positive sample to the mean value of negative sample) was calculated. The results were shown in the table below.
TABLE-US-00005 TABLE 4-1 P/N values of pairing detection of magnetic bead-coated monoclonal antibody 18B2-2 and acridinium ester-labeled monoclonal antibody Coated-antibody (18B2-2) Positive Positive Positive Positive Positive sample sample sample sample sample Negative Labeled (1*10.sup.7) (1*10.sup.6) (1*10.sup.5) (1*10.sup.4) (1*10.sup.3) sample antibody RLU P/N RLU P/N RLU P/N RLU P/N RLU P/N RLU 1B11 8692 16.9 2563 5.0 1660 3.2 520 1.0 504 1.0 515 5H4 7849 10.2 3918 5.1 1759 2.3 684 0.9 601 0.8 773 6E1 45203 20.1 12266 5.4 8256 3.7 3316 1.5 2549 1.1 2253 14C6 129622 140.1 45445 49.1 11281 12.2 4830 5.2 1595 1.7 925 14C7 153900 165.3 54505 58.5 12296 13.2 5882 6.3 2468 2.7 931 2A7 649026 318.7 173474 85.2 54199 26.6 17853 8.8 6981 3.4 2037
TABLE-US-00006 TABLE 4-2 P/N values of pairing detection of magnetic bead-coated monoclonal antibody 1F9 and acridinium ester-labeled monoclonal antibody Coated-antibody (1F9) Positive Positive Positive Positive Positive sample sample sample sample sample Negative Labeled (1*107) (1*106) (1*105) (1*104) (1*103) sample antibody RLU P/N RLU P/N RLU P/N RLU P/N RLU P/N RLU 1B11 1566 13.86 773 6.84 263 2.33 166 1.47 164 1.45 113 5H4 2279 11.23 857 4.22 531 2.62 268 1.32 306 1.51 203 6E1 1910 13.26 658 4.57 183 1.27 174 1.21 200 1.39 144 14C6 24910 54.99 8443 18.64 3630 8.01 1457 3.22 357 0.79 453 14C7 29077 118.20 8357 33.97 2355 9.57 1452 5.90 340 1.38 246 2A7 94536 243.65 18329 47.24 4353 11.22 2164 5.58 1435 3.70 388
[0212] Tables 4-1 and 4-2 showed the detection results of samples with different DNA loads (P/N>3 represented positive) by using the antibody that recognized HBcAg aa150-183 (i.e., recognized the Arginine Rich Domain (ARD) of HBcAg) to coat magnetic beads and using the monoclonal antibodies that recognized different epitopes in 1-149aa of HBcAg as labeling antibodies. The results showed that when the three antibodies (2A7, 14C6, 14C7) that recognized the HBcAg 141-154aa epitope were used as the labeling antibodies, the detection effect of the samples with different HBV DNA loads was significantly better than that of other antibody pairs.
Example 6: Enzyme Immunoassay and Detection Reagent for Detecting HBcAg
[0213] The monoclonal antibody 18B2-2 was diluted with phosphate buffer (20 mmol/LPB, pH 7.4) and coated on a polyvinyl chloride plate, and the monoclonal antibody 2A7 was labeled with horseradish peroxidase (Beijing Wantai Bio-pharmaceuticals, Co., Ltd.). The samples to be tested included: C183 antigen dilution with a concentration of 1 ug/ml, C149 antigen (developed by the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center of Xiamen University) dilution with a concentration of 1 ug/ml, positive sample 1/2, negative sample 1/2, and 20% nbs.
[0214] The samples were treated with the same method as in Example 5 to lysis viruses. Subsequently, 2A7-HRP (1/500 dilution) was added and incubated for 40 min, the plate was washed 5 times, and 50 ul each of chromogenic solution A and B (Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added and incubated for 15 min. Finally, the stop solution (2M H.sub.2SO.sub.4) was added, shaken gently and mixed well, and the value at the wavelength of 450-620 was read on a microplate reader. The results were shown in
Example 7: Chemiluminescence Detection Method and Reagent for Detecting HBcAg
[0215] 7.1 Preparation of detection kit
[0216] 7.1.1 Preparation of magnetic bead-coated monoclonal antibody
[0217] The magnetic microparticles were magnetic beads coated with hydrophilic polymers and carboxyl groups on the surface with the particle size of 1.5-3 um. The preparation method thereof was as follows: the magnetic microparticles, EDC and NHS in a mass ratio of 1:1:1 were added with 50 min MES solution at pH 5.0 to make the magnetic microparticles to have a concentration of 4 mg/mL, then loaded on a vertical rotator for activation at environmental temperature of 25° C. for 20 min. The activated magnetic microparticles and the 18B2-2 monoclonal antibody at a ratio of 15 ug of HBcAg monoclonal antibody per mg of magnetic microparticles were loaded on a vertical rotator for labeling, and the reaction was performed at an environmental temperature of 25° C. for 3 hours. The reacted magnetic microparticles were washed three times with washing solution, added with a phosphate buffer at pH 7.4 containing glycine, 0.5% bovine serum albumin, 0.05% Triton X-100 to make the magnetic particles to have a concentration of 4 mg/mL, loaded on a vertical rotator to perform termination at a reaction environment temperature of 25° C. for 2 h. The magnetic particles after the termination were washed 3 times with washing solution, added with a phosphate buffer at pH 7.4 containing 0.5% (w/v) bovine serum albumin, 0.5% (w/v) casein, 0.05% (w/v) Triton X-100, and preservative to make the magnetic microparticles to have a concentration of 4 mg/mL, and stored at 2-8° C. for later use.
[0218] 7.1.2 Preparation of acridinium ester-labeled monoclonal antibody
[0219] The preparation method was as follows: 50 ug of the 2A7 monoclonal antibody to be labeled was added with a phosphate buffer containing NaCl to reach a volume of 300 μL, then added with 5 μL of acridinium ester stock solution, shaken and mixed well, and the reaction was performed at room temperature for 30 min in the dark. After the reaction, 200 μL of a phosphate buffer containing NaCl and glycine was added, mixed by manually turning upside down for 20 times, and the reaction was performed at room temperature for 30 min in the dark. After the reaction, the product was transferred to a dialysis bag, and dialyzed at 2-8° C. in the dark against a dialysate that was 20 mM PBS buffer at pH 7.4, the PBS buffer was changed every 2 h for a total of 3 times to remove the unlabeled acridinium ester. The labeled product was added with 10% (W/V) bovine serum albumin according to the actual volume so that the bovine serum albumin had a final concentration of 0.1% (V/V, 1:100), and added with an equal volume of glycerol, mixed by manually turning upside-down and stored at −15° C. in the dark for later use.
[0220] 7.2 Detection method
[0221] 1. Preparation: The kit obtained in 7.1 was allowed to stand and equilibrate at room temperature (18-30° C.) for 15-30 min.
[0222] 2. Liquid preparation: 50 ml of concentrated washing solution (20×) was diluted with distilled water or deionized water to 1000 ml for later use.
[0223] 3. Adding sample: 25 ul of samples to be tested were added to the corresponding wells respectively.
[0224] 4. Lysis: The same method as described in Example 5 was adopted to lyse viruses.
[0225] 5. Reaction: 50 ul of magnetic bead-coated monoclonal antibody 18B2-2 was added to the sample well; after being mixed well, the plate was sealed with sealing film, and incubated at 37±1° C. for 15 min. After the incubation for 15˜20 min, washing was performed with a phosphate buffer containing 0.05˜0.08% Tween 20, then 50 ul of acridinium ester-labeled antibody 2A7 was added, and incubated for 10˜15 min. After incubation, washing was performed with a phosphate buffer containing 0.05˜0.08% Tween 20, then 100˜200 ul of pre-trigger solution was added to perform pre-trigger. Then, the pre-trigger solution was removed, and 100˜200 ul of trigger solution was added to perform trigger and detection.
[0226] Result Judgment
[0227] Threshold: Cut Off (C.O.)=9000
[0228] Result judgment: (S=luminescence value of each well)
[0229] Negative result: (S/C.O.<1): Negative was determined when the luminescence value of the sample was less than the Cut Off value, which meant that the HBV core antigen was not detected in the sample.
[0230] Positive result: (S/C.O.>1): Positive was determined when the luminescence value of the sample was greater than or equal to the Cut Off value, which meant that the HBV core antigen was detected in the sample.
Example 8: Specificity and Sensitivity Analysis of HBcAg Detection Kit
[0231] 8.1 Specificity analysis of core antigen detection kit
[0232] 8.1.1 Preparation of kit
[0233] The luminescence diagnostic kit (luminescence detection reagent method) for detecting HBV core antigen was prepared according to the method as described in Example 7.
[0234] 8.1.2 Detection of samples
[0235] A total of 80 samples collected from April 2019 to the present, which had negative results in all the items of HBsAg, HBsAb, HBeAg, HBeAb and HBcAbS of Hepatitis B serologic test, were cryopreserved at −20° C.
[0236] 8.1.3 Detection items
[0237] Each of the serum samples was subjected to chemiluminescence detection for hepatitis B virus core antigen, and the method was shown in Example 7.
[0238] 8.1.4 Detection results
[0239] After all samples were detected, the results were analyzed for the specificity of the kit. The detection results of each sample were shown in the table below.
TABLE-US-00007 TABLE 7 Detection results Sample No. RLU S/C.O. 1 4042 0.45 2 7762 0.86 3 2415 0.27 4 3289 0.37 5 3934 0.44 6 2659 0.30 7 4208 0.47 8 3353 0.37 9 5665 0.63 10 5729 0.64 11 6535 0.73 12 4042 0.45 13 4074 0.45 14 4845 0.54 15 7663 0.85 16 7635 0.85 17 3915 0.44 18 3263 0.36 19 3359 0.37 20 4642 0.52 21 6425 0.71 22 2001 0.22 23 6185 0.69 24 6711 0.75 25 4260 0.47 26 3622 0.40 27 2002 0.22 28 6600 0.73 29 3373 0.37 30 3506 0.39 31 4236 0.47 32 5977 0.66 33 2003 0.22 34 4441 0.49 35 5622 0.62 36 3348 0.37 37 2247 0.25 38 5231 0.58 39 2703 0.30 40 4687 0.52 41 4250 0.47 42 7862 0.87 43 7690 0.85 44 4921 0.55 45 5134 0.57 46 2610 0.29 47 2011 0.22 48 4551 0.51 49 7942 0.88 50 4328 0.48 51 3660 0.41 52 3398 0.38 53 4108 0.46 54 6807 0.76 55 4818 0.54 56 4525 0.50 57 3793 0.42 58 5096 0.57 59 4639 0.52 60 4899 0.54 61 4482 0.50 62 5347 0.59 63 7227 0.80 64 5773 0.64 65 4618 0.51 66 6705 0.75 67 5271 0.59 68 3337 0.37 69 5560 0.62 70 4115 0.46 71 6320 0.70 72 5610 0.62 73 4806 0.53 74 3181 0.35 75 7675 0.85 76 6643 0.74 77 4273 0.47 78 6221 0.69 79 6740 0.75 80 6391 0.71
[0240] 8.1.5 Results and analysis
[0241] It could be seen from the results in Table 7 that S/C.O.<1 indicated that the HBV core antigen was not detected in the sample, and the specificity was good.
[0242] 8.2 Sensitivity analysis of core antigen detection kit 8.2.1 Preparation of kit
[0243] The luminescence diagnostic kit for detecting HBV core antigen (by luminescence detection reagent method) was prepared according to the method as described in Example 7.
[0244] 8.2.2 Detection of samples
[0245] 8.2.2.1. One fresh serum sample with a DNA load of 1.60E+08 copies/mL detected by hepatitis B virus nucleic acid quantitative PCR detection reagent was subjected to linear dilution with 20% NBS at 11 points with 3-fold gradient, and 20% NBS was used as a negative control; the detection was carried out according to the method described in Example 8, and a reference curve was made.
[0246] 8.2.2.2. The C183B antigen was diluted with 20% NBS to 1 ug/ml, and subjected to dilution at 11 points with 3-fold gradient, and 20% NBS without C183B antigen was used as a negative control; the detection was carried out according to the method described in Example 8, and the above reference curve was used to convert the value corresponding to the antigen detection at each point. The results were shown in the table below.
TABLE-US-00008 TABLE 8 Detection results C183B ng/ml RLU1 S/C.O. DNA load RLU2 S/C.O. 1000.00 10272307 1141.37 5.19E+08 1233807 137.09 333.33 3047322 338.59 1.73E+08 906003 100.67 111.11 1297299 144.14 5.77E+07 389994 43.33 37.04 459415 51.05 1.92E+07 164105 18.23 12.35 177478 19.72 6.41E+06 64594 7.18 4.12 85184 9.46 2.14E+06 25633 2.85 1.37 39003 4.33 7.12E+05 14108 1.57 0.46 19941 2.22 2.37E+05 10453 1.16 0.15 11277 1.25 7.91E+04 9244 1.03 0.05 9368 1.04 2.64E+04 6975 0.78 0.02 6532 0.73 8.79E+03 3558 0.40 20% nbs 2780 0.31 20% nbs 2195 0.24
[0247] 8.2.2.3. Results and analysis
[0248] In the data of Table 8, S/C.O greater than 1 represented positive, while less than 1 represented negative. The results showed that the antigen detection sensitivity of c183 was 0.05 ng/ml, and the sample detection sensitivity was about 10.sup.4 copies/ml (DNA load).
Example 9: Comparison of HBcAg Detection Kit and PCR Detection Method
[0249] 9.1 Preparation of kit
[0250] 9.1.1 The luminescence diagnostic kit for detecting HBV core antigen (by luminescence detection reagent method) was prepared according to the method described in Example 7.
[0251] 9.1.2 The hepatitis B virus nucleic acid quantitative PCR detection reagents were purchased from Shenzhen Piji Bioengineering Co., Ltd.
[0252] 9.2 Detection samples
[0253] A total of 82 hepatitis B virus-infected serum samples collected since April 2019 to the present were cryopreserved at −20° C.
[0254] 9.3 Detection items
[0255] Quantitative PCR detection of hepatitis B virus nucleic acid was performed on each serum sample.
[0256] Chemiluminescence detection of hepatitis B virus core antigen was performed on each serum sample.
[0257] 9.4 Detection results
[0258] The correlation between HBV core antigen detection and HBV virus nucleic acid detection was analyzed by comparing the detection results of all items. The results were shown in the table below.
TABLE-US-00009 TABLE 9 Detection results of HBsAg-positive hepatitis B virus-infected serum samples Sample No. DNA load Lg(DNA load) RLU S/C.O. Lg(RLU) 1 5.22E+02 2.72 7058 0.78 3.85 2 5.54E+02 2.74 9611 1.07 3.98 3 8.61E+02 2.94 5454 0.61 3.74 4 9.35E+02 2.97 7074 0.79 3.85 5 1.10E+03 3.04 7360 0.82 3.87 6 1.22E+03 3.09 7795 0.87 3.89 7 1.57E+03 3.20 6627 0.74 3.82 8 1.65E+03 3.22 8167 0.91 3.91 9 2.40E+03 3.38 6574 0.73 3.82 10 2.68E+03 3.43 5818 0.65 3.76 11 2.85E+03 3.45 8450 0.94 3.93 12 3.23E+03 3.51 9279 1.03 3.97 13 1.33E+04 4.12 9822 1.09 3.99 14 1.53E+04 4.18 11797 1.31 4.07 15 1.57E+04 4.20 11252 1.25 4.05 16 3.03E+04 4.48 18600 2.07 4.27 17 4.24E+04 4.63 11099 1.23 4.05 18 6.94E+04 4.84 13330 1.48 4.12 19 2.08E+05 5.32 5862 0.65 3.77 20 2.60E+05 5.41 29362 3.26 4.47 21 9.62E+05 5.98 25736 2.86 4.41 22 1.19E+06 6.08 51702 5.74 4.71 23 4.93E+06 6.69 17820 1.98 4.25 24 2.11E+07 7.32 99756 11.08 5.00 25 2.68E+07 7.43 283919 31.55 5.45 26 3.47E+07 7.54 152230 16.91 5.18 27 3.74E+07 7.57 1067414 118.60 6.03 28 4.79E+07 7.68 455741 50.64 5.66 29 1.01E+08 8.00 1434375 159.38 6.16 30 1.05E+08 8.02 74442 8.27 4.87 31 1.26E+08 8.10 160285 17.81 5.20 32 1.48E+08 8.17 1017496 113.06 6.01 33 1.54E+08 8.19 205803 22.87 5.31 34 1.64E+08 8.21 252638 28.07 5.40 35 3.28E+08 8.52 1503273 167.03 6.18 36 5.38E+08 8.73 2669259 296.58 6.43 37 1.15E+03 3.06 9936 1.10 4.00 38 9.14E+07 7.96 170179 18.91 5.23 39 1.26E+08 8.10 940798 104.53 5.97 40 5.39E+07 7.73 110586 12.29 5.04 41 9.02E+07 7.96 124868 13.87 5.10 42 1.96E+04 4.29 10500 1.17 4.02 43 2.04E+03 3.31 9001 1.00 3.95 44 5.19E+08 8.72 1053667 117.07 6.02 45 1.09E+06 6.04 14822 1.65 4.17 46 4.09E+04 4.61 9546 1.06 3.98 47 1.25E+03 3.10 4749 0.53 3.68 48 1.92E+03 3.28 9042 1.00 3.96 49 2.96E+08 8.47 810430 90.05 5.91 50 1.36E+08 8.13 327209 36.36 5.51 51 9.75E+03 3.99 7899 0.88 3.90 52 4.38E+03 3.64 4702 0.52 3.67 53 6.35E+03 3.80 9578 1.06 3.98 54 1.26E+05 5.10 22687 2.52 4.36 55 1.73E+05 5.24 16642 1.85 4.22 56 1.73E+05 5.24 10389 1.15 4.02 57 2.11E+03 3.32 8117 0.90 3.91 58 1.96E+03 3.29 7350 0.82 3.87 59 2.83E+06 6.45 22523 2.50 4.35 60 1.25E+03 3.10 10673 1.19 4.03 61 1.06E+09 9.03 1432767 159.20 6.16 62 9.48E+06 6.98 28928 3.21 4.46 63 1.35E+04 4.13 9640 1.07 3.98 64 1.88E+04 4.27 9524 1.06 3.98 65 8.82E+07 7.95 292615 32.51 5.47 66 8.82E+07 7.95 110758 12.31 5.04 67 3.29E+03 3.52 6017 0.67 3.78 68 7.18E+06 6.86 27373 3.04 4.44 69 7.75E+04 4.89 9986 1.11 4.00 70 3.03E+05 5.48 13518 1.50 4.13 71 7.43E+07 7.87 233848 25.98 5.37 72 3.84E+07 7.58 148066 16.45 5.17 73 1.33E+08 8.12 439697 48.86 5.64 74 2.36E+05 5.37 18871 2.10 4.28 75 2.72E+08 8.43 460449 51.16 5.66 76 3.78E+04 4.58 10103 1.12 4.00 77 6.50E+07 7.81 255839 28.43 5.41 78 7.35E+05 5.87 15580 1.73 4.19 79 3.57E+07 7.55 255944 28.44 5.41 80 8.58E+04 4.93 9963 1.11 4.00 81 1.50E+04 4.18 12018 1.34 4.08 82 4.29E+03 3.63 6577 0.73 3.82
[0259] 9.5 Analysis of results
[0260] In Table 9, S/C.O.>1 represented that the core antigen was detected in the sample, and S/C.O.<1 represented that the core antigen was not detected in the sample. Correlation analysis was carried out between the results of HBcAg detection and the DNA load results obtained by PCR method; specifically, the logarithm of the virus content and the luminescence intensity of each sample were taken and the linear correlation analysis was carried out. As shown in in
Example 10: Use of 2A7 Monoclonal Antibody in Other Immunological Assays
[0261] 10.1 Use of 2A7 as HBcAg immunofluorescence detection antibody
[0262] HepG2 (obtained from the Laboratory of National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center, Xiamen University) and HepG2-N10 cells stably integrated with a HBV 1.1-fold genome (obtained from the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University) were plated in a 24-well plate with 60,000 cells per well. After 12 hours for cell adhesion, the medium was removed, washing was performed once with 20 min PBS. After fixing with 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.02% Triton x-100 for 10 min, and blocked with 2% BSA for 1 hour. And then the 2A7 monoclonal antibody (1 mg/ml) diluted by 2% BSA at a dilution ratio of 1:1000, was added and incubated at room temperature for 1 hour, and washed 4 times with PBS. The fluorescent secondary antibody, goat-anti-mouse-Alexa488 (Beyotime, Cat. No: A0428), was added and the incubation was carried out at room temperature for 40 min After washing 4 times with PBS, DAPI Staining was performed for nucleus. After the experiment was completed, the cells were photographed with a 63× water objective with an Opera phenix laser confocal high-content imaging system.
[0263] The results were shown in
[0264] 10.2 Use of 2A7 as monoclonal antibody for HBcAg immunohistochemical detection
[0265] At present, the anti-HBcAg antibodies used in HBcAg immunohistochemical detection are polyclonal antibodies, but polyclonal antibodies often have high background and low specificity, and the immunohistochemical results using them are not easy to standardize. There is no report on the use of anti-HBcAg monoclonal antibody for immunohistochemical detection. In this experiment, the performance of 2A7 monoclonal antibody as an immunohistochemical detection antibody was investigated.
[0266] Hepatic tissue paraffin sections of HBV transgenic mice HBV-TG (obtained from the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University) and normal C57BL/6 mice (obtained from Shanghai SLAC Laboratory Animal Co., Ltd.) were subjected to dewaxing, rehydration, antigen retrieval, washing, blocking, and then added with the anti-HBc commercial polyclonal antibody and the 2A7 monoclonal antibody, for reaction at room temperature for 1 hour. After washing, a secondary antibody was added and reacted at room temperature for 10 minutes. After washing, color developing solution was added to perform staining, followed by hydrochloric acid differentiation, counterstaining, and mounting.
[0267] The results were shown in
[0268] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.