CN113683684A

CN113683684A - Anti-hepatitis B virus surface antigen antibody, antibody pair, detection reagent containing same and kit

Info

Publication number: CN113683684A
Application number: CN202110683093.2A
Authority: CN
Inventors: 李亚丽; 何海华; 周莉; 陈清浦; 杜凯; 李婷华
Original assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Current assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2021-11-23
Anticipated expiration: 2041-06-21
Also published as: CN113683684B

Abstract

The invention discloses an anti-hepatitis B virus surface antigen antibody, an antibody pair, a detection reagent containing the same 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 hepatitis B virus surface antigen, can be specifically combined with the hepatitis B virus surface antigen, 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 antibody pair and a kit.

Background

Hepatitis B Virus (HBV) is a very small virus that belongs to a member of the hepadnavirus (DNA) group. The virus particle is composed of two parts of outer membrane and inner core, the complete HBV particle is a spherical particle with the diameter of 42nm, the outer membrane is 7nm thick, and the particle is composed of protein and membrane lipid. The main infectious agents of hepatitis B are patients and HBV antigen carriers. The 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 virus.

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

With the continuous development of the world medical detection technology, the chemiluminescence method diagnosis detection system obtains better detection effect and quality in the detection of hepatitis B surface antigen, but the high-specificity and high-sensitivity antibody pair applied to the chemiluminescence immune sandwich method is still deficient, so that the production of the high-specificity and high-sensitivity antibody raw material suitable for the chemiluminescence system to detect hepatitis B surface antigen becomes more important.

Disclosure of Invention

The invention mainly aims to provide an anti-hepatitis B virus surface antigen antibody (HBsAb) to solve the problem that the anti-hepatitis B virus surface antigen antibody raw material is deficient in the existing chemiluminescence detection technology of hepatitis B virus surface antigen.

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 having the amino acid sequence of CDR-L1 of SEQ ID NO. 1, the amino acid sequence of CDR-L2 of SEQ ID NO. 2, the amino acid sequence of CDR-L3 of SEQ ID NO. 3, the amino acid sequence of CDR-H1 of the heavy chain variable region of SEQ ID NO. 4, the amino acid sequence of CDR-H2 of SEQ ID NO. 5, and the amino acid sequence of CDR-H3 of SEQ ID NO. 6;

further, an anti-hepatitis B virus surface antigen antibody 7G5 is provided, which 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 CDR-H3 amino acid sequence is SEQ ID NO. 12;

further, 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; 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.

Further, the constant regions of the above-mentioned antibody, including the light chain constant region and/or the heavy chain constant region, are also provided, specifically, the 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 encoding 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 encoding 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 7G 5.

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

according to another aspect of the present invention, there is provided a hepatitis B virus surface antigen detection reagent, further comprising a stabilizing diluent, the stabilizing diluent comprising a buffer solution, a base component and a protective component; preferably, the buffer solution is phosphate buffer solution, and the basic components comprise bovine serum albumin BSA and sodium azide NaN₃Said protective componentIncluding penicillin and glycerol; more preferably, the pH of the phosphate buffer is 7.2; the BSA content was 1.5 m/v%; NaN₃The content of (A) is 0.03 m/v%; the content of penicillin is 0.6 m/v%; the content of glycerol is 0.5 v/v%

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

By applying the technical scheme of the invention, the anti-hepatitis B virus surface antigen antibody can be prepared quickly and efficiently, the problem of antibody deficiency of the hepatitis B virus surface antigen detected by an immune sandwich method in the current chemiluminescence field is solved, and a high-sensitivity and high-specificity raw material is provided for detecting the hepatitis B virus surface antigen.

Drawings

FIG. 1 is a graph of the degradation rate of antibody 2D8 over time in comparative example buffers and stabilizing solutions A-F provided in Table 7 of example 2;

FIG. 2 is a graph of the degradation rate of antibody 2D8 over time in comparative example buffer and stabilization solutions J-L provided in Table 7 of example 2.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

The invention can quickly and efficiently screen out the anti-hepatitis B surface antigen antibody by utilizing the phage surface display technology, and solves the problems of high sensitivity and deficient raw material of specific antibody in the traditional hepatitis B surface antigen detection.

Except where expressly indicated to the contrary, the practice of the present invention 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. The embodiments of the present description may be applied to other embodiments mutatis mutandis, 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 exhibits an extremely low cross-over rate, and superior performance compared to commercially available antibody pairs.

As is well known in the art, an antibody is an immunoglobulin molecule that is capable of specific 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 a portion that binds an antibody with an antigen-binding fragment of a desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or a fragment of a desired specificity.

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

In some embodiments, the antibodies and antigen-binding fragments thereof described herein comprise heavy and light chain CDR sets embedded between heavy and light chain framework regions, respectively, that provide support for the CDRs and determine the spatial relationship of the CDRs to each other. As used herein, the term "CDR regions" refers to the three hypervariable regions of a heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are denoted as "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen binding site includes six CDRs, including CDRs located in each of the heavy and light chain V regions.

The embodiment of the invention relates to a method for directly obtaining genes from peripheral blood of a hepatitis B virus patient by utilizing a phage display method and obtaining an antibody with high titer by using a molecular cloning technology. The term "phage display technology" (PDT) is a novel technique for screening specific proteins or polypeptides by fusion of foreign proteins or polypeptides to phage coat proteins, display on the surface of phage while maintaining a specific spatial conformation, and using specific affinity. Smith, G.P in 1985 proposed the insertion of a foreign gene into a modified coat protein gene of a bacteriophage to express a fusion protein containing a foreign protein or polypeptide, and such a bacteriophage with the fusion protein is called a fusion bacteriophage. Through repeated adsorption-elution-amplification processes, phage containing exogenous protein capable of being specifically combined with target protein is screened from phage library expressing various exogenous proteins, and then enrichment, amplification and gene sequence determination are carried out to deduce amino acid composition of the exogenous protein.

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

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

In another exemplary embodiment of the present application, 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 the amino acid sequence of CDRL1 of SEQ ID NO. 7, the amino acid sequence of CDRL2 of SEQ ID NO. 8, the amino acid sequence of CDRL3 of SEQ ID NO. 9, the heavy chain variable region has the amino acid sequence of CDRH1 of SEQ ID NO. 10, the amino acid sequence of CDRH2 of SEQ ID NO. 11, and the amino acid sequence of CDRH3 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 regions, the sequences of immunoglobulin constant regions are conserved sequences that are responsible for binding to a variety of native proteins to elicit specific physiological functions. In certain preferred embodiments, the antibody or antigen-binding fragment thereof may further comprise a constant region whose amino acid sequence may be searched by NCBI or may be obtained 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, there is provided a nucleotide molecule encoding anti-hepatitis B virus surface antigen antibody 2D8 or 7G5, wherein the nucleotide sequence of the light chain variable region encoding 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 of the encoding 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 synthetically produced or any combination thereof.

In another exemplary embodiment of the present application, there is provided a method for screening an anti-hepatitis b virus surface antigen antibody, comprising the steps of: a) using peripheral blood of hepatitis B patient to obtain total mRNA, reverse transcribing to obtain cDNA and amplifying antibody variable region segment; b) constructing a phage display antibody expression library; c) screening the library by using HBsAg to obtain the antibody or antibody fragment.

Further, a method for preparing the screened antibody is provided, and the specific preparation steps are as follows: comprising a) carrying out gene recombination on the anti-hepatitis B virus surface antigen antibody or antibody fragment gene 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, the present application provides an anti-hepatitis b virus surface antigen antibody pair comprising antibody 2D8 or/and antibody 7G 5.

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 stable diluent, wherein the stable diluent comprises a buffer solution, a base component and a protective component; preferably, the buffer solution is phosphate buffer solution, and the basic components comprise bovine serum albumin BSA and sodium azide NaN₃The protective component comprises penicillin and glycerol; more preferably, the pH of the phosphate buffer is 7.2; the BSA content was 1.5 m/v%; NaN₃The content of (A) is 0.03 m/v%; the content of penicillin is 0.6 m/v%; the content of glycerol was 0.5 v/v%.

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

The advantageous effects of the present application will be further described with reference to specific examples. In the following examples and comparative examples, reagents and consumables used in the examples are commercially available products unless otherwise specified.

Example 1: materials and methods

1. Experimental materials and instruments

1.1 materials

HBsAg antigen, source: china institute for testing biological products;

anti-HBsAg monoclonal antibody 1 (HbsAb-1), origin: RA Biosources, USA; anti-HBsAg monoclonal antibody 2 (HbsAb-2), origin: RA Biosources, USA;

magnetic microspheres, ABEI: shenzhen new industry biomedicine shares, produced by Limited.

1.2. Instrument for measuring the position of a moving object

The full-automatic chemical spectroscopic analyzer Maglumi 2000Plus developed and produced by Shenzhen City New Productivity biomedical engineering shares, serial number: 2000200068.

preparation of anti-hepatitis B virus surface antigen antibody

2.1 acquisition of hepatitis B Virus antigens

Extraction of total mRNA from hepatitis B patients: extracting mRNA from peripheral blood of hepatitis B patient; directly using 200uL of fresh blood, adding 20 uLProteineASeK solution, and uniformly mixing; adding 200uL buffer solution GB, fully reversing and uniformly mixing, standing at 70 ℃ for 10Min to enable the solution to become clear, and centrifuging to remove water drops on the inner wall of the tube cover; adding 200uL of absolute ethyl alcohol, fully shaking 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 a buffer solution GD into the adsorption column, and centrifuging to remove 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 air-drying the residual rinsing liquid in the adsorption column; adding elution buffer solution, and centrifuging to obtain total hepatitis B mRNA.

RT-PCR: to a 0.5ml microcentrifuge tube, 1-5. mu.g of total RNA was added, supplemented with appropriate amounts of DEPC H2O to make a total volume of 11. mu.l. Adding 12-181 μ l of 10 μ M Oligo (dT) into the tube, gently mixing, and centrifuging; heating at 70 deg.C for 10min, immediately inserting the microcentrifuge tube into ice bath for at least 1 min; then a mixture of 10 XPCR buffer, 2. mu.l was added; 25mM MgCl2, 2. mu.l; 10mM dNTPmix, 1. mu.l; 0.1M DTT, 2. mu.l gently mixed and centrifuged. Incubating at 42 deg.C for 2-5 min; adding 1 μ l Superscript II, and incubating in 42 deg.C water bath for 50 min; heating at 70 deg.C for 15min to terminate the reaction; the tube was inserted into ice, RNase H1. mu.l was added, and incubation at 37 ℃ for 20min degraded residual RNA. Storing at-20 deg.C for use.

After the PCR reaction, all PCR products were subjected to 1% agarose gel electrophoresis, and the gel containing the target fragment was excised and recovered with a gel recovery kit.

2.2 construction of phage vectors

The purified light chain gene PCR product and the pComb3H vector are purified and recovered after being respectively digested by SacI/XbaI double enzymes, and the recovered product is connected by T4 DNA ligase; after the enzyme digestion is finished, the light chain fragment and the enzyme digestion product of the pComb3H vector are directly purified and recovered by using a gel recovery kit. After ligation with the light chain gene library which had been similarly double-digested, XL1-B1ue strain was electrotransformed, 1mL SOC medium was added rapidly, shaking culture was carried out at 37 ℃ for 1h, 10mL SB medium (containing 20mg/L Amp and 10mg/LTet) was added, shaking culture was carried out at 37 ℃ for 1h, then Amp was supplemented to 50mg/L, shaking culture was continued for 1h, 100mL SB (containing 50mg/L Amp and 10mg/L Tet) medium and 1012pfu helper phage VCSM13 were added, shaking culture was carried out at 37 ℃ for 2h, Kana was added to 70mg/L, and shaking culture was carried out at 37 ℃ overnight. The next day, 40g/L PEG8000 and 30g/L NaC1 were added to the centrifuged supernatant to precipitate phage, the precipitate was suspended in 2mL of 10g/LBSA-TBS, and the supernatant was collected by centrifugation as Fab phage antibody library.

2.3 phage specificity screening

(a) Preparing a 6-hole enzyme label plate coated by the human hepatitis B virus surface antigen by using 10mg/L hepatitis B virus surface antigen and 0.05mo1/L sodium bicarbonate buffer solution, and sealing the 6-hole enzyme label plate by using calf serum protein at 4 ℃ for overnight; (b) add 1m 1/well Fab phage antibody library, incubate 2 hours at 37 ℃ and wash once with PBST (lg/LTween-20). (the first round of washing is 1 time, the second round of washing is 5 times, and the third, fourth and fifth rounds of washing are 10 times); (c) adding 1ml of 0.lmo1/LHC1 (pH value is adjusted to 2.2 by glycine and containing 1g/LBSA) into each well to elute phage, standing for 10min at room temperature, and neutralizing by adding 2mol/LTris after slightly blowing; (d) 2m1 of freshly prepared E.coli XL1-b1ue were infected with the eluate from the wells, incubated at 37 ℃ for 2 hours with shaking, 1012pfu of the helper phage VCSM13 (unloaded phage, helper phage) was added, the incubation with shaking was continued for 1 hour, kanamycin was added to a final concentration of 70mg/L, and the incubation with shaking was carried out at 30 ℃ overnight. Centrifuging and collecting supernatant; (e) and (d) performing 5 rounds in total from the step (a) to the step (d) 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 antibodies

The titer of the anti-hepatitis B virus surface antigen antibody fusion protein was measured and compared with a commercially available antibody pair (RA Biosources, USA).

The direct method is adopted to detect the titer of the antibody, the antibody marks ABEI, and the antigen coats the magnetic ball, and the detection method comprises the following steps: adding 150 mu L of sample, 20uL of magnetic microspheres and 100 mu L of Buffer into a reaction cup, incubating for 20min at 37 ℃, applying a magnetic field for precipitation, removing supernatant, washing a precipitation complex for 3 times by using washing liquor, adding 200uLABEI, 100 mu L of Buffer, incubating for 5min at 37 ℃, entering a sample measuring chamber, and automatically detecting the light intensity (RLU).

10 effective antibodies were obtained and sequenced, and named 2D1, 2D4, 2D8, 2D6, 7G5, 6G5, 7G3, 8D5, 9G6, and 6G4, respectively.

Purification of high potency antibodies

3.1 construction of insect expression vectors

The in vitro synthesized light chain gene and the vector pCHO1.0 (from Youbao) were digested with AvrII and Bst117I, purified and ligated. The ligation product was transformed into Escherichia coli JM109, the transformed strain was spread evenly on a plate containing resistance, cultured overnight at 37 ℃ and verified for positive strain by PCR. The positive plasmid L-pCHO1.0 was obtained.

The in vitro synthesized heavy chain gene and the vector pCHO1.0 (purchased from Youbao) are digested with EcoRV and PacI, purified and ligated. The ligation product was transformed into Escherichia coli JM109, the transformed strain was spread evenly on a plate containing resistance, cultured overnight at 37 ℃ and verified for positive strain by PCR. The positive plasmid L-H-pCHO1.0 was obtained.

3.2 expression of recombinant proteins

CHO-K1 cells in good growth state were grown at 1.5X 10⁶one/mL of the cells were inoculated into a 500mL shake flask with a total cell volume of 100mL, incubated at 37 ℃ on a 5% CO2 shaker at 100rpm for 4 hours. Transfection: the transfection complex was prepared at a ratio of DNA to transfection reagent =1:3, and added dropwise to the above prepared cells while gently shaking the cells to sufficiently disperse the complex. After the addition, the cap was screwed down, and the cells were cultured at 37 ℃ under 5% CO2 at 100 rpm. The day of transfection was recorded as day 0. The next day (i.e., 48h post-transfection culture) was supplemented with 5% medium volume and transferred to culture at 32 ℃ under 5% CO2 at 100 rpm. On the fourth and sixth days, 5% of the medium volume was fed with the medium, and the culture conditions were maintained. Depending on the timing and cell viability, samples may be collected on day eight or day nine.

3.3 purification of recombinant proteins

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

Example 2 results and analysis

1. Screening to obtain antibody titer detection

As shown in Table 1, the light intensity of 2D1, 2D8, 7G5, 6G5 and 9G6 was much higher than that of the existing antibody, and the light intensity of 2D8 was the highest and reached 7024584.

TABLE 1 potency assay (RLU) for the antibodies screened

2 screening of paired antibodies

And selecting 5 antibodies with higher titer after affinity purification to respectively carry out ABEI marking and magnetic sphere coating, respectively taking the antibodies as a detection antibody and a capture antibody, and carrying out primary screening on paired antibodies by adopting a chessboard double-antibody sandwich pairing experiment.

Detecting by adopting a chemiluminescence immune sandwich method, and measuring the concentration of five anti-hepatitis B virus surface antigen antibodies on a protein spectrophotometer; the magnetic beads are used for coating one strain of anti-hepatitis B virus surface antigen antibody, the other strain of anti-hepatitis B virus surface antigen antibody is used for marking a luminescent marker ABEI, and different coating or marking solutions are numbered.

Adding 150 mu l of sample into a reaction cup, then adding 20 mu l of magnetic microsphere solution for coating the antibody, simultaneously adding 100 mu l of antibody solution for marking ABEI, uniformly mixing, carrying out warm bath at 37 ℃ for 10min, adding a magnetic field to precipitate the reaction product, removing supernatant, washing for 3 times by using buffer solution, adding 200 mu l of antibody solution for marking ABEI into the precipitate, uniformly mixing, carrying out warm bath at 37 ℃ for 20min, and fully reacting to form a double-antibody sandwich compound; 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)₂O₂) The relative light intensity (RLU) emitted is detected. Under the condition of adding the same amount of antigen, the higher the light intensity is, the better the pairing effect is.

As shown in table 2, of the 25 (5 × 5) antibody pairing combinations, 10 combinations can form a double antibody sandwich pairing, and of these, there are 3 combinations in total with better pairing effect: 2D8-7G5, 2D8-6G5, 6G5-7G 5.

TABLE 2 results of the paired detection of the five antibodies

3 characterization of the mateable antibodies

3.1 evaluation of Positive detection Rate of paired antibody

And (3) collecting 307 serum samples with positive hepatitis B virus surface antigen nucleic acid detection by taking a commercially available antibody pair as a comparative example, detecting the samples by using 3 candidate paired antibodies, and determining the positive coincidence rate of the serum of the patient to be detected by the paired antibodies. The results are shown in Table 3.

Table 3 shows the detection rate of the paired antibodies against positive hepatitis B surface antigen samples

The results are shown in table 3, and the detection rate of all three antibody pairs is better for 307 positive samples, wherein the detection rate of the antibody pair 2D8-7G5 is the highest and is 99.02%.

3.2 evaluation of specificity

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

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

Sample(s)	Hepatitis C	Syphilis	Normal human serum
				Number of samples	159	108	243
Negative rate of HbsAb-1-2	98.66%	95.21%	93.47%
				2D8-7G5 negative rate	99.37%	100%	100%
2D8-6G5 negative rate	93.71	94.44%	99.59%
				6G5-7G5 negative rate	96.23%	96.33%	98.77%

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

3.3 evaluation of sensitivity

The antibody pair with the highest specificity in the experiment is subjected to sensitivity evaluation on 2D8-7G5, and the antibody pair 2D8-7G5 is used for detection aiming at a national negative serum reference sample plate, so that all sample types are negative and meet the requirements, and the results are shown in Table 5.

Because the known hepatitis B virus surface antigen has different variants and wild types, the antibody has the detection capacity for the hepatitis B virus surface antigen with different phenotypes, therefore, the experiment carries out gradient dilution on the antigens with different phenotypes, carries out sensitivity verification on a pair of antibodies with the highest positive detection rate and specificity obtained by screening, and determines the sensitivity of the paired antibodies to be detected, and in the following experiment results, the detection is carried out when the relative light intensity is higher than 2000, and the mark is plus.

For the national reference subtypes and mutants of hepatitis B surface antigen, different antigens were diluted in a gradient to the following concentrations (0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28), and the results were shown in Table 6 and Table 7. The experimental result shows 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 reference

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 subtype for 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

4 antibody stability assay

Respectively preparing antibody stabilizing solutions according to formulas in a table 8, preparing the obtained antibody 2D8 according to the method, respectively diluting the antibody 2D8 to 1500 pg/mL by using the stabilizing solutions in the table, averagely dividing the diluted antibody solution into 7 parts, placing the parts in a dark place at 40 ℃, taking 1 part every 5 days according to the detected antibody concentration, and drawing a curve of the degradation rate of the antibody 2D8 along with the change of time so as to enable the result to be more visual as shown in the table 9.

TABLE 8 Diluent component formulations

TABLE 9 degradation Rate Change of different stabilizing solutions for 30 days

The detection results are shown in fig. 1 and fig. 2, and the change curve of the degradation rate of 2D8 with time reflects the protective effect of the stable diluent on the antibody, so that the stable solutions a-L in the experiment have a certain protective effect on the antibody 2D8 compared with the comparative example. Wherein the stabilizing solution L contains NaN when the pH of the buffer solution is =7.2, BSA is 1.5 percent₃0.03 percent, 0.6 percent of penicillin and 0.5 percent of glycerol, the degradation rate of the antibody is lower, and the stable liquid L has good stabilizing effect on another antibody 7G5 and has the degradation rate of only 8.33 percent after 30 days, so the stable liquid L can be used as a stable preservation liquid for two antibodies.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement 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 City New industry biomedical engineering shares Limited

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

<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

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

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

3. an anti-hepatitis B virus surface antigen antibody 2D8, comprising light chain variable region and heavy chain variable region, the amino acid sequence of its light chain variable region is SEQ ID NO: 13, the amino acid sequence of its heavy chain variable region is SEQ ID NO:14.

4. an anti-hepatitis B virus surface antigen antibody 7G5, comprising light chain variable region and heavy chain variable region, the amino acid sequence of its light chain variable region is SEQ ID NO: 15, the amino acid sequence of its heavy chain variable region is SEQ ID NO:16.

5. The antibody according to any one of claims 1-4, further comprising a light chain constant region and/or a heavy chain constant region, the amino acid sequence of the light chain constant region is SEQ ID NO: 17, the heavy chain The amino acid sequence of the constant region is 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, 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 7G5 is SEQ ID NO: 21, the heavy chain variable region The nucleotide sequence of the region is SEQ ID NO:22.

7. An anti-HBV 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, wherein the detection reagent comprises the antibody pair of claim 7.

9. a kind of hepatitis B virus surface antigen detection reagent according to claim 8, is characterized in that, also comprises stable diluent, and described stable diluent comprises buffer solution, basic component and protection component; Preferably, described The buffer is a phosphate buffer, the basic components include bovine serum albumin BSA and sodium azide NaN ₃ , and the protective components include penicillin and glycerol; more preferably, the pH of the phosphate buffer is 7.2; BSA The content of NaN 3 was 1.5 m/v %; the content of NaN ₃ was 0.03 m/v %; the content of penicillin was 0.6 m/v %; the content of glycerol was 0.5 v/v %.

10. A hepatitis B virus surface antigen detection kit, wherein the kit comprises the detection reagent according to any one of claims 8 or 9.