Antibodies, products, preparation methods, and applications thereof for HBV genotyping

Abstract

The present invention provides antibodies, products, preparation methods, and applications for HBV genotyping, relating to the field of hepatitis B virus detection. The present invention provides an antibody, B11, for HBV genotyping, capable of simultaneously identifying HBV genotypes B and C. The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO. 15, and the amino acid sequence of the light chain being shown in SEQ ID NO. 17. The present invention also provides a colloidal gold immunochromatography kit based on the B11 antibody, which can be directly used for serum sample testing, simplifying the hepatitis B virus typing diagnostic procedure and achieving high detection accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of hepatitis B virus detection and relates to hepatitis B virus genotyping, and specifically relates to antibodies, products, preparation methods and applications thereof for HBV genotyping. Background Art

[0002] Hepatitis B, caused by infection with the hepatitis B virus (HBV), is a serious global disease that can ultimately lead to cirrhosis and hepatocellular carcinoma. Currently, 10 HBV sequences have been reported, and different HBV genotypes are associated with disease progression, treatment options, and prognosis. Studies have found that genotype B has a better therapeutic effect than genotype C in interferon treatment of hepatitis B. Individuals with genotypes B and C exhibit different seroconversion behaviors. Genotype C replicates more actively and mutates more frequently, making it more likely to cause hepatocellular carcinoma than genotype B. Similarly, clinically isolated genotype C exhibits higher in vitro transmission efficiency than genotype B. Patients with genotype B exhibit earlier seroconversion and less portal vein inflammation and fibrosis. Viral genotyping in HBV-infected individuals can effectively guide clinical precision medication and predict the efficacy of antiviral treatment.

[0003] HBV genotyping is based on HBV genome-wide differences of >7.5% or surface protein (S) gene differences of ≥4%. Therefore, existing HBV genotyping methods primarily include DNA-based methods such as polymerase chain reaction (PCR)-based whole-genome sequencing and S gene sequencing, nested PCR, real-time quantitative PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP).

[0004] The prior art CN101560576A discloses a specific probe for identifying HBV genotypes and a method and kit for detecting HBV genotypes using the same. The process includes steps such as preparing specific primers and specific probes, extracting PCR templates, performing PCR, performing fluorescent PCR reactions, and interpreting the results. It involves operations such as PCR and uses professional equipment such as clean workbenches and PCR amplifiers. The steps are cumbersome, time-consuming, and labor-intensive.

[0005] Prior art CN107475444A discloses a PCR primer, a kit and a method for selectively amplifying RNA from the total nucleic acid of hepatitis B virus. It includes PCR preparation of HBV viral RNA standard, extraction of HBV total nucleic acid including DNA and RNA from patient serum, and quantitative PCR detection. This invention requires the extraction of DNA and RNA from patient serum, and multiple PCR amplifications. Under normal circumstances, PCR amplification also has deletions or mutations in the amplified sequence. Multiple PCR amplifications are not only time-consuming and labor-intensive, but also affect the accuracy of the result determination. Sequencing after the ordinary PCR method needs to be sent to a sequencing company for sequencing of the amplified gene. The genotype of the virus can only be determined after comparison. This process is time-consuming and labor-intensive, with cumbersome operations, high requirements on the operator and the environment, and high costs. It takes at least 1-2 days to obtain the test results, which is time-consuming and has not been used in clinical medicine.

[0006] PCR-RFLP requires special design and processing of fragment primers, etc. Although genotype-specific linear probe detection is a highly specific method, it is more suitable for personalized customization, is costly, and is not suitable for large-scale application. Some researchers have developed protein-based rapid genotyping methods using antigen-antibody recognition, such as enzyme-linked immunosorbent assay (ELISA) and fluorescent lateral flow immunoassay (LFIA). The ELISA test time is relatively long, about 2.5-4 hours, and both require specific detection instruments. Therefore, there is an urgent need for a clinical point-of-care test (POCT) and self-service test kit that can meet the needs of hepatitis B virus typing detection. This allows hepatitis B patients to quickly and independently test, allowing doctors to fully understand the patient's condition in a timely manner, prescribe symptomatic treatment plans, and promote accurate medication for hepatitis B treatment. Summary of the Invention

[0007] The present invention addresses the problems of the prior art and provides an antibody, product, preparation method, and application thereof for HBV genotyping. The present invention provides an antibody, B11, for HBV genotyping, capable of simultaneously identifying HBV genotypes B and C. The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO. 15, and the amino acid sequence of the light chain being shown in SEQ ID NO. 17. The present invention also provides a colloidal gold immunochromatography kit based on the B11 antibody, which can be directly used for serum sample testing to perform rapid hepatitis B virus typing.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides an antibody for HBV genotyping, wherein the antibody can simultaneously recognize HBV genotype B and genotype C, and the antibody comprises a heavy chain and a light chain; the complementary determining region CDR in the heavy chain comprises HCDR1 shown in SEQ ID NO.1, HCDR2 shown in SEQ ID NO.2, and HCDR3 shown in SEQ ID NO.3, and the complementary determining region CDR in the light chain comprises LCDR1 shown in SEQ ID NO.4, LCDR2 shown in SEQ ID NO.5, and LCDR3 shown in SEQ ID NO.6.

[0010] Preferably, the heavy chain further includes a framework region FR, and the framework region FR of the heavy chain includes HFR1 shown in SEQ ID NO.7, HFR2 shown in SEQ ID NO.8, HFR3 shown in SEQ ID NO.9, and HFR4 shown in SEQ ID NO.10; the light chain further includes a framework region FR, and the framework region FR of the light chain includes LFR1 shown in SEQ ID NO.11, LFR2 shown in SEQ ID NO.12, LFR3 shown in SEQ ID NO.13, and LFR4 shown in SEQ ID NO.14.

[0011] Preferably, the amino acid sequence of the heavy chain is shown as SEQ ID NO.15, and the amino acid sequence of the light chain is shown as SEQ ID NO.17.

[0012] In some embodiments, the amino acid sequence of the heavy chain has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity to SEQ ID NO.15 and the CDR regions are identical; the amino acid sequence of the light chain has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity to SEQ ID NO.17 and the CDR regions are identical.

[0013] On the other hand, the present application provides a nucleic acid molecule encoding the above-mentioned antibody.

[0014] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain and a nucleotide sequence encoding a light chain, the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.16, and the nucleotide sequence encoding the light chain is shown as SEQ ID NO.18.

[0015] In another aspect, the present invention provides a product comprising the above-mentioned antibody or the above-mentioned nucleic acid molecule.

[0016] Preferably, the product comprises a medicine or a kit.

[0017] Specifically, the kit includes an HBV genotyping kit.

[0018] On the other hand, the present invention provides the use of the above-mentioned antibody, nucleic acid molecule or product in HBV genotype detection, which is a non-disease diagnosis or treatment application.

[0019] Preferably, the application includes application in preparing an HBV genotyping kit.

[0020] Preferably, the application is to prepare a colloidal gold chromatography immunoassay kit for HBV genotyping.

[0021] On the other hand, the present invention provides a method for preparing an HBV genotyping kit, which is prepared using the above-mentioned antibody, the above-mentioned nucleic acid molecule or the above-mentioned product.

[0022] Preferably, the preparation method comprises the following steps:

[0023] 1) preparing colloidal gold nanoparticles;

[0024] 2) The above antibodies and chicken IgY antibodies were conjugated to colloidal gold nanoparticles, respectively, and resuspended in colloidal gold preservation solution to prepare gold label pads;

[0025] 3) Prepare the sample pad, NC membrane and absorbent pad, and stick the sample pad, gold label pad, NC membrane and absorbent pad on the back plate with adhesive film in sequence to assemble into a test strip (see the schematic structure diagram). Figure 1 ), and then prepared into a kit.

[0026] Preferably, the preparation method comprises the following steps:

[0027] 1) preparing colloidal gold nanoparticles, optimizing the pH value for binding of the above-mentioned antibody to the colloidal gold, and optimizing the optimal concentration for binding of the above-mentioned antibody to the colloidal gold;

[0028] 2) Prepare colloidal gold blocking solution, colloidal gold preservation solution, gold label pad solution, coating diluent and sample pad solution;

[0029] 3) Conjugating chicken IgY antibodies to colloidal gold nanoparticles and resuspending them in colloidal gold preservation solution; according to the pH value and optimal concentration optimized in step 1), conjugating the antibodies to colloidal gold nanoparticles and resuspending them in colloidal gold preservation solution; preparing a gold label pad;

[0030] 4) Prepare a sample pad, NC membrane, and absorbent pad, and sequentially adhere the sample pad, gold label pad, NC membrane, and absorbent pad to a backing plate with adhesive film. After cutting and packaging, assemble the test strips to prepare a test kit.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention provides an antibody B11 for HBV genotyping, which can simultaneously recognize HBV genotype B and genotype C. The antibody comprises a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain is shown in SEQ ID NO. 17.

[0033] 2. The present invention also provides a colloidal gold immunochromatography kit based on B11 antibody, which can be directly used for serum sample detection, simplifies the procedure of hepatitis B virus typing diagnosis, shortens the detection time and has a high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Diagram of the structure of the HBV colloidal gold immunochromatographic typing kit test strip.

[0035] Figure 2 B11 antibody SDS-PAGE test results.

[0036] Figure 3 Color change diagram of colloidal gold solution.

[0037] Figure 4 UV absorption characterization image of colloidal gold solution and TEM characterization image of gold nanoparticles; A is the UV absorption characterization image; B is the TEM characterization image, and the scale bar in the figure is 100 nm.

[0038] Figure 5 Screening chart of pH value when colloidal gold is used to label B11 antibody conjugate, where A is color change and B is UV absorption characterization.

[0039] Figure 6 Screening diagram for the optimal concentration of B11 antibody binding, where A represents color change and B represents UV absorption characterization.

[0040] Figure 7 The sensitivity of HBV colloidal gold immunochromatographic typing kit was determined.

[0041] Figure 8Specificity test chart of HBV colloidal gold immunochromatographic typing kit; among them, the negative control is a healthy person serum test group that does not contain HBV B and C genotype antigens, B represents the HBV B genotype serum test group, C represents the HBV C genotype serum test group, B&C represents the mixed serum test group of HBV B genotype and C genotype, HAV represents the hepatitis A virus sample test group, HCV represents the hepatitis C virus sample test group, CMV represents the cytomegalovirus sample test group, TP represents the syphilis virus sample test group, HIV represents the human immunodeficiency virus sample test group, HBV-A represents the HBV genotype A sample test group, and HBV-D represents the HBV genotype D sample test group.

[0042] Figure 9 Batch repeatability test chart of the HBV colloidal gold immunochromatographic typing kit for HBV B and C genotype mixed antigens.

[0043] Figure 10 Batch repeatability test chart of HBV colloidal gold immunochromatographic typing kit for healthy human serum.

[0044] Figure 11 Batch repeatability test chart of the HBV colloidal gold immunochromatographic typing kit for high-concentration HBV B genotype antigen.

[0045] Figure 12 Batch repeatability test chart of HBV colloidal gold immunochromatographic typing kit for low-concentration HBV B genotype antigen.

[0046] Figure 13 Batch repeatability test chart of HBV colloidal gold immunochromatographic typing kit for HBV C genotype high-concentration antigen.

[0047] Figure 14 Batch repeatability test chart of HBV colloidal gold immunochromatographic typing kit for low-concentration HBV C genotype antigen.

[0048] Figure 15 HBV colloidal gold immunochromatographic typing kit thermal stability test chart.

[0049] Figure 16 HBV colloidal gold immunochromatographic typing kit transportation stability test chart.

[0050] Figure 17 Real-time stability test chart of HBV colloidal gold immunochromatographic typing kit. DETAILED DESCRIPTION

[0051] Unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0052] Example 1: Preparation of antibodies

[0053] Balb / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animals were housed and handled according to the approved protocol of Beijing Anbiqi Biotechnology Co., Ltd. (approval number: ABACE20151115-001).

[0054] The P94B peptide fragment (SEQ ID NO.19: PASTNRQSGRQPTPLSPPLRDTHP) corresponding to amino acids 94-117 of the pre-S1 region of HBV genotype B (GenBank ID AAF87838.1) and the P94C peptide fragment (SEQ ID NO.20: PASSNRQSGKQPTPISPPLRDSHP) corresponding to amino acids 94-117 of the pre-S1 region of HBV genotype C (GenBank ID AAL25952.1) were prepared by Beijing Anbiqi Biotechnology Co., Ltd. using solid-phase peptide synthesis technology. TM The maleimide-activated mcKLH kit (purchased from ThermoFisher) was used to couple the P94B and P94C peptides to KLH, named P94B-KLH and P94C-KLH, respectively, and mixed with Complete Freund's adjuvant (purchased from MP Biomedicals) in equal volumes. The resulting mixed adjuvant was used to immunize six Balb / C mice (60 μg, 200 μL / mouse). On days 14, 30, and 55, the mice were injected with three more doses of the mixture (30 μg, 200 μL / mouse) to boost immunity. 7-10 days after each immunization, the corresponding antibody levels in the serum of the immunized mice were titrated until high titers of the corresponding antibodies were reached. 50 ng of shock immune protein were injected intraperitoneally into mice immunized with P94B-KLH and mice immunized with P94C-KLH, respectively. Three days after shock immunization, mouse spleen cells were extracted and fused with SP2 / 0 cells (purchased from Sigma-Aldrich). After 12 days of fusion, single cell clones were obtained and cultured in 96-well plates. Antibodies in the culture supernatant were screened by indirect ELISA using P94B and P94C peptide-coated plates, and then counter-screened using P94C and P94B peptide-coated plates. Ten cell lines specific for the HBVB genotype and C genotype were identified, and the monoclonal antibodies produced are shown in Table 1. Among them, the hybridoma cell line B11 showed the best dual response results (the highest OD values). The monoclonal antibodies before and after DTT reduction were reduced with DTT (2 μg) and detected by SDS-PAGE to verify the purified monoclonal antibodies.

[0055] Table 1 Hybridoma cell line ELISA screening results

[0056]

[0057]

[0058] The antibody subclass identification of the hybridoma cell line was performed (using the Southern Biotech antibody subclass identification kit). The results are shown in Table 2. The antibody subclass of B11 is mainly IgG2b. Antibody B11 was prepared using hybridoma cell line B11. The B11 antibody was reduced with DTT, and the monoclonal antibody before and after DTT reduction was analyzed by SDS-PAGE. Two protein bands were obtained, which were approximately 50kd and 25kd, respectively. Figure 2 As shown, lane M is a DNA marker, lane 1 is after DTT reduction, and lane 2 is before DTT reduction, indicating that the monoclonal antibody was successfully prepared.

[0059] Table 2 IgG subclone identification results of subcloned cell lines

[0060] cell lines M G1 G2a G2b G3 A κ λ B11 0.062 0.054 0.033 0.798 0.036 0.033 0.316 0.055

[0061] The purified B11 antibody was sequenced by Beijing Chiyao Biotechnology. The B11 antibody consists of a heavy chain and a light chain. The specific amino acid sequences of the variable region CDRs (HCDR1-3) and FRs (HFR1-4) of the B11 antibody heavy chain are shown in Table 3, and the specific amino acid sequences of the variable region CDRs (LCDR1-3) and FRs (LFR1-4) of the B11 antibody light chain are shown in Table 4. The amino acid sequence of the B11 antibody heavy chain is shown in SEQ ID NO.15, and the nucleotide sequence is shown in SEQ ID NO.16. The amino acid sequence of the B11 antibody light chain is shown in SEQ ID NO.17, and the nucleotide sequence is shown in SEQ ID NO.18.

[0062] Table 3 Description of CDRs and FRs in the heavy chain variable region of B11 antibody

[0063] Position in the heavy chain Amino acid sequence Sequence number HCDR1 NYWMH SEQ ID NO.1 HCDR2 AVYPGNGDTSYNQKFKG SEQ ID NO.2 HCDR3 TGGLSWFVY SEQ ID NO.3 HFR1 QVQLQQSGTVLARPGASVKMSCKTSGYTFT SEQ ID NO.7 HFR2 WVKQRPGQGLEWIG SEQ ID NO.8 HFR3 KAKFTAVTSASTAYMEVNSLTSEDSAVYYCTT SEQ ID NO.9 HFR4 WGQGTLVAVSA SEQ ID NO.10

[0064] Table 4 Description of CDRs and FRs in the light chain variable region of antibody B11

[0065]

[0066]

[0067] Example 2: Preparation of colloidal gold test strips and kit

[0068] In this example, colloidal gold test strips were prepared based on the B11 antibody prepared in Example 1.

[0069] (1) Preparation of colloidal gold solution

[0070] Colloidal gold nanoparticles were prepared according to the classic trisodium citrate reduction method (Frens.Colloids-Preparation of gold dispersions of varying particle size[J].Nature,1973.). The specific method is as follows: 100 mL of ultrapure water was added to a siliconized conical flask, the water was heated to 90°C, and then 1 mL of 10% HAuCl4 (w / v) was added. The conical flask was heated to boiling under temperature-controlled magnetic stirring, and 1.56 mL of freshly prepared 10% trisodium citrate was added dropwise. The stirring speed was set to 1000 r / min, and the colloidal gold solution was heated and stirred. The color first darkened to purple, then gradually lightened, and finally turned into a translucent red (see Figure 3 ), stop heating, and continue stirring for 10 minutes. Turn off the power, transfer the above glass container to an unheated stirrer, and keep stirring at low speed for 20 minutes. After cooling to room temperature, add water to the original volume. Then filter it with a 0.22μm disposable filter membrane and store it at 4℃ for use. The ultraviolet absorption peak of the colloidal gold solution tested by ultraviolet spectrophotometer is about 520nm (see Figure 4 A), TEM images of the particles show that the particle size is about 20 nm (see Figure 4 B).

[0071] (2) Determination of pH value when colloidal gold labels B11 antibody conjugate

[0072] Use dilute hydrochloric acid and NaOH solution to adjust the pH value of the colloidal gold solution to 7, 8, 8.5, 9, 9.5, 10, 11, and 12. Take 8 EP tubes, add 500 μL of colloidal gold solution with adjusted pH value to each tube, add 100 μL of B11 antibody with a concentration of 100 μg / mL to each EP tube, and incubate at room temperature for 30 minutes. Then add 500 μL of 10% NaCl solution to each of the above EP tubes, observe the change in the color of the liquid in each EP tube to determine whether the antibody is successfully coupled to the gold nanoparticles, thereby preventing NaCl from causing the gold nanoparticles to aggregate, and find the optimal reaction pH value. Subsequently, perform UV-visible spectroscopic analysis, and the results are shown in Figure 2. Figure 5 The color of the mixture of antibody and gold nanoparticles increases with pH (see Figure 5 A), the color change gradually becomes smaller (see Figure 5 B), it can be found that when the pH is 10, the binding performance of B11 antibody and gold nanoparticles is the best.

[0073] (3) Determination of the optimal concentration of B11 antibody binding to colloidal gold

[0074] Use NaOH solution to adjust the pH value of the colloidal gold solution to 10, take 10 EP tubes, add 500 μL of colloidal gold solution with adjusted pH value to each tube, add 100 μL of B11 antibody with concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, and 100 μg / mL to each EP tube, and incubate at room temperature for 30 minutes. Subsequently, 500 μL of 10% NaCl solution was added to each of the above EP tubes, and the color change of the liquid in each EP tube was observed to determine whether the antibody was successfully coupled to the gold nanoparticles, thereby preventing NaCl from causing the gold nanoparticles to aggregate (see Figure 6 A). UV-visible spectroscopic analysis shows (see Figure 6 B), when the antibody binding concentration was 80 μg / mL, the absorption peak no longer shifted, and the antibody coupling concentration was 1.2 times the lowest concentration value. 96 μg / mL was used as the optimal concentration for coupling to ensure coupling efficiency.

[0075] (4) Preparation of colloidal gold BSA blocking solution (calculated based on 1L)

[0076] Use a graduated cylinder to measure 0.8 L of ultrapure water into a clean beaker. Add 100 g of bovine serum albumin and 0.2 g of sodium azide, and stir until completely dissolved. Bring the volume up to 1 L with ultrapure water. Transfer the solution to a storage container, label it, and store it at room temperature for 3 months.

[0077] (5) Preparation of colloidal gold preservation solution (calculated based on 1L)

[0078] Use a graduated cylinder to measure 0.8 L of ultrapure water into a clean beaker. Add 8 g Triz-base, 0.5 g PVP40, 2.86 g trisodium citrate dihydrate, 12 g casein sodium salt, 25 g sucrose, 10 g trehalose, 3.5 mL Tween 20, and 0.2 g sodium azide, and stir until completely dissolved. Bring the volume to 1 L with ultrapure water. Transfer the solution to a storage container, label it, and store at 2-8°C for 3 months.

[0079] (6) Preparation of gold label pad solution (calculated based on 1L)

[0080] Mix 250 mL of colloidal gold preservation solution with 750 mL of ultrapure water.

[0081] (7) Preparation of B-line, C-line, and quality control line coating diluent (calculated based on 1L)

[0082] Use a graduated cylinder to measure 0.9 L of ultrapure water into a clean beaker. Add 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of sodium dihydrogen phosphate, and stir until completely dissolved. Adjust the pH to 3.4 with 1 mol / L hydrochloric acid or 6 mol / L sodium hydroxide. Bring the volume to 1 L with ultrapure water. Transfer the solution to a storage container, label it, and store it at room temperature for 3 months.

[0083] (8) Preparation of sample pad solution (calculated based on 1 L)

[0084] Take 500 mL of ultrapure water and add 250 mL of colloidal gold preservation solution, add 5 g of ethylenediaminetetraacetic acid tetrasodium salt hydrate, stir evenly, then add 300 mg of anti-RBC (purchased from Sigma) and 300 mg of blocking agent (purchased from Aladdin), and dilute to 1 L with ultrapure water.

[0085] (9) Preparation of B11 antibody solution labeled with colloidal gold nanoparticles

[0086] Adjust the pH of 200 mL of colloidal gold solution to 10 with 0.2 mol / L NaOH solution. Add B11 antibody to the colloidal gold solution (final concentration: 96 μg / mL) at a stirring speed of 1000 rpm and stir for 30 minutes. Add 2 mL of BSA blocking solution to the above solution and stir for 20 minutes. Centrifuge at 10,000 rpm for 35 minutes. Discard the supernatant and resuspend the collected gold precipitate in colloidal gold preservation solution. The resuspended volume is approximately 2 mL and filtered through a filter with a pore size of 0.45 μm.

[0087] (10) Preparation of Chicken IgY Antibody Solution Labeled with Colloidal Gold Nanoparticles

[0088] Adjust the pH of 200 mL of colloidal gold solution to 9 with 0.2 mol / L potassium carbonate solution. Add 1 mg of chicken IgY antibody (purchased from Dening Biotechnology) to the colloidal gold solution at maximum stirring speed and stir for 30 minutes. Add 2 mL of BSA blocking solution to the above solution and stir for 20 minutes. Centrifuge at 10,000 rpm for 35 minutes. Discard the supernatant and suspend the collected gold precipitate in colloidal gold preservation solution. The volume after suspension is approximately 2 mL. Filter through a filter with a pore size of 0.45 μm.

[0089] (11) Preparation of gold label pad

[0090] The material of the gold label pad is glass fiber cotton. Spray 3% BSA (w / v) solution (8 μL / cm) on the cut glass fiber cotton (7×300 mm) and place it in a 37°C oven to dry for 30 minutes. Then, spray 5 μL / cm of a mixture of B11 antibody and chicken IgY antibody labeled with colloidal gold nanoparticles (the volume ratio of B11 antibody solution to chicken IgY antibody solution is 1:1) on the glass fiber cotton, place it in a 50°C oven to dry for at least 1 hour, and then seal it and store it away from light.

[0091] (12) Preparation of sample pad

[0092] The material of the sample pad is also glass fiber cotton. The cut glass fiber cotton (15×300 mm) is soaked in the sample pad solution, placed in a 50°C oven to dry for 1 hour, and then sealed and stored away from light.

[0093] (13) Preparation of NC membrane

[0094] Frozen 16D12 antibody (specifically binding to HBV genotype B antigen, purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number M10513) and 6H3 antibody (specifically binding to HBV genotype C antigen, purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number M10514) were thawed at 4°C and diluted to 2 mg / mL with purified water. The resulting mixtures were centrifuged at 10,000 rpm for 35 minutes to remove precipitated impurities and then filtered through a 0.22 μm disposable microporous filter membrane. The 16D12 antibody solution was used to spray the B test line (line B) on the NC membrane, and the 6H3 antibody solution was used to spray the C test line (line C) on the NC membrane. Chicken antigen (purchased from Dening Biotechnology) was treated similarly and applied to the control line on the NC membrane. The spray line width was 1 mm, and the spray volume was 1 μL / cm (50 dots / mL / cm). After drying in a 40°C oven for 1 hour, the tubes were immersed in a 2% BSA (w / v) solution for full sealing. After natural drying at room temperature, desiccant was added, the tubes were vacuum sealed, and stored at room temperature away from light for future use.

[0095] (14) Assembly of colloidal gold immunochromatographic test paper

[0096] The sample pad, gold label pad, NC membrane, and absorbent pad are sequentially pasted on the backing plate with adhesive film (the overlapping area is 2 mm, see the structural diagram for details). Figure 1 ) to form a semi-finished test strip. Cut the test strip to a width of 3 mm. After cutting, place the assembled semi-finished test strip into the test strip plastic card holder and press it tightly. Place the test strip and a small package of silica gel desiccant into an aluminum foil bag and seal it for storage to prepare the test kit.

[0097] Effect example: Performance test of colloidal gold test strips

[0098] This effect example performs a performance test on the test strip prepared in Example 2.

[0099] (1) Sensitivity test

[0100] Different concentrations of protein standard solutions were tested with colloidal gold kits, and the T-line color development results were observed with the naked eye. The detection limit (visual inspection) was the lowest concentration of the standard solution that could clearly be judged as a positive result. HBV genotype B antigen and genotype C antigen were diluted with sample diluent to form standard solutions with different concentrations (see the specific concentration settings for details). Figure 7 ), take 100 μL of each and drop them onto the sample pad of the test strip. After 15 minutes, observe the color development results of the test strip with naked eyes (see Figure 7 ), indicating that the detection limit of the test strip prepared with the B11 antibody of the present application is 0.1 μg / mL.

[0101] (2) Specificity test

[0102] HBV B genotype serum, HBV C genotype serum, and mixed serum of genotypes B and C were used as the main research objects for the specificity of the test paper. The serum of healthy people without HBV B and C genotypes was used as the negative control. After 15 minutes, the color development of line B and line C was observed. In addition, 7 virus samples were tested, including hepatitis A virus (HAV), hepatitis C virus (HCV), cytomegalovirus (CMV), syphilis virus (TP), human immunodeficiency virus (HIV), HBV genotype A (HBV-A), and HBV genotype D (HBV-D). The results are as follows Figure 8 As shown, after 15 minutes, the B line and C line of the test strips for detecting serum of other diseases did not show color, indicating that the test strips prepared with the B11 antibody of the present invention had no cross reaction with these viruses and had good specificity.

[0103] (3) Repeatability test

[0104] Six batches of test strips (named Batch 1, 2, 3, 4, 5 and 6) were used to test two different concentrations of HBV B genotype and C genotype serum, and mixed serum of genotypes B and C (the antigen concentration was 30 μg / mL, the results are shown in Table 1). Figure 9 ) and healthy human serum (without HBV B genotype or C genotype antigens, see Figure 10 ), where the HBV B genotype antigen concentration in HBV B genotype serum was 30 μg / mL (high concentration, see Figure 11 ) and 1 μg / mL (low concentration, see Figure 12 ), HBV C genotype antigen concentrations in HBV C genotype serum were 30 μg / mL (high concentration, see Figure 13) and 1 μg / mL (low concentration, see Figure 14 ), the test results of different batches are all effective color development, and the test results are consistent, indicating that the test strips have good reproducibility between batches.

[0105] (4) Accelerated stability test

[0106] Take the colloidal gold test kit prepared in the same batch and put it into the 37℃ incubator. Test the serum before, 3 days after, 7 days after and 10 days after. The results are as follows: Figure 15 As shown, it is shown that the kit for preparing the B11 antibody of the present invention is still effective when placed in a 37°C environment for 10 days and has good stability.

[0107] (5) Transportation stability test

[0108] The colloidal gold test strips prepared in the same batch were used for transport stability testing. The colloidal gold test kit simulated transport testing by shaking the test strips at 60 r / min for 7 days on a rocker arm and then dropping them from a height of 1.5 meters three times. The serum was tested before and after the simulated transport test. The results are as follows: Figure 16 As shown, lines B and C are clear, indicating that the colloidal gold test kit has good transportation stability.

[0109] (6) Real-time stability test

[0110] Take the test strips prepared in the same batch and store them in a dry and sealed state at room temperature and away from light. Test the mixed serum of B and C (HBV DNA load of serum species less than 1000 IU / mL) on the day of preparation and 3, 6, 9 and 12 months after storage. The results are as follows: Figure 17 As shown, it is shown that the colloidal gold kit prepared by the B11 antibody of the present invention has a shelf life of at least one year.

[0111] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An antibody for HBV genotyping, characterized in that: The antibody can simultaneously recognize HBV genotype B and genotype C, and the antibody includes a heavy chain variable region and a light chain variable region; the complementary determining region CDR in the heavy chain variable region includes HCDR1 shown in SEQ ID NO.1, HCDR2 shown in SEQ ID NO.2, and HCDR3 shown in SEQ ID NO.3, and the complementary determining region CDR in the light chain variable region includes LCDR1 shown in SEQ ID NO.4, LCDR2 shown in SEQ ID NO.5, and LCDR3 shown in SEQ ID NO.

6.

2. The antibody according to claim 1, characterized in that The heavy chain variable region also includes a framework region FR, which includes HFR1 shown in SEQ ID NO.7, HFR2 shown in SEQ ID NO.8, HFR3 shown in SEQ ID NO.9, and HFR4 shown in SEQ ID NO.10; the light chain variable region also includes a framework region FR, which includes LFR1 shown in SEQ ID NO.11, LFR2 shown in SEQ ID NO.12, LFR3 shown in SEQ ID NO.13, and LFR4 shown in SEQ ID NO.

14.

3. The antibody according to claim 1 or 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

17.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.16, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

18.

6. A product comprising the antibody according to any one of claims 1 to 3 or the nucleic acid molecule according to any one of claims 4 to 5.

7. The product according to claim 6, characterized in that The product includes a medicine or a kit, and the kit includes an HBV genotyping kit.

8. Use of the antibody according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 5, or the product according to any one of claims 6 to 7 in HBV genotype detection, characterized in that: The application is a non-disease diagnosis or treatment application, and includes the application in preparing an HBV genotyping kit.

9. A method for preparing an HBV genotyping kit, characterized in that: The following steps are involved: 1) preparing colloidal gold nanoparticles; 2) coupling the antibody of claim 1 and the chicken IgY antibody to colloidal gold nanoparticles, respectively, and resuspending them in colloidal gold preservation solution to prepare a gold label pad; 3) preparing a sample pad, an NC membrane, and a water-absorbing pad, and sequentially pasting the sample pad, the gold-labeled pad, the NC membrane, and the water-absorbing pad on a backing plate with an adhesive film to form a test strip, thereby preparing a test kit.

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