Antibody for HBV genotyping, product and preparation method and application thereof

Abstract

The invention provides an antibody for HBV genotyping, a product and a preparation method and application thereof, and relates to the field of hepatitis B virus detection. The invention provides an antibody B11 for HBV genotype typing, the antibody B11 can simultaneously recognize B genotype and C genotype of HBV, the antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain is as shown in SEQ ID NO.17; the invention further provides a colloidal gold immunochromatography kit based on the B11 antibody, the colloidal gold immunochromatography kit can be directly used for serum sample detection, the procedure of hepatitis B virus typing diagnosis is simplified, and the detection accuracy is high.

Description

Technical Field

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

[0002] Hepatitis B caused by hepatitis B virus (HBV) infection is a serious global disease that may ultimately lead to liver cirrhosis and hepatocellular carcinoma. Currently, 10 HBV sequences have been reported, and different HBV genotypes are related to disease progression, treatment option selection and prognosis. Some studies have found that in the interferon treatment of hepatitis B, genotype B has a better treatment effect than genotype C. Individuals with genotypes B and C show different serum conversions. Genotype C replicates more actively and mutates more frequently, so it is more likely to cause hepatocellular carcinoma than genotype B. Similarly, clinically isolated genotype C shows a higher in vitro transmission efficiency than genotype B. Patients with genotype B show earlier serum conversion and less portal inflammation and fibrosis. Viral genotyping of HBV-infected patients can effectively guide clinical precision medication and predict the antiviral treatment effect of patients.

[0003] The genotype classification of HBV is based on a difference of >7.5% in the whole HBV genome or a difference of ≥4% in the surface protein (S) gene. Therefore, the existing HBV genotyping methods mainly include DNA technology-based genotyping methods such as whole genome sequencing and S gene sequencing based on polymerase chain reaction (PCR), nested PCR, real-time quantitative PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), etc.

[0004] The prior art CN101560576A discloses specific probes for identifying HBV genotypes and methods and kits 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 fluorescence PCR reactions and result interpretation, etc. It involves operations such as PCR and uses professional equipment such as clean benches and PCR amplifiers. The steps are cumbersome and time-consuming and laborious.

[0005] The prior art CN107475444A discloses a PCR primer, kit and method for selectively amplifying RNA from total hepatitis B virus nucleic acid. It includes the preparation of HBV virus RNA standard by PCR, the total HBV nucleic acid extracted from patient serum including DNA and RNA, and quantitative PCR detection. This invention needs to extract DNA and RNA from patient serum and uses PCR amplification multiple times. In normal circumstances, there are also deletions or mutations in the amplified sequences during PCR amplification. Multiple PCR amplifications not only consume time and effort, but also affect the accuracy of result determination. After the ordinary PCR method, sequencing needs to be sent to a sequencing company for sequencing of the amplified gene, and the genotype of the virus can be determined only after comparison. This process is time-consuming, laborious, and cumbersome, has high requirements for operators and the environment, and costs a lot. It takes at least 1-2 days to obtain the test results, with a long time-consuming, and is not used in clinical medicine.

[0006] PCR-RFLP requires special design and treatment of fragment primers, etc. Although genotype-specific linear probe detection is a method with relatively high specificity, it is more suitable for personalized customization, with high costs and not suitable for large-scale applications. Some researchers have developed protein-based rapid genotyping methods using antigen-antibody recognition, such as enzyme-linked immunosorbent assay (ELISA), fluorescence lateral flow immunoassay (LFIA), etc. The detection time of ELISA is relatively long, about 2.5 - 4 hours, and specific detection instruments are required for both. Therefore, there is an urgent need for a kit for point-of-care testing (POCT) and self-testing in clinics that can meet the needs of hepatitis B virus genotyping detection. So that hepatitis B patients can quickly and independently test, enabling doctors to comprehensively understand the patient's condition in a timely manner, give symptomatic treatment plans, and promote the precise use of drugs for hepatitis B treatment. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides an antibody, product, preparation method and application thereof for HBV genotype typing. The present invention provides an antibody B11 for HBV genotype typing, which can simultaneously recognize genotype B and genotype C of HBV. This antibody includes a heavy chain and a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain is as shown in SEQ ID NO.17. The present invention also provides a colloidal gold immunochromatographic kit based on the B11 antibody, which can be directly used for the detection of serum samples and quickly type the hepatitis B virus.

[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, which can simultaneously recognize genotype B and genotype C of HBV, and the antibody comprises a heavy chain and a light chain; the complementarity-determining regions CDRs in the heavy chain include HCDR1 shown in SEQ ID NO.1, HCDR2 shown in SEQ ID NO.2, and HCDR3 shown in SEQ ID NO.3, and the complementarity-determining regions CDRs in the light chain include 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 comprises 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 comprises 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 as shown in SEQ ID NO.15, and the amino acid sequence of the light chain is as shown in 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 with SEQ ID NO.15 and the CDR regions are the same; 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 with SEQ ID NO.17 and the CDR regions are the same.

[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 the heavy chain and a nucleotide sequence encoding the light chain, the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO.16, and the nucleotide sequence encoding the light chain is as shown in SEQ ID NO.18.

[0015] On the other hand, the present invention provides a product comprising the above-mentioned antibody or the above-mentioned nucleic acid molecule.

[0016] Preferably, the product includes a drug or a kit.

[0017] Specifically, the kit includes an HBV genotype typing kit.

[0018] On the other hand, the present invention provides the use of the above-mentioned antibody, the above-mentioned nucleic acid molecule or the above-mentioned product in the detection of HBV genotypes, and the use is for non-disease diagnosis or treatment applications.

[0019] Preferably, the use includes the use in the preparation of an HBV genotype typing kit.

[0020] Preferably, the use is for the preparation of a colloidal gold chromatography immunoassay kit for HBV genotype typing.

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

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

[0023] 1) Prepare colloidal gold nanoparticles;

[0024] 2) Couple the above-mentioned antibody and chicken IgY antibody with colloidal gold nanoparticles respectively, resuspend with colloidal gold preservation solution, and prepare a gold-labeled pad;

[0025] 3) Prepare a sample pad, an NC membrane and a water absorption pad, and paste the sample pad, the gold-labeled pad, the NC membrane and the water absorption pad on the back plate with a glue film in sequence to assemble a test strip (see the schematic structural diagram in Figure 1 ), and then prepare a kit.

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

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

[0028] 2) Prepare colloidal gold blocking solution, colloidal gold preservation solution, gold-labeled pad solution, coating dilution solution and sample pad solution;

[0029] 3) Couple chicken IgY antibody with colloidal gold nanoparticles and resuspend with colloidal gold preservation solution; according to the optimized pH value and optimal concentration in step 1), couple the above-mentioned antibody with colloidal gold and resuspend with colloidal gold preservation solution; prepare a gold-labeled pad;

[0030] 4) Prepare the sample pad, NC membrane, and absorbent pad, and sequentially paste the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto the backplane with a glue film. After cutting and packaging, assemble them into a test strip, and then prepare a 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 genotype B and genotype C of HBV. The antibody includes a heavy chain and a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain is as shown in SEQ ID NO.17.

[0033] 2. The present invention also provides a colloidal gold immunochromatographic kit based on the B11 antibody, which can be directly used for the detection of serum samples, simplifies the procedure of hepatitis B virus genotyping diagnosis, has a short detection time and a high detection accuracy rate. Description of the Drawings

[0034] Figure 1 Structural diagram of the test strip of the HBV colloidal gold immunochromatographic genotyping kit.

[0035] Figure 2 SDS-PAGE detection result of the B11 antibody.

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

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

[0038] Figure 5 pH value screening diagram when the colloidal gold labels the B11 antibody conjugate, where A is the color change and B is the UV absorption characterization.

[0039] Figure 6 Optimal concentration screening diagram of the B11 antibody binding, where A is the color change and B is the UV absorption characterization.

[0040] Figure 7 Determination of the sensitivity of the HBV colloidal gold immunochromatographic genotyping kit.

[0041] Figure 8Specificity test chart of HBV colloidal gold immunochromatographic genotyping kit; among them, the negative control is a healthy human serum test group without 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 HBV colloidal gold immunochromatographic genotyping kit for mixed antigens of HBV B and C genotypes.

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

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

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

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

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

[0048] Figure 15 Thermal stability test chart of HBV colloidal gold immunochromatographic genotyping kit.

[0049] Figure 16 Transport stability test chart of HBV colloidal gold immunochromatographic genotyping kit.

[0050] Figure 17 Real-time stability test chart of HBV colloidal gold immunochromatographic genotyping kit. Detailed implementation manners

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

[0052] Example 1: Preparation of Antibodies

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

[0054] Using solid-phase peptide synthesis technology, Beijing Anbige Biotechnology Co., Ltd. prepared the P94B peptide (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 (SEQ ID NO.20: PASSNRQSGKQPTPISPPLRDSHP) corresponding to amino acids 94 - 117 of the pre-S1 region of HBV genotype C (GenBank ID AAL25952.1). Then use Imject TM Maleimide-activated mcKLH kit (purchased from ThermoFisher) was used to conjugate 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 volume. The obtained mixed adjuvant was immunized into 6 Balb / C mice (60 μg, 200 μL / mouse). At day 14, day 30 and day 55, the mice were injected with 3 doses of the mixture (30 μg, 200 μL / mouse) to boost the immunity. 7 - 10 days after each immunization, the corresponding antibody levels in the sera of the immunized mice were titrated until a high titer of the corresponding antibody was reached. 50 ng of shock immunoprotein was intraperitoneally injected into the P94B-KLH immunized mice and P94C-KLH immunized mice respectively. 3 days after shock immunization, the spleen cells of the mice were extracted and fused with SP2 / 0 cells (purchased from Sigma-Aldrich). 12 days after fusion, single cell clones were taken and cultured in 96-well plates. Antibodies in the culture supernatant were screened by indirect ELISA using plates coated with P94B and P94C peptides, and then re-screened using plates coated with P94C and P94B peptides respectively. 10 cell lines specific for HBV genotype B and C were identified. The monoclonal antibodies produced are shown in Table 1. Among them, the hybridoma cell line B11 showed the best dual response results (both OD values were the highest). The monoclonal antibodies before and after reduction with DTT (2 μg) were detected by SDS-PAGE to verify the purified monoclonal antibodies.

[0055] Table 1 ELISA Screening Results of Hybridoma Cell Lines

[0056]

[0057]

[0058] The antibody subclass of the hybridoma cell line was identified (using the antibody subclass identification kit from Southern Biotech). The results are shown in Table 2. The main antibody subclass of B11 is IgG2b. Antibody B11 was prepared using the hybridoma cell line B11. The B11 antibody was reduced with DTT, and the monoclonal antibody before and after DTT reduction was detected by SDS-PAGE, obtaining two protein bands, approximately 50 kd and 25 kd respectively. As Figure 2 shown, lane M is the DNA marker, lane 1 is after DTT reduction, and lane 2 is before DTT reduction, indicating the successful preparation of the monoclonal antibody.

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

[0060] Cell line 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 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 light chain are shown in Table 4; the amino acid sequence of the B11 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 light chain is shown in SEQ ID NO.17, and the nucleotide sequence is shown in SEQ ID NO.18.

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

[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 Explanation of CDRs and FRs in the variable region of the B11 light chain

[0065]

[0066]

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

[0068] This example is based on the B11 antibody prepared in Example 1 to prepare a colloidal gold test strip.

[0069] (1) Preparation of colloidal gold solution

[0070] Referring to the classic sodium citrate reduction method for preparing colloidal gold nanoparticles (Frens. Colloids - Preparation of gold dispersions of varying particle size[J]. Nature, 1973.), the specific method is as follows: Add 100 mL of ultrapure water into a silanized conical flask, heat the water to 90 °C, and then add 1 mL of 10% HAuCl 4 (w / v). Heat the conical flask to boiling under the condition of temperature - controlled magnetic stirring, and dropwise add 1.56 mL of freshly prepared 10% sodium citrate. Set the stirring speed to 1000 r / min, heat and stir. The color of the colloidal gold solution first becomes darker to purple and then gradually fades until it finally becomes a transparent red (see Figure 3 ). Stop heating and continue to stir for 10 min. Turn off the power, transfer the above glass container to an unheated stirrer, and keep stirring at a low speed for 20 min. After cooling to room temperature, add water to make up the original volume. Then filter it with a 0.22 - μm disposable filter membrane and store it at 4 °C for later use. Use an ultraviolet spectrophotometer to measure that the ultraviolet absorption peak of the colloidal gold solution is about 520 nm (see Figure 4 A), and TEM photograph shows that the particle size of the particles is about 20 nm (see Figure 4 B).

[0071] (2) Determination of the pH value when colloidal gold labels the 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, 12. Take 8 EP tubes, add 500 μL of the colloidal gold solution with the 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 with shaking for 30 min. Subsequently, add 500 μL of 10% NaCl solution to each of the above EP tubes, observe the color change of the liquid in each EP tube to judge whether the antibody is successfully conjugated to the gold nanoparticles to prevent the aggregation of gold nanoparticles caused by NaCl, and find the optimal reaction pH value. Subsequently, perform ultraviolet - visible spectrophotometric analysis, and the results are shown in Figure 5 , the color of the mixture of antibody and gold nanoparticles changes with the increase of pH (see Figure 5 A), and 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] Adjust the pH value of the colloidal gold solution to 10 with NaOH solution. Take 10 EP tubes, add 500 μL of the colloidal gold solution with the adjusted pH value to each tube, and 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 respectively, and incubate with shaking at room temperature for 30 min. Subsequently, add 500 μL of 10% NaCl solution to each of the above EP tubes, and observe the color change of the liquid in each EP tube to determine whether the antibody is successfully conjugated to the gold nanoparticles, thereby preventing the aggregation of gold nanoparticles caused by NaCl (see Figure 6 A). It can be known from ultraviolet-visible spectrophotometric analysis (see Figure 6 B) that when the antibody binding concentration is 80 μg / mL, the absorption peak no longer shifts, and the antibody conjugation concentration is 1.2 times the lowest concentration value. A concentration of 96 μg / mL is used as the optimal concentration for conjugation to ensure the conjugation efficiency.

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

[0076] Measure 0.8 L of ultrapure water with a measuring cylinder and add it to a clean beaker. Add 100 g of bovine serum albumin and 0.2 g of sodium azide, and stir until completely dissolved. Make up the volume to 1 L with ultrapure water, transfer the solution to a storage container, label the container, and it can be stored at room temperature for 3 months.

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

[0078] Measure 0.8 L of ultrapure water with a measuring cylinder and add it to a clean beaker. Add 8 g of Triz-base, 0.5 g of PVP40, 2.86 g of trisodium citrate dihydrate, 12 g of sodium caseinate, 25 g of sucrose, 10 g of trehalose, 3.5 mL of Tween 20, and 0.2 g of sodium azide, and stir until completely dissolved. Make up the volume to 1 L with ultrapure water, transfer the solution to a storage container, label the container, and it can be stored at 2 - 8 °C for 3 months.

[0079] (6) Preparation of gold conjugate pad solution (calculated by 1 L)

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

[0081] (7) Preparation of coating dilution for line B, line C, and control line (calculated by 1 L)

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

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

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

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

[0086] Use 0.2mol / L NaOH solution to adjust the pH of 200mL colloidal gold solution to 10. Add B11 antibody to the colloidal gold solution (final concentration is 96μg / mL) at a stirring speed of 1000r / min and stir for 30min. Add 2mL of BSA blocking solution to the above solution and stir for 20min. Centrifuge at 10000r / min for 35min. Discard the supernatant and suspend the collected gold precipitate in the colloidal gold preservation solution. The volume after suspension is about 2mL, and filter with a filter with a pore size of 0.45μm.

[0087] (10) Preparation of chicken IgY antibody solution labeled with colloidal gold nanoparticles

[0088] Use 0.2mol / L potassium carbonate solution to adjust the pH of 200mL colloidal gold solution to 9. At the maximum stirring speed, add 1mg chicken IgY antibody (purchased from Dening Bio) to the colloidal gold solution and stir for 30 minutes. Add 2mL 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 the colloidal gold preservation solution. The volume after suspension is about 2mL. Filter with a filter with a pore size of 0.45μm.

[0089] (11) Preparation of gold label pad

[0090] The material of the gold label pad is selected as glass fiber cotton. 3% BSA (w / v) solution (8 μL / cm) is sprayed on the cut glass fiber cotton (7×300 mm), and it is placed in an oven at 37 °C for drying for 30 min; then, a mixture of B11 antibody labeled with colloidal gold nanoparticles and chicken IgY antibody (the volume ratio of B11 antibody solution to chicken IgY antibody solution is 1:1) at 5 μL / cm is sprayed on the glass fiber cotton, and it is placed in an oven at 50 °C for drying for at least 1 h, and then it is sealed and stored away from light.

[0091] (12) Preparation of the sample pad

[0092] The material of the sample pad is also selected as glass fiber cotton. The cut glass fiber cotton (15×300 mm) is soaked in the sample pad solution, placed in an oven at 50 °C for drying for 1 h, and then it is sealed and stored away from light.

[0093] (13) Preparation of the NC membrane

[0094] The frozen 16D12 antibody (which can specifically bind to HBV genotype B antigen, purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd., product number M10513) and 6H3 antibody (which can specifically bind to HBV genotype C antigen, purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd., product number M10514) are thawed at 4 °C, diluted with pure water to 2 mg / mL respectively, centrifuged at a speed of 10000 r / min for 35 min to remove precipitation impurities, and then filtered through a 0.22 μm disposable microporous membrane. The 16D12 antibody solution is used for spraying the B test line (B line) on the NC membrane, and the 6H3 antibody solution is used for spraying the C test line on the NC membrane (C line); chicken antigen (purchased from Dening Biology) is treated in the same way and used for spraying the quality control line on the NC membrane. The spraying line width is 1 mm, and the spraying amount is 1 μL / cm (50 dots / mL / cm). After being placed in an oven at 40 °C for drying for 1 h, it is immersed in 2% BSA (w / v) solution for sufficient blocking, naturally dried at room temperature, desiccant is added, and it is vacuum-sealed and stored away from light at room temperature for standby.

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

[0096] The sample pad, gold label pad, NC membrane, and absorbent pad are sequentially pasted on the back plate with a glue film (the overlapping area is 2 mm for all, and the structural schematic diagram is shown in Figure 1 ) to form a semi-finished test strip. The cutting width is 3 mm. After cutting, the assembled semi-finished test strip is put into the plastic cartridge of the test strip and pressed tightly. The test strip and a small package of silica gel desiccant are put into an aluminum foil bag and sealed for storage, and then a kit is prepared.

[0097] Effect example: Performance test of the colloidal gold test strip

[0098] This example of results conducts performance tests on the test strips prepared in Example 2.

[0099] (1) Sensitivity test

[0100] Protein standard solutions of different concentrations are detected through a colloidal gold kit, and the color development result of the T line is observed with the naked eye. The detection limit (visual inspection) is the lowest concentration of the standard solution that can be clearly judged as a positive result. Dilute HBV genotype B antigen and genotype C antigen with sample diluent to different concentration standard solutions with gradients (for specific concentration settings, see Figure 7 ), take 100 μL of each and drop them onto the sample pad of the test strip respectively. After 15 minutes, observe the color development result of the test strip with the naked eye (see Figure 7 ), indicating that the detection limit of the test strip prepared with the B11 antibody of this application is 0.1 μg / mL.

[0101] (2) Specificity test

[0102] Use HBV genotype B serum, HBV genotype C serum, and the mixed serum of genotype B and genotype C as the main research objects for the specificity of the test strip. The serum of healthy people without HBV genotype B and C is used as a negative control. After 15 minutes, observe the color development of the B line and C line. In addition, 7 virus samples are 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 Figure 8 shown. After 15 minutes, for the test strips detecting the sera of other diseases, the B line and C line do not show color, indicating that the test strip prepared with the B11 antibody of the present invention has no cross-reaction with these several viruses and has good specificity.

[0103] (3) Repeatability test

[0104] Take test strips of 6 batches (named Batch1, 2, 3, 4, 5, and 6 respectively), and detect two different concentrations of HBV genotype B and genotype C sera, the mixed serum of genotype B and genotype C (the antigen concentration is 30 μg / mL for all, and the results are shown in Figure 9 ) and the serum of healthy people (without HBV genotype B or C antigen, and the results are shown in Figure 10 ) under the same conditions. Among them, the HBV genotype B antigen concentration in the HBV genotype B serum is 30 μg / mL (high concentration, and the results are shown in Figure 11 ) and 1 μg / mL (low concentration, and the results are shown in Figure 12 ), and the HBV genotype C antigen concentration in the HBV genotype C serum is 30 μg / mL (high concentration, and the results are shown in Figure 13) and 1 μg / mL (low concentration, results are shown in Figure 14 ), the detection results of different batches are all effectively colored, and the detection results are consistent, indicating that the strip has good repeatability between batches.

[0105] (4) Accelerated stability test

[0106] Take the same batch of prepared colloidal gold kits and put them in an incubator at 37 °C. Serum tests are carried out before putting them in, 3 days, 7 days, and 10 days after putting them in. The results are as Figure 15 shown, indicating that the kit prepared with the B11 antibody of the present invention is still effective after being placed at 37 °C for 10 days and has good stability.

[0107] (5) Transportation stability test

[0108] Take the same batch of prepared colloidal gold test strips for transportation stability test. The method for simulating transportation test of the colloidal gold kit is: shake on the rocker arm at a speed of 60 r / min for 7 days, and then drop it 3 times from a height of 1.5 m. Serum is detected before and after the simulated transportation test. The results are as Figure 16 shown, the B line and C line are clear, indicating that the colloidal gold 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 manner at room temperature in the dark. Detect the mixed serum of B and C (the HBV DNA load in the serum is less than 1000 IU / mL) on the day of preparation and at 3 months, 6 months, 9 months, and 12 months of storage. The results are as Figure 17 shown, indicating that the colloidal gold kit prepared with 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 a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart 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 genotype B and genotype C of HBV, and the antibody includes a heavy chain and a light chain; the complementary determining region CDR in the heavy chain 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 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 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 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 is shown in SEQ ID NO.15, and the amino acid sequence of the light chain is shown in SEQ ID NO.

17.

4. The antibody according to claim 3, characterized in that 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 with 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 with SEQ ID NO.17, and the CDR regions are identical.

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

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

18.

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

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

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

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

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