CN113683684B - Anti-hepatitis B virus surface antigen antibody, antibody pair, detection reagent containing same and kit - Google Patents

Abstract

The invention discloses an anti-hepatitis B virus surface antigen antibody, an antibody pair, a detection reagent containing the antibody pair and a kit, wherein the antibody comprises an antibody light chain and an antibody heavy chain, the antibody light chain comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3, and the antibody heavy chain comprises complementarity determining regions CDR-H1 and CDR-H, CDR-H3. The antibody disclosed by the invention is used for detecting the surface antigen of the hepatitis B virus, can specifically bind to the surface antigen of the hepatitis B virus, and has high detection specificity and sensitivity.

Description

Anti-hepatitis B virus surface antigen antibody, antibody pair, detection reagent containing same and kit

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-hepatitis B virus surface antigen antibody, an antibody pair, a detection reagent containing the same and a kit.

Background

Hepatitis B Virus (HBV) is a very small virus that belongs to one member of the hepadnavirus group (DNA). The virus particle consists of an outer membrane and an inner core, the complete HBV particle is a spherical particle with the diameter of 42nm, and the outer membrane thickness of the particle is 7nm, and the particle consists of protein and membrane lipid. The main sources of infection for hepatitis B are patients and HBV antigen carriers. Viral hepatitis caused by Hepatitis B Virus (HBV) has the characteristics of strong infectivity, high carrying rate, wide epidemic range and serious chronic tendency, and belongs to oncogenic viruses.

The hepatitis B virus surface antigen HBsAg is the coat protein of HBV and is not infectious per se. Since the presence of HBsAg is often accompanied by the presence of HBV, HBsAg is a marker of HBV infection in the body. It was found that HBsAg could be detected in serum within 2-6 months after HBV infection in humans. The quantitative detection of HBsAg of the hepatitis B patient has higher application value in the aspects of preparing a treatment scheme for the hepatitis B patient, evaluating the clinical curative effect and prognosis.

With the continuous development of the world medical detection technology, the chemiluminescence diagnostic detection system obtains better detection effect and quality in detecting the hepatitis B surface antigen, but the antibody pair with high specificity and high sensitivity applied to the chemiluminescence immune sandwich method is still deficient, so that the production of the antibody raw material with high specificity and sensitivity which can be suitable for detecting the hepatitis B surface antigen by the chemiluminescence system becomes particularly important.

Disclosure of Invention

The invention mainly aims to provide an anti-hepatitis B virus surface antigen antibody (HBsAb) to solve the problem of lack of anti-hepatitis B virus surface antigen antibody raw materials in the existing technology for detecting the hepatitis B virus surface antigen by chemiluminescence.

In order to achieve the above object, according to one aspect of the present invention, there is provided an anti-hepatitis B virus surface antigen antibody 2D8 comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDR-L1 amino acid sequence of SEQ ID NO:1, a CDR-L2 amino acid sequence of SEQ ID NO:2, a CDR-L3 amino acid sequence of SEQ ID NO:3, the heavy chain variable region has a CDR-H1 amino acid sequence of SEQ ID NO:4, a CDR-H2 amino acid sequence of SEQ ID NO:5, and a CDR-H3 amino acid sequence of SEQ ID NO:6.

Further, there is provided an anti-hepatitis B virus surface antigen antibody 7G5 comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDR-L1 amino acid sequence of SEQ ID NO. 7, a CDR-L2 amino acid sequence of SEQ ID NO. 8, a CDR-L3 amino acid sequence of SEQ ID NO. 9, the heavy chain variable region has a CDR-H1 amino acid sequence of SEQ ID NO. 10, a CDR-H2 amino acid sequence of SEQ ID NO. 11, and a CDR-H3 amino acid sequence of SEQ ID NO. 12.

Further, antibody 2D8 comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is SEQ ID NO. 13, and the amino acid sequence of the heavy chain variable region is SEQ ID NO. 14; antibody 7G5 comprising a light chain variable region having the amino acid sequence SEQ ID NO. 15 and a heavy chain variable region having the amino acid sequence SEQ ID NO. 16.

Further, there is provided a constant region of the above antibody comprising a light chain constant region and/or a heavy chain constant region, specifically, an amino acid sequence of the light chain constant region is SEQ ID NO. 17; the amino acid sequence of the heavy chain constant region is SEQ ID NO. 18.

Further, the nucleotide sequence of the light chain variable region of the coded antibody 2D8 is SEQ ID NO. 17, and the nucleotide sequence of the heavy chain variable region is SEQ ID NO. 18; the nucleotide sequence of the light chain variable region of the coded antibody 7G5 is SEQ ID NO. 19, and the nucleotide sequence of the heavy chain variable region is SEQ ID NO. 20.

According to another aspect of the present invention, there is provided an anti-hepatitis B virus surface antigen antibody pair comprising antibody 2D8 or/and antibody 7G5.

According to another aspect of the present invention, there is provided a hepatitis B virus surface antigen detection reagent comprising the antibody pair.

According to another aspect of the present invention, there is provided a hepatitis B virus surface antigen detection reagent further comprising a stabilizing diluent comprising a buffer solution, a base component and a protective component; preferably, wherein the buffer is phosphate buffer, the base component comprises bovine serum albumin BSA and sodium azide NaN ₃ The protective component comprises penicillin and glycerol; more preferably, the pH of the phosphate buffer is 7.2; the BSA content was 1.5. 1.5 m/v%; naN (NaN) ₃ The content of (C) is 0.03 m/v%; penicillin content is 0.6 m/v%; the glycerol content was 0.5. 0.5 v/v%.

According to another aspect of the present invention, there is provided the use of the hepatitis B virus surface antigen detection reagent of any one of the above in the preparation of a clinical detection kit for HBsAg.

By applying the technical scheme of the invention, the anti-hepatitis B virus surface antigen antibody can be quickly and efficiently prepared, the problem of lack of antibody which can be applied to the detection of hepatitis B virus surface antigen by an immune sandwich method in the current chemiluminescence field is solved, and high-sensitivity and high-specificity raw materials are provided for the detection of hepatitis B virus surface antigen.

Drawings

FIG. 1 is a graph of degradation rate of antibody 2D8 over time in the comparative buffers and stabilizers A-F provided in Table 8 of example 2.

FIG. 2 is a graph of degradation rate of antibody 2D8 over time in the comparative buffer and stabilizer J-L provided in Table 8 of example 2.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

The invention can rapidly and efficiently screen the anti-hepatitis B surface antigen antibody by utilizing phage surface display technology, and solves the problems of high sensitivity and lack of specific antibody raw materials in the current hepatitis B surface antigen detection.

The practice of the present invention, except where explicitly indicated to the contrary, involves conventional virological, immunological, microbiological and molecular biological methods and DNA recombination techniques well known to those skilled in the art, some of which are described below for illustrative purposes. Other embodiments may be practiced in other embodiments, mutatis mutandis, of embodiments of the present description unless explicitly stated otherwise.

Embodiments of the present invention relate to an anti-hepatitis b virus surface antigen antibody. In particular, the antibody of the present invention can detect hepatitis B virus surface antigen with unexpectedly high specificity and sensitivity, and shows extremely low crossing rate, and shows excellent performance compared with the commercial antibody pair.

As is well known in the art, an antibody is an immunoglobulin molecule that is capable of specifically binding to a target site through an epitope recognition site of a variable region. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof such as Fab, fab ', F (ab') 2, fv, single chain ScFv, synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion that binds to a specific desired antigen binding fragment, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen binding site or a specific desired fragment.

The term "epitope" includes any determinant, preferably a polypeptide determinant, which is capable of specifically binding to an immunoglobulin or T-cell receptor, and is an antigen region that binds to an antibody, and in this application includes in particular all CDR regions of VH and VL sequences of an antibody that binds to HBsAg.

In some embodiments, antibodies and antigen binding fragments thereof described herein include sets of heavy and light chain CDRs embedded between heavy and light chain framework regions, respectively, which provide support for the CDRs and determine the spatial relationship of the CDRs to one another. As used herein, the term "CDR region" refers to three hypervariable regions of either the heavy or light chain V regions. These regions are denoted as "CDR1", "CDR2", and "CDR3", respectively, starting from the N-terminus of the heavy or light chain. Thus, the antigen binding site comprises six CDRs comprising the CDRs located at each of the heavy and light chain V regions.

The embodiment of the invention relates to a phage display method, which is used for directly obtaining genes from peripheral blood of a hepatitis B virus patient and obtaining high-titer antibodies by using a molecular cloning technology. The term "phage display technology" (phage displayed technology, PDT) is a novel technology that fuses a foreign protein or polypeptide with a phage coat protein, displays it on the phage surface and maintains a specific spatial conformation, and uses specific affinity to screen specific proteins or polypeptides. Smith, G.P in 1985 proposed that a fusion protein containing a foreign protein or polypeptide was expressed by inserting a foreign gene into the engineered coat protein gene of a phage, and this phage with the fusion protein was called a fusion phage. Through repeated adsorption-elution-amplification processes, phage containing exogenous proteins capable of specifically binding to target proteins are selected from phage libraries expressing various exogenous proteins, and then enrichment, amplification and gene sequence determination are performed to infer the amino acid composition of the exogenous proteins.

Embodiments of the present invention also relate to the term "chemiluminescent immunoassay" which is based on the principle of combining a chemiluminescent assay technology with high sensitivity with a highly specific immune reaction for detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. The term "capture antibody" is used herein to refer to an antibody that can be immobilized to a solid medium such as an elisa plate, magnetic beads, colloidal gold, etc., for capturing an antigen in solution. The term "labeled antibody" refers to an antibody on which a chromogenic compound such as alkaline phosphatase, horseradish peroxidase, luminol or the like is labeled, whereby the binding properties of the antibody are detected.

Based on the above research results, the applicant proposes the technical scheme of the present application. In a typical embodiment of the present application, there is provided an anti-hepatitis B virus surface antigen antibody 2D8 comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDR-L1 amino acid sequence of SEQ ID NO:1, a CDR-L2 amino acid sequence of SEQ ID NO:2, a CDR-L3 amino acid sequence of SEQ ID NO:3, and the heavy chain variable region has a CDR-H1 amino acid sequence of SEQ ID NO:4, a CDR-H2 amino acid sequence of SEQ ID NO:5, and a CDR-H3 amino acid sequence of SEQ ID NO:6.

In another exemplary embodiment of the present application, there is also provided an anti-hepatitis B virus surface antigen antibody 7G5 comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDRL1 amino acid sequence of SEQ ID NO. 7, a CDRL2 amino acid sequence of SEQ ID NO. 8, a CDRL3 amino acid sequence of SEQ ID NO. 9, and the heavy chain variable region has a CDRH1 amino acid sequence of SEQ ID NO. 10, a CDRH2 amino acid sequence of SEQ ID NO. 11, and a CDRH3 amino acid sequence of SEQ ID NO. 12.

In another exemplary embodiment of the present application, there is also provided an anti-hepatitis B virus surface antigen antibody 2D8 comprising a light chain variable region having the amino acid sequence of SEQ ID NO. 13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO. 14.

In another exemplary embodiment of the present application, there is also provided an anti-hepatitis B virus surface antigen antibody 7G5 comprising a light chain variable region having the amino acid sequence of SEQ ID NO. 15 and a heavy chain variable region having the amino acid sequence of SEQ ID NO. 16.

In contrast to the variable region, the sequence of the immunoglobulin constant region is a conserved sequence that is responsible for binding to a variety of natural proteins to stimulate a specific physiological function. In certain preferred embodiments, the antibody or antigen binding fragment thereof may further comprise a constant region, the amino acid sequence of which may be obtained by NCBI search, as well as by other means known to those skilled in the art.

In another exemplary embodiment of the present application, the amino acid sequence of the constant region is provided, including a light chain constant region having the amino acid sequence of SEQ ID NO:17 and/or a heavy chain constant region having the amino acid sequence of SEQ ID NO:18.

In another exemplary embodiment of the present application, a nucleotide molecule encoding anti-hepatitis B virus surface antigen antibody 2D8 or 7G5 is provided, the nucleotide sequence encoding the light chain variable region of said antibody 2D8 is SEQ ID NO. 19, and the nucleotide sequence of the heavy chain variable region is SEQ ID NO. 20; the nucleotide sequence of the light chain variable region of the antibody 7G5 is SEQ ID NO. 21, and the nucleotide sequence of the heavy chain variable region is SEQ ID NO. 22.

The nucleic acid molecules of the invention may exist in the form of RNA or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthetic production or any combination thereof.

In another exemplary embodiment of the present application, there is provided a screening method of anti-hepatitis b virus surface antigen antibodies, comprising the steps of: a) Obtaining total mRNA by using peripheral blood of a hepatitis B patient, obtaining cDNA by reverse transcription, and amplifying an antibody variable region section; b) Constructing a phage display antibody expression library; c) The library is screened with HBsAg, and the antibody or antibody fragment is obtained by screening.

Further, a method for preparing and screening the antibody is provided, which comprises the following specific preparation steps: comprises a) carrying out gene recombination on the anti-hepatitis B virus surface antigen antibody or antibody fragment genes screened by the screening method and a mammalian cell expression vector; b) Recombinant expression in a mammalian cell expression system; c) Separating to obtain the anti-hepatitis B virus surface antigen antibody.

In another exemplary embodiment of the present application, an anti-hepatitis B virus surface antigen antibody pair is provided, comprising antibody 2D8 or/and antibody 7G5.

In another exemplary embodiment of the present application, there is provided a hepatitis b virus surface antigen detection reagent comprising the antibody pair.

In another exemplary embodiment of the present application, there is provided a hepatitis b virus surface antigen detection reagent further comprising a stabilizing diluent comprising a buffer, a base component, and a protective component; preferably, wherein the buffer is phosphate buffer, the base component comprises bovine serum albumin BSA and sodium azide NaN ₃ The protective component comprises penicillin and glycerol; more preferably, the pH of the phosphate buffer is 7.2; the BSA content was 1.5. 1.5 m/v%; naN (NaN) ₃ The content of (C) is 0.03 m/v%; penicillin content is 0.6 m/v%; the glycerol content was 0.5. 0.5 v/v%.

In another exemplary embodiment of the present application, a kit for clinical detection of hepatitis b virus surface antigen is provided, which comprises any one of the above-mentioned reagents for detection of hepatitis b virus surface antigen, and has good detection sensitivity, specificity and/or storage stability due to the presence of the above-mentioned reagents.

The beneficial effects of the present application will be further described below in conjunction with specific embodiments. In the following examples and comparative examples, reagents and consumables used were commercially available products unless otherwise specified.

Example 1: materials and methods

1. Experimental material and instrument

1.1 materials

HBsAg antigen, source: chinese medicine biological products institute;

anti-HBsAg monoclonal antibody 1 (HbsAb-1), source: RA Biosources, usa; anti-HBsAg monoclonal antibody 2 (HbsAb-2), source: RA Biosources, usa;

magnetic microspheres, ABEI: shenzhen New industry biomedical Co., ltd.

1.2. Instrument for measuring and controlling the intensity of light

Full-automatic optical spectrum analyzer Maglumi 2000Plus, serial number developed and produced by Shenzhen New industry biomedical engineering Co., ltd.): 2000200068.

2 preparation of anti-hepatitis B Virus surface antigen antibody

2.1 acquisition of hepatitis B Virus antigen

Extraction of total mRNA of hepatitis B patient: taking peripheral blood of a patient with hepatitis B, and extracting mRNA; directly using 200uL of fresh blood, adding 20uLProteinaseK solution, and uniformly mixing; adding 200uL buffer solution GB, fully and reversely mixing, standing at 70 ℃ for 10Min, clearing the solution, and centrifuging to remove water drops on the inner wall of the tube cover; adding 200uL absolute ethyl alcohol, fully vibrating and uniformly mixing, and centrifuging briefly to remove water drops on the inner wall of the tube cover; adding the solution obtained in the last step into an adsorption column for centrifugation, and pouring out waste liquid; adding buffer solution GD into an adsorption column, and centrifuging to pour out waste liquid; adding a rinsing liquid into the adsorption column, centrifuging to remove waste liquid, and washing twice; centrifuging for two minutes, standing at room temperature for two minutes, and airing residual rinsing liquid in the adsorption column; adding elution buffer solution, and centrifuging to obtain total hepatitis B mRNA.

RT-PCR: 1-5. Mu.g of total RNA was added to a 0.5ml microcentrifuge tube, and an appropriate amount of DEPC H2O was added to make the total volume 11. Mu.l. Add 10. Mu.M Oligo (dT) 12-18. Mu.l to the tube, mix gently, centrifuge; heating at 70deg.C for 10min, and immediately inserting the microcentrifuge tube into ice bath for at least 1min; then a mixture of 10 XPCR buffer, 2. Mu.l, was added; 25mM MgCl2, 2. Mu.l; 10mM dNTPMix,1 μl;0.1M DTT, 2. Mu.l gently mix and centrifuge. Incubating at 42 ℃ for 2-5min; adding Superscript II 1 μl, and incubating in a water bath at 42deg.C for 50min; heating at 70deg.C for 15min to terminate the reaction; the tube was inserted into ice, 1. Mu.l of RNase H was added, and incubated at 37℃for 20min to degrade the residual RNA. Preserving at-20 ℃ for standby.

After the PCR reaction is finished, all the PCR products are subjected to 1% agarose gel electrophoresis, a gel block containing the target fragment is cut off, and the target fragment is recovered by using a gel recovery kit.

2.2 construction of phage vectors

The purified light chain gene PCR product and pComb3H vector are respectively purified and recovered after double enzyme digestion by SacI/XbaI, and the recovered products are connected by T4 DNA ligase; and directly purifying and recovering enzyme digestion products of the light chain fragment and the pComb3H vector by using a gel recovery kit after enzyme digestion is finished. The strain XL1-B1ue is electrically transformed by being connected with a light chain gene library which is subjected to double enzyme digestion, 1mL of SOC culture medium is rapidly added, the culture is carried out for 1h at 37 ℃ in a shaking way, 10mL of SB culture medium (containing 20mg/L Amp and 10 mg/LTet) is added, the culture is carried out for 1h at 37 ℃ in a shaking way, amp is added to 50mg/L, shaking is continued for 1h, 100mL of SB (containing 50mg/L Amp and 10mg/L Tet) culture medium and 1012pfu of helper phage VCSM13 are added, and the culture is carried out for 2h at 37 ℃ in a shaking way, kana to 70mg/L is added, and the culture is carried out for overnight at 37 ℃ in a shaking way. 40g/L PEG8000 and 30g/L NaC1 precipitate phage are added into the supernatant after centrifugation the next day, the precipitate is suspended in 2mL 10g/LBSA-TBS, and the supernatant is collected by centrifugation to obtain the Fab phage antibody library.

2.3 phage-specific screening

(a) Preparing a 6-hole ELISA plate coated with human hepatitis B virus surface antigen by using 10mg/L of hepatitis B virus surface antigen and 0.05mo1/L of sodium bicarbonate buffer solution, and blocking overnight by using calf serum protein at 4 ℃; (b) The 1m 1/well Fab phage antibody library was added and incubated at 37℃for 2 hours and washed once with PBST (lg/LTwen-20). (first round of washing 1, second round of washing 5, third, fourth, five round of washing 10 times); (c) 1ml of 0.lmo1/LHC1 (pH adjusted to 2.2 with glycine, 1 g/LBSA) was added to each well to elute phage, left to stand at room temperature for 10min, and after gentle blowing, 2mol/LTris was added to neutralize; (d) The freshly prepared E.coli XL1-b1ue was infected with 2m1 of the eluate from the wells and incubated with shaking at 37℃for 2 hours, 1012pfu of helper VCSM13 (no-load phage, helper phage) was added, the incubation was continued with shaking for 1 hour, kanamycin was added to a final concentration of 70mg/L and incubated with shaking at 30℃overnight. Centrifuging and collecting a supernatant; (e) And (3) taking one round from the steps (a) - (d) and carrying out 5 rounds altogether to obtain the high-concentration specific phage and anti-hepatitis B virus surface antigen antibody fusion protein.

2.4 Direct method for detecting and screening antibody

The resulting anti-hepatitis B virus surface antigen antibody fusion protein was subjected to titer detection and compared with a commercially available antibody pair (RA Biosources Co., USA).

The antibody titer is detected by a direct method, the antibody is marked with ABEI, and the antigen is coated with magnetic balls, and the detection method is as follows: 150. Mu.L of sample, 20. Mu.L of magnetic microspheres, 100. Mu.L of Buffer, add to a reaction cup for 20min incubation at 37℃and magnetic field precipitation, remove supernatant, wash the precipitated complex 3 times with wash solution, add 200. Mu.L of Buffer, 100. Mu.L of Buffer for 5min incubation at 37℃and enter a sample measurement chamber to automatically detect its light intensity (RLU).

10 antibodies with good effect were obtained and sequenced, and the sequences were designated as 2D1, 2D4, 2D8, 2D6, 7G5, 6G5, 7G3, 8D5, 9G6, and 6G4, respectively.

3. Purification of high titer antibodies

3.1 construction of insect expression vectors

The in vitro synthesized light chain gene and vector pCHO1.0 (purchased from Ubbelopsis) were digested with AvrII and Bst117I, purified and ligated. Coli JM109 was transformed with the ligation product, the transformed bacteria were uniformly spread on a plate containing resistance, cultured overnight at 37℃and PCR-verified to be positive. The positive plasmid L-pCHO1.0 was obtained.

The heavy chain gene synthesized in vitro and the vector pCHO1.0 (purchased from Youbao organism) were digested with EcoRV and PacI, purified and ligated. Coli JM109 was transformed with the ligation product, the transformed bacteria were uniformly spread on a plate containing resistance, cultured overnight at 37℃and PCR-verified to be positive. The positive plasmid L-H-pCHO1.0 was obtained.

3.2 expression of recombinant proteins

CHO-K1 cells with good growth state were cultured at 1.5X10 ⁶ The cells were inoculated at a volume of 100mL in 500mL shake flasks, placed at 37℃on a 5% CO2 shaker, and incubated at 100rpm for 4 hours. Transfection: the transfection complexes were prepared in the ratio of DNA: transfection reagent=1:3, added drop-wise to the cells prepared above, and gently shaken while adding to allow the complexes to spread out well. After the completion of the dropwise addition, the cap was screwed, and the cells were cultured at 37℃and 5% CO2 at 100 rpm. Transfection was scored as day 0. The following day (i.e., 48h post-transfection) was supplemented with 5% of the medium volume and transferred to 32℃with 5% CO2 at 100 rpm. On the fourth and sixth days, 5% of the medium volume of the feed was added, and the culture conditions were maintained. Samples may be collected on either day eight or day nine depending on the schedule and cell viability.

3.3 purification of recombinant proteins

The recombinant protein is purified by adopting affinity chromatography, and then the protein is further purified by adopting an ion exchange column, so that the purity of the recombinant protein is more than or equal to 95 percent.

Example 2 results and analysis.

1. Screening to obtain antibody titer detection.

The results are shown in Table 1, where the intensity of 2D1, 2D8, 7G5, 6G5, 9G6 is far higher than the intensity of the existing antibody, and the intensity of 2D8 is highest, reaching 7024584.

TABLE 1 detection of titers of antibodies obtained by screening (RLU)

2 screening of paired antibodies

And 5 antibodies with higher titers after affinity purification are selected to be respectively subjected to ABEI labeling and magnetic sphere coating, and are respectively used as detection antibodies and capture antibodies, and a chessboard double-antibody sandwich pairing experiment is adopted to perform primary screening of paired antibodies.

Detecting by adopting a chemiluminescent immune sandwich method, and measuring the concentration of five anti-hepatitis B virus surface antigen antibodies on a protein spectrophotometer; the magnetic bead is coated with one anti-hepatitis B virus surface antigen antibody, and the other anti-hepatitis B virus surface antigen antibody marks the luminous marker ABEI and numbers different coatings or marking solutions.

Adding 150 μl sample into a reaction cup, adding 20 μl of antibody-coated magnetic microsphere solution, adding 100 μl of antibody solution of labeled ABEI, mixing, incubating at 37deg.C for 10min, precipitating the reaction product with an external magnetic field, removing supernatant, washing with buffer solution for 3 times, adding 200 μl of antibody solution of labeled ABEI into the precipitate, mixing, incubating at 37deg.C for 20min, and reacting to obtain double antibody sandwich complex; precipitating the double antibody sandwich complex with an external magnetic field, removing supernatant, washing with buffer solution for 3 times, and adding luminescent substrate (NaOH and H) ₂ O ₂ ) The emitted relative light intensity (RLU) is detected. The higher the intensity of the light, the better the pairing effect, given the same amount of antigen added.

Pairing results as shown in table 2, of the 25 (5×5) antibody pairing combinations, 10 combinations formed a double antibody sandwich pairing, of which there were a total of 3 combinations with better pairing effect: 2D8-7G5, 2D8-6G5, 6G5-7G5.

Table 2 pairing test results of five antibodies.

3 characterization of the paired antibodies

3.1 evaluation of Positive detection Rate of paired antibodies

Taking a commercial antibody pair as a comparative example, collecting 307 serum samples with positive hepatitis B virus surface antigen nucleic acid detection, detecting the samples by using 3 candidate paired antibodies, and determining the positive coincidence rate of the candidate paired antibodies to detect the serum of patients. The results are shown in Table 3.

TABLE 3 detection rate of positive samples of the paired antibodies against hepatitis B surface antigen obtained by screening

The results are shown in Table 3, and for 307 positive samples, all three antibody pairs have good detection rates, with the highest detection rate of the antibody pair 2D8-7G5 being 99.02%.

3.2 evaluation of specificity

Three pairs of antibodies with high positive detection rate are obtained by screening, and the collected 159 serum samples of the hepatitis C patient, 108 serum samples of the syphilis patient and 243 serum samples of the normal person subjected to nucleic acid detection are used for detection, and the results are shown in table 4.

TABLE 4 evaluation of Cross-reactivity for three antibody pairs with high Positive detection Rate

Sample of	Hepatitis C	Syphilis	Serum for normal person
				Number of samples	159	108	243
HbsAb-1-2 negative Rate	98.66%	95.21%	93.47%
				2D8-7G5 negative Rate	99.37%	100%	100%
2D8-6G5 negative Rate	93.71	94.44%	99.59%
				Negative rate of 6G5-7G5	96.23%	96.33%	98.77%

The results show that the positive detection rate, the sensitivity and the negative rate of the cross reaction of the antibodies to the 2D8-7G5 are superior to those of the comparative example and the other two pairs of antibodies.

3.3 sensitivity evaluation

The highest specificity antibody in the experiment is subjected to sensitivity evaluation on 2D8-7G5, and the 2D8-7G5 antibody is used for detecting a national negative serum reference sample tray, wherein all sample types are negative, and the results meet the requirements and are shown in Table 5.

Since the known hepatitis B virus surface antigen has different variant and wild type, the antibody has detection force to the hepatitis B virus surface antigen with different phenotypes, therefore, the experiment performs gradient dilution to the antigen with different phenotypes, performs sensitivity verification to the pair of antibodies with highest positive detection rate and specificity obtained by the screening, and determines the sensitivity of the paired antibody to be detected, and the relative light intensity is higher than 2000 in the following experimental results, and is marked as "+".

For the national reference subtypes and mutants of hepatitis B surface antigen, the different antigens were diluted in gradient to the following concentrations (0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28), and then assayed, with the results shown in Table 6 and Table 7. Experimental results show that the antibody pairs are detected with higher sensitivity aiming at hepatitis B surface antigens with different phenotypes.

TABLE 5 sensitivity to national negative serum references

Sample type	N1	N2	N3	N4	N5	N6	N7	N8	N9	N10
											RLU	1083	1038	1032	815	786	1002	875	1189	1041	1079
Sample type	N11	N12	N13	N14	N15	N16	N17	N18	N19	N20
											RLU	1007	888	892	1108	701	769	997	922	917	980

TABLE 6 sensitivity to national reference subtypes of hepatitis B surface antigen

Antigen (IU/mL)	1.28	0.64	0.32	0.16	0.08	0.04	0.02	0
									adr1 0.05	+	+	+	+	+	+	-	-
adr2 0.1	+	+	+	+	-	-	-	-
									adr3 0.2	+	+	-	-	-	-	-	-
adw1 0.05	+	+	+	+	+	-	-	-
									adw2 0.1	+	+	+	+	-	-	-	-
adw3 0.2	+	+	+	+	+	-	-	-
									ay1 0.1	+	+	+	+	+	-	-	-
ay2 0.2	+	+	+	+	-	-	-	-
									ay3 0.4	+	+	-	-	-	-	-	-

TABLE 7 sensitivity to hepatitis B surface antigen mutants

Antigen (IU/mL)	1.28	0.64	0.32	0.16	0.08	0.04	0.02	0
									T123N	+	+	+	-	-	-	-	-
T123N + T124S	+	+	+	+	+	-	-	-
									P124L-F/Y1543H- D144E-G145R	+	+	+	+	+	-	-	-
I110R-S1171-G119R-T123N	+	+	+	+	-	-	-	-
									122+DT	+	+	+	-	-	-	-	-
122+DT-G145R	+	+	+	+	+	-	-	-
									G145R	+	+	+	+	-	-	-	-
D144A	+	+	+	-	-	-	-	-
									P142L-G145R	+	+	+	-	-	-	-	-
P142S-G145R	+	+	+	-	-	-	-	-

4 antibody stability assay

The antibody stabilizing solutions are prepared according to the formula of the table 8, the antibody 2D8 prepared according to the method is diluted to 1500 pg/mL by the stabilizing solutions, the diluted antibody solution is evenly divided into 7 parts, the solution is placed in a dark place at 40 ℃,1 part of the solution is taken every 5 days according to the concentration of the detected antibody, the results are shown in the table 9, and in order to make the results more visual, a curve of the degradation rate of the antibody 2D8 changing with time is drawn.

Table 8 Diluent composition formulation

TABLE 9 degradation rate Change of antibodies stored in different stabilizers for 30 days

The detection result is shown in FIG. 1 and FIG. 2, and the degradation rate of 2D8 is changed along with timeReflecting the protection effect of the stable diluent on the antibody, compared with the comparative example, the stable liquid A-L in the experiment has a certain protection effect on the antibody 2D 8. Wherein the stabilizing solution L comprises buffer solution pH=7.2, BSA 1.5%, naN ₃ When 0.03%, 0.6% of penicillin and 0.5% of glycerol, the antibody degradation rate is lower, and the stabilizing solution L has good stabilizing effect on the other antibody 7G5, and the degradation rate is only 8.33% after 30 days, so that the stabilizing solution L can be used as a stable preservation solution for two antibodies.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shenzhen New product biomedical engineering Co., ltd

<120> anti-hepatitis B virus surface antigen antibody, antibody pair, detection reagent and kit containing the same

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Ser Ala Ser Ser Ser Val Ser Tyr Leu Tyr

1 5 10

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Asp Thr Ser Asn Leu Ala Ser

1 5

<210> 3

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Gln Gln Trp Ser Thr Tyr Pro Pro Trp Thr

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Asn Tyr Gly Val Asn

1 5

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Ile Ile Trp Ala Asn Arg Thr Asn Tyr Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 6

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

His Asp Asp Asp Gln Met Asp Tyr

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Gln Gln Gly Asn Thr Leu Pro Phe Thr

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Ser Tyr Trp Met His

1 5

<210> 11

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Gln Ile Tyr Pro Gly Ser Gly Thr Thr His Tyr Asp Glu Lys Phe Lys

1 5 10 15

<210> 12

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Ser Gly His Tyr Phe Asp Tyr

1 5

<210> 13

<211> 129

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Leu Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser

50 55 60

Pro Arg Leu Leu Thr Phe Asp Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Thr Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Lys

<210> 14

<211> 134

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Met Ala Gly Leu Gly Leu Leu Phe Cys Leu Val Ala Phe Pro Ser Cys

1 5 10 15

Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Asn Asn Tyr Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Ile Ile Trp Ala Asn Arg Thr Asn Tyr Asn Ser Ala

65 70 75 80

Leu Met Ser Thr Leu Thr Ile Ser Lys Asp Asn Asn Glu Thr Val Leu

85 90 95

Phe Leu Lys Met Asn Ser Leu Gln Ser Asp Asp Thr Ala Met Tyr Phe

100 105 110

Cys Ala Arg His Asp Asp Asp Gln Met Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Ser Val Thr Val Ser Ser

130

<210> 15

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn

100 105 110

Thr Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 16

<211> 135

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Met Gly Trp Ser Cys Ile Met Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Leu Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg His Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Gln Ile Tyr Pro Gly Ser Gly Thr Thr His Tyr Asp

65 70 75 80

Glu Lys Phe Lys Ser Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser

85 90 95

Thr Ala Phe Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Gly His Tyr Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Leu Thr Val Ser Ser

130 135

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 18

<211> 252

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Arg Gly Leu Thr Phe Leu Lys Asn Val Ser Ser Thr Cys Ala Ala Ser

1 5 10 15

Pro Ser Thr Asp Ile Leu Thr Phe Thr Ile Pro Pro Ser Phe Ala Asp

20 25 30

Ile Phe Leu Ser Lys Ser Ala Asn Leu Thr Cys Leu Val Ser Asn Leu

35 40 45

Ala Thr Tyr Glu Thr Leu Asn Ile Ser Trp Ala Ser Gln Ser Gly Glu

50 55 60

Pro Leu Glu Thr Lys Ile Lys Ile Met Glu Ser His Pro Asn Gly Thr

65 70 75 80

Phe Ser Ala Lys Gly Val Ala Ser Val Cys Val Glu Asp Trp Asn Asn

85 90 95

Arg Lys Glu Phe Val Cys Thr Val Thr His Arg Asp Leu Pro Ser Pro

100 105 110

Gln Lys Lys Phe Ile Ser Lys Pro Asn Glu Val His Lys His Pro Pro

115 120 125

Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu

130 135 140

Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser Pro Ala Asp Ile

145 150 155 160

Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro Gln Glu Lys Tyr

165 170 175

Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro Gly Phe Tyr Phe

180 185 190

Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp Asn Ser Gly Glu

195 200 205

Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro His Leu Val Thr

210 215 220

Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val

225 230 235 240

Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr

245 250

<210> 19

<211> 387

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

atggacttcc aagtgcagat cttcagcttc ctgctgatca gcgctagcgt gatcctgagc 60

agaggacaga tcgtgctgac acagagccct gccatcatga gcgctagccc tggcgagaag 120

gtgaccatga cctgcagcgc tagcagcagc gtgagctacc tgtactggta tcagcagaag 180

cctggcagca gccctagact gctgaccttc gacacaagca acctggctag cggcgtgcct 240

gtgagattca gcggcagcgg cagcggcaca agctacagcc tgaccatcag cagaatggag 300

gccgaggacg ccgccaccta ctactgtcag cagtggagca cctaccctcc ttggaccttc 360

ggcggaggca ccaagctgga gatcaag 387

<210> 20

<211> 402

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

atggccggcc tgggcctgct gttctgcctg gtggccttcc ctagctgcgt gctgagccaa 60

gtgcagctga aggagagcgg ccctggcctg gtggccccta gccaaagcct gagcatcacc 120

tgcaccgtga gcggcttcag cctgaacaac tacggcgtga actgggtgag acagcctcct 180

ggcaagggcc tggagtggct gggcatcatc tgggccaaca gaaccaacta caacagcgcc 240

ctgatgagca ccctgaccat cagcaaggac aacaacgaga ccgtgctgtt cctgaagatg 300

aacagcctgc agagcgacga caccgccatg tacttctgcg ctagacacga cgatgatcag 360

atggactact ggggccaagg cacaagcgtg accgtgagca gc 402

<210> 21

<211> 368

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

atgatgagca gcgctcagtt cctgggcctg ctcctgctgt gcttccaagg cacaagatgc 60

gacattcaga tgacacagac cacaagcagc ctgagcgcta gcctgggcga cagagtgacc 120

atcagctgca gagctagcca agacatcagc aactacctga actggtatca gcagaagcct 180

gacggcaccg tgaagctgct gatctactac acaagcagac tgcacagcgg cgtgcctagc 240

agattcaccg gcagcggcag cggaaccgac tacagcctga ccatcagcaa cctggagcaa 300

gaggacatcg ccacctactt ctgtcagcaa ggcaacaccc tgcctttcac cttcggcagc 360

ggcaccaa 368

<210> 22

<211> 405

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

atgggctgga gctgcatcat gctgttcctg gtggccaccg ccaccggcgt gcacagccaa 60

gtgcagctgc agcagcctgg cagcgagctg ctgagacctg gcgctagcgt gaagctgagc 120

tgcaaggcta gcggctacac cttcacaagc tactggatgc actgggtgaa gcagagacac 180

ggccaaggcc tggagtggat cggacagatc taccctggca gcggcaccac ccactacgac 240

gagaagttca agagcaaggg caccctgaca gtggacacaa gcagcagcac cgccttcatg 300

cacctgagca gcctgacaag cgaggacagc gccgtgtact actgcacaag aagcggccac 360

tacttcgact actggggcca aggcaccacc ctgaccgtca gcagc 405

Claims (12)

1. An anti-hepatitis B virus surface antigen antibody 2D8 comprises a light chain variable region and a heavy chain variable region, wherein the CDR-L1 amino acid sequence of the light chain variable region is SEQ ID NO. 1, the CDR-L2 amino acid sequence is SEQ ID NO. 2, the CDR-L3 amino acid sequence is SEQ ID NO. 3, the CDR-H1 amino acid sequence of the heavy chain variable region is SEQ ID NO. 4, the CDR-H2 amino acid sequence is SEQ ID NO. 5, and the amino acid sequence of the CDR-H3 is SEQ ID NO. 6.

2. An anti-hepatitis B virus surface antigen antibody 7G5 comprises a light chain variable region and a heavy chain variable region, wherein the CDR-L1 amino acid sequence of the light chain variable region is SEQ ID NO 7, the CDR-L2 amino acid sequence is SEQ ID NO 8, the CDR-L3 amino acid sequence is SEQ ID NO 9, the CDR-H1 amino acid sequence of the heavy chain variable region is SEQ ID NO 10, the CDR-H2 amino acid sequence is SEQ ID NO 11, and the amino acid sequence of the CDR-H3 is SEQ ID NO 12.

3. The anti-hepatitis b virus surface antigen antibody 2D8 according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID No. 13 and a heavy chain variable region having the amino acid sequence of SEQ ID No. 14.

4. The anti-hepatitis B virus surface antigen antibody 7G5 according to claim 2, comprising a light chain variable region having the amino acid sequence of SEQ ID NO. 15 and a heavy chain variable region having the amino acid sequence of SEQ ID NO. 16.

5. The antibody of any one of claims 1-4, further comprising a light chain constant region having the amino acid sequence of SEQ ID No. 17 and/or a heavy chain constant region having the amino acid sequence of SEQ ID No. 18.

6. A nucleotide molecule encoding the antibody 2D8 of claim 3 or the antibody 7G5 of claim 4, wherein the nucleotide sequence encoding the light chain variable region of the antibody 2D8 is SEQ ID No. 19 and the nucleotide sequence of the heavy chain variable region is SEQ ID No. 20; the nucleotide sequence of the light chain variable region encoding the antibody against the body 7G5 is SEQ ID NO. 21, and the nucleotide sequence of the heavy chain variable region is SEQ ID NO. 22.

7. An anti-hepatitis b virus surface antigen antibody pair comprising the antibody 2D8 of claim 1 or 3 or/and the antibody 7G5 of claim 2 or 4.

8. A hepatitis b virus surface antigen detection reagent comprising the antibody pair of claim 7.

9. The reagent for detecting hepatitis B virus surface antigen according to claim 8, further comprising a stabilizing diluent comprising a buffer, a base component and a protective component.

10. The reagent for detecting hepatitis B virus surface antigen according to claim 9, wherein the buffer solution is phosphate buffer solution, and the basic components comprise bovine serum albumin BSA and sodium azide NaN ₃ The protective component comprises penicillin and glycerol.

11. The reagent for detecting hepatitis b virus surface antigen according to claim 10, wherein the pH of the phosphate buffer solution is 7.2; the BSA content was 1.5 m/v%; the NaN is ₃ The content of (C) is 0.03 m/v%; the penicillin content is 0.6 m/v%; the glycerol content was 0.5 v/v%.

12. A kit for detecting a hepatitis b virus surface antigen, characterized in that the kit comprises the detection reagent according to any one of claims 8 to 11.