An antibody or antigen-binding fragment against the venom of the five-step snake and its application

Abstract

The application discloses an antibody or antigen binding fragment against Agkistrodon acutus venom and application thereof. The antibody or antigen binding fragment against Agkistrodon acutus venom comprises three heavy chain complementarity determining regions which can specifically bind to Agkistrodon acutus venom and the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8; and three light chain complementarity determining regions which the amino acid sequences of which are shown in SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16. The antibody or antigen binding fragment against Agkistrodon acutus venom can specifically bind to Agkistrodon acutus venom and does not bind to Vipera venom, Vipera russelli venom, Naja naja atra venom and Bungarus candidus venom, and can be used for detection of Agkistrodon acutus venom. The monoclonal antibody of the application has neutralizing and protective effects on Agkistrodon acutus venom, and has important significance for assisting clinical treatment, reducing complications and improving survival rate of patients.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to an antibody or antigen-binding fragment against the venom of the five-step snake and its application, specifically to an antibody or antigen-binding fragment against the venom of the five-step snake, a test strip, and its application. Background Technology

[0002] The sharp-nosed viper (Deinagkistrodon acutus), commonly known as the five-step snake, is a medium to large-sized venomous snake belonging to the genus Deinagkistrodon in the family Viperidae. It has a large, distinctly triangular head with an upturned snout and loreal pits. Juveniles have a light brown head and back, a pinkish-brown body, and a light yellow tail tip. It is widely distributed in China. The venom of the five-step snake mainly consists of snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs), C-type lectins (CLECs), 5'-nucleotidases, nucleases, and snake venom phospholipase A2 (SVPLA2). The protein components are particularly high in SVMPs, CLECs, PLA2, SVSPs, and L-amino-acid oxidases (LAAOs).

[0003] China is one of the countries with the richest snake resources, with snakes distributed throughout the entire country. Cases of bites from venomous snakes such as vipers, pit vipers, and cobras are common. The types of venomous snakes vary significantly across provinces and regions, leading to inconsistent levels of treatment for snake bites and a continued high rate of death and disability. Venomous snake bites are acute injuries, with rapid onset and progression of the disease. Severe cases can result in severe poisoning and even death, significantly correlated with the amount of venom injected and the time between the bite and medical attention. Therefore, rapid and accurate venom detection is crucial for assisting clinical treatment, reducing complications, and improving patient survival rates. In the field of snake venom diagnostics, the biggest challenge is distinguishing one snake venom from others. First, snake venom is not a single protein or polypeptide but possesses multiple components; second, the same components in different types of snake venom can interfere with diagnostic results. Therefore, it is essential to detect components specific to pit viper venom. Developing a monoclonal antibody that specifically binds to pit viper venom is key to solving these problems.

[0004] Currently, the detection of snake venom using monoclonal antibody-corresponding hybridoma cells suffers from problems such as low antibody yield, unknown antibody sequences, and easy changes in cell stability leading to loss of positivity. Summary of the Invention

[0005] Firstly, in order to obtain antibodies or antigen-binding fragments against the venom of the five-step snake that differ from existing technologies, and to obtain independently developed neutralizing antibodies or detection reagents specifically binding to the venom of the five-step snake, this invention provides the following technical solutions:

[0006] An antibody against the venom of the five-step snake or an antigen-binding fragment that binds to the venom of the five-step snake, characterized in that it can specifically bind to the venom of the five-step snake and comprises a heavy chain variable region and a light chain variable region:

[0007] c1) The three heavy chain complementarity-determining regions of the heavy chain variable region—HC-CDR1, HC-CDR2, and HC-CDR3—have amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively; and

[0008] c2) The three light chain complementarity-determining regions of the light chain variable region—LC-CDR1, LC-CDR2, and LC-CDR3—have amino acid sequences as shown in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively:

[0009] The antibody against the venom of the five-step snake is a monoclonal antibody;

[0010] The antigen-binding fragment that binds to the venom of the five-step snake is:

[0011] d1) Single-chain Fv; or

[0012] d2) Fv linked by disulfide bonds; or

[0013] d3) Fab fragment; or

[0014] d4)F(ab')2 fragment; or

[0015] d5)Fab' fragment.

[0016] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively.

[0017] In some embodiments, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO:22, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:24.

[0018] In some embodiments, the antibody against the venom of the five-step snake or the antigen-binding fragment that binds to the venom of the five-step snake has a guide peptide with an amino acid sequence as shown in SEQ ID NO:18 and SEQ ID NO:20, located at the N-terminus of its heavy chain variable region and the N-terminus of its light chain variable region, respectively.

[0019] In some embodiments, the antibody against the venom of the five-step snake or the antigen-binding fragment that binds to the venom of the five-step snake is a single-specific, bispecific, or multispecific antibody or antigen-binding fragment.

[0020] Secondly, the present invention also discloses a nucleic acid comprising a nucleic acid fragment encoding an antibody against the venom of the five-step snake as described in the first aspect, or an antigen-binding fragment that binds to the venom of the five-step snake; the nucleic acid fragment includes a heavy chain variable region nucleic acid fragment and a light chain variable region nucleic acid fragment, respectively encoding the heavy chain variable region and the light chain variable region. The heavy chain variable region nucleic acid fragment and the light chain variable region nucleic acid fragment may or may not be connected. When connected, the nucleic acid fragment corresponds to one sequence that encodes both the heavy chain variable region and the light chain variable region; when not connected, the nucleic acid fragment corresponds to two sequences that respectively encode the heavy chain variable region and the light chain variable region.

[0021] In some embodiments, HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 have nucleotide sequences as shown in SEQ ID NO:3, 5, 7, 11, 13, and 15 to respectively encode the antibody against the five-step snake venom or the antigen-binding fragment that binds to the five-step snake venom.

[0022] In some embodiments, the nucleotide sequences are represented as shown in SEQ ID NO:1 (heavy chain variable region) and SEQ ID NO:9 (light chain variable region).

[0023] Thirdly, the present invention also discloses one or more expression vectors, wherein the inserted target gene comprises the nucleic acid described in the second aspect. The expression vector here simultaneously inserts the nucleic acid encoding both the heavy chain variable region and the light chain variable region; or comprises a heavy chain variable region plasmid and a light chain variable region plasmid, respectively inserting the nucleic acid encoding the heavy chain variable region and the nucleic acid encoding the light chain variable region.

[0024] Fourthly, the present invention also discloses a host cell comprising one or more expression vectors as described in the third aspect.

[0025] Fifthly, the present invention also discloses a composition comprising the antibody against the venom of the five-step snake as described in the first aspect or an antigen-binding fragment that binds to the venom of the five-step snake.

[0026] In a sixth aspect, the present invention also discloses a test strip, wherein the detection line is coated with the antibody against the venom of the five-step snake as described in the first aspect or an antigen-binding fragment that binds to the venom of the five-step snake; the test strip can be used for the detection of the venom of the five-step snake.

[0027] In a seventh aspect, the present invention also discloses the use of the antibody against the venom of the five-step snake or the antigen-binding fragment of the five-step snake venom described in the first aspect in the preparation of a drug for treating five-step snake bites.

[0028] This invention discloses an antibody or antigen-binding fragment that can specifically distinguish the venom of the five-step snake (Sinocyclocheilus fragilis) without interference from other types of snake venom. The antibody or antigen-binding fragment, composed of six CDRs (amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16), specifically binds to the venom of the five-step snake and does not bind to the venom of vipers, cobras, or kraits, and can be used for the detection of the five-step snake venom. Test strips prepared using the monoclonal antibody of this invention can rapidly detect and determine whether a patient has been poisoned by the five-step snake. The monoclonal antibody of this invention has a neutralizing and protective effect against the venom of the five-step snake, which is of great significance for adjuvant clinical treatment, reducing complications, and improving patient survival rates.

[0029] This invention uses snake venom, a complex substance, as an antigen to immunize mice, screens for antibodies or antigen-binding fragments with specific CDRs, and demonstrates their specific binding to the venom of the five-step snake, which has significant practical implications. Furthermore, the antibodies of this invention can be prepared using Chinese hamster ovary cells (CHO), effectively solving problems such as low antibody yield, unknown antibody sequences, and easy changes in cell stability leading to positivity rates in hybridoma cells.

[0030] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is the result of the titer assay for the anti-five-step snake venom monoclonal antibody W14B5G5.

[0033] Figure 2 This is the electrophoresis result of the anti-five-step snake venom monoclonal antibody W14B5G5. Where M represents the marker, R represents reducing, and NR represents non-reducing.

[0034] Figure 3 These are the results of plasmid experiments.

[0035] Figure 4 This is the result of a sensitivity test on the rapid test strip for the venom of the five-step snake.

[0036] Figure 5 This is the result of cross-venom detection using the five-step snake venom rapid test strip. Detailed Implementation

[0037] To facilitate understanding by those skilled in the art, some terms appearing in this document are explained and clarified.

[0038] In this document, the singular forms “an,” “an,” and “the” include their plural forms unless the context otherwise requires. Thus, for example, “an agent” can be understood to include multiple agent components.

[0039] In this document, unless otherwise stated, the terms “comprising,” “including,” or “containing” mean that the listed values, steps, or ingredients are included, but do not exclude the inclusion of other values, steps, or ingredients.

[0040] In this document, the terms "individual" or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. A mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.

[0041] In this article, the term "antibody" refers to an immunoglobulin that specifically recognizes and binds to an antigen, encompassing a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

[0042] In this paper, the term "fragment of variable region (Fv)" refers to a domain that recognizes and specifically binds to an antigenic epitope, consisting of a short peptide linking the variable regions of the antibody heavy chain and light chain.

[0043] In this document, the CDR region, or "complementarity-determining region," refers to a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop and / or contains antigen-contacting amino acid residues. The CDR is primarily responsible for the binding of the antibody to the antigen epitope, determining the antibody's specificity. In a given heavy or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering rules, including, for example, Kabat, Contact, AbM, and Chothia. The CDR of the antibodies of this invention can be determined according to any rule or combination thereof in the art.

[0044] Because viper venom and five-step snake venom are complex, multi-component snake venoms, although this invention yielded the monoclonal antibody W14B5G5, it is currently unclear which component of the five-step snake venom this monoclonal antibody specifically detects. However, experiments have shown that this monoclonal antibody can indeed specifically bind to five-step snake venom from various snake venoms. Therefore, in this paper, the six CDRs of the monoclonal antibody W14B5G5 are referred to as "antibodies against five-step snake venom or antigen-binding fragments binding to five-step snake venom." In this paper, "specifically binding to five-step snake venom" does not mean specifically binding to all components of the five-step snake venom, but rather that it can specifically bind to a component of the five-step snake venom, without specifically analyzing which component it targets.

[0045] In this paper, both the light chain variable region (VL) and the heavy chain variable region (VH) include three complementary determinant regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4). The three CDRs of the light chain variable region (VL) are LC-CDR1, LC-CDR2, and LC-CDR3; the three CDRs of the heavy chain variable region (VH) are HC-CDR1, HC-CDR2, and HC-CDR3.

[0046] The anti-five-step snake venom antibody or its antigen-binding fragment of the present invention comprises substitution, insertion, or deletion. The anti-five-step snake venom antibody of the present invention includes modifications to the light chain variable region, the heavy chain variable region, the light chain, or the heavy chain, such that the modified amino acid sequence differs from the amino acid sequence from which the antibody is derived. For example, the amino acid sequence derived from the same specified protein can be similar to the starting sequence, for example, having a certain percentage identity, such as 90%, 92%, 96%, or 98% of the percentage identity with the starting sequence.

[0047] In this invention, "identity" refers to the percentage of bases (or amino acids) that are identical in the two sequences compared during sequence alignment between two peptides or two nucleic acid molecules. This alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as BLASTN and BLASTP.

[0048] The “antibodies and their antigen-binding fragments” applicable to this invention include, but are not limited to, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, deimmunogenic antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies, and epitope-binding fragments of any of the above.

[0049] The Fab fragment (Antigen-binding fragment), also known as the antigen-binding fragment, refers to the antigen-binding region of an antibody, which contains a complete light chain and a CH1 domain of the variable and constant regions of a heavy chain.

[0050] The F(ab')2 segment contains two Fab segments and a hinge region segment, with the heavy chains connected by disulfide bonds. The disulfide bonds between the heavy chains of the F(ab')2 segment break to form two Fab' segments.

[0051] The terms “treatment,” “curing,” “relief,” or “improvement” are used interchangeably herein and refer to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic and / or preventative benefits. As used herein, a therapeutic benefit generally refers to the eradication or reduction of the severity of the underlying condition being treated. Furthermore, a therapeutic benefit is achieved by eradicating, reducing the severity, or decreasing the incidence of one or more physiological symptoms associated with the underlying condition, so that improvement is observed in the animal (although the animal may still suffer from the underlying condition). For preventative benefits, the risk of developing a specific disease in an animal at risk of developing the disease is reduced. As used herein, the term “therapeutic effect” generally includes therapeutic and / or preventative benefits as described above. Preventative effects include delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition, or any combination thereof.

[0052] As used in this application, the terms "subject," "individual," "animal," or "patient" refer to a human or non-human animal, including mammals or primates, that requires diagnosis, prognosis, relief, prevention, and / or treatment of a disease or condition. Mammal subjects include humans, domesticated animals, farm animals, and zoo or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0053] As used in this article, the term "in vivo" generally refers to events that occur inside an animal's body.

[0054] As used herein, the term "in vitro" generally refers to an event that occurs outside of an animal. Examples include in vitro cell function assays or any in vitro animal assay. In vitro assays include cell-based assays that use dead or live cells. In vitro assays also include cell-free assays that do not use intact cells.

[0055] As used herein, the term "administration" refers to the delivery of a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein or fusion protein of the present invention to a subject. Administration can be systemic or local. Administration can be performed using an administration device, such as a syringe. Methods of administration include, but are not limited to, implantation, nasal inhalation, spraying, and injection. Routes of administration include inhalation, intranasal administration, oral administration, intravenous administration, subcutaneous administration, or intramuscular administration.

[0056] The nucleotide sequences shown in this article are arranged from the 5' end to the 3' end; the amino acid sequences are arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0057] In this article, for the sake of brevity, "antibody against five-step snake venom or antigen-binding fragment that binds to five-step snake venom" will be abbreviated as "antibody against five-step snake venom or antigen-binding fragment".

[0058] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0059] Example 1. Obtaining mouse monoclonal antibodies.

[0060] 1.1 Immunization schedule:

[0061] (1) Five-step snake venom (from Shanghai Seren Biotechnology Co., Ltd.) was used as the immunogen (antigen). All Balb / c mice used for immunization (Shanghai Shengchang Biotechnology Co., Ltd.) were healthy purebred mice that were 6-8 weeks old, female and weighed 18-20g.

[0062] (2) First immunization: Mix 20 μg of antigen with 200 μl of complete Freund's adjuvant to prepare a 400 μl solution, which is then injected subcutaneously at multiple sites into Ba1b / c mice. Second-to-fourth immunizations: Two weeks after the previous immunization, mix 20 μg of antigen (200 μl) with 200 μl of incomplete Freund's adjuvant to prepare a 400 μl solution, which is then injected subcutaneously at multiple sites into Ba1b / c mice. Seven days after the fourth immunization, blood is collected from the tail vein of the mice. The antibody titer in the mouse serum is determined using an enzyme-linked immunosorbent assay (ELISA). When the antibody titer reaches 1:1,000,000 or higher, cell fusion is prepared. A booster immunization is performed three days before cell fusion, using 20 μg of antigen to immunize the mice.

[0063] 1.2 Cell Fusion:

[0064] (1) SP2 / 0 mouse myeloma cells were purchased from ATCC, USA. The culture medium was changed the day before fusion to keep the SP2 / 0 myeloma cells in good growth condition.

[0065] (2) Spleen cells: One mouse immunized with five-step snake venom was bled, its neck was severed and it died suddenly. The cells were then soaked in 75% alcohol for 3-4 minutes. The mouse was transferred to a clean bench. The mouse spleen was removed and placed in a cell culture dish. 15 ml of serum-free RPM1640 medium was added, and the cells were gently crushed with a glass rod until there were no tissue clumps and the cells were homogeneous. Then, the mouse spleen cells were washed three times with serum-free RPM1640 medium and counted for later use.

[0066] (3) Take Sp2 / 0 myeloma cells in the logarithmic growth phase, wash them three times with serum-free RPM1640 medium, and count them for later use.

[0067] (4) Mix mouse spleen cells and Sp2 / 0 myeloma cells at a ratio of 10:1, centrifuge at 1500 rpm for 7 min, wash away the supernatant, and prepare for fusion.

[0068] (5) Slowly add 1 ml of PEG (1450) within 1 min and shake gently for 90 s; then add 5 ml of serum-free RPM1640 culture medium within 2.5 min, and finally add 5 ml of serum-free RPM1640 culture medium to stop the reaction. After standing for 5 min, centrifuge at 1280 rpm for 8 min, discard the supernatant, add regular RPM1640 culture medium (containing 10% fetal bovine serum) to prepare a cell suspension.

[0069] (6) The above cell suspension was prepared at 2 x 10⁻⁶ cells per well. 4 200 μl of cells were seeded into each well of a 96-well plate at a density of 100 μL and incubated at 37°C in a 5% CO2 cell culture incubator. After 5 days of culture, the medium was replaced with standard RPM1640 medium containing HAT (25X), and the cells were incubated again at 37°C in a 5% CO2 cell culture incubator. HAT refers to hypoxanthine (H), aminopterin (A), and thymidine (T).

[0070] 1.3 Obtaining single-cell clones using the limiting dilution method

[0071] (1) Screening was performed using the limiting dilution method. The hybridoma cells to be cloned were gently pipetted into the culture wells and counted; the cell density was adjusted to 3-10 cells / ml. A new cell culture plate was prepared, and 100 μl of diluted cells was added to each well. The plate was incubated at 37°C in a 5% CO2 cell culture incubator. The medium was changed on day 7, and then every 2-3 days thereafter. On days 8-9, single cell clones were observed, and the supernatant from the wells was collected for ELISA detection. The ELISA detection method is shown in 1.4.

[0072] (2) The identified cell monoclonal cells are subcloned 2-3 times to ensure that the cell colony comes from the same hybridoma cell.

[0073] 1.4 Identification of positive clones using ELISA

[0074] (1) Antigen coating: The ELISA plates were coated with 0.2 μg / ml solutions of viper venom, five-step snake venom, viper venom, cobra venom, and silver-ringed snake venom. The ELISA plates were washed after coating.

[0075] (2) Blocking: Block the ELISA plate with 2% bovine serum albumin solution. Clean the ELISA plate after blocking.

[0076] (3) Primary antibody incubation: Take the supernatant from the single-cell cloning wells and add it to the ELISA plate. Incubate at 37°C for 90 min. Wash the ELISA plate after incubation.

[0077] (4) Secondary antibody incubation: Add goat anti-mouse secondary antibody HRP (Southernbiotech-USA) to the ELISA plate and incubate at 37℃ for 90 min. Clean the ELISA plate after incubation.

[0078] (5) Color development: Add 3,3',5,5'-tetramethylbenzidine (TMB) single-component color development solution to the ELISA plate and incubate at 37°C for 5 min.

[0079] (6) Termination: Add 50 μl of 2 mol / L sulfuric acid to the ELISA plate.

[0080] (7) OD value detection: The ELISA plate was tested at a wavelength of 450nm.

[0081] (8) Criteria for determining positive wells: For supernatant from the same well, when the antigen being tested is the venom of the five-step snake, the OD value is... 检测孔 / OD value 阴性对照 >2.1; When detecting other snake venoms, the OD value 检测孔 / OD value 阴性对照 <2.1. Then the single-cell clone corresponding to the supernatant is the desired positive hybridoma cell.

[0082] 1.5 Screening Results

[0083] (1) After screening, one monoclonal antibody against the venom of the five-step snake was obtained. The monoclonal antibody against the venom of the five-step snake was named W14B5G5. Figure 1 As shown, the monoclonal antibody W14B5G5 specifically binds to the venom of the five-step snake, but does not bind to other snake venoms.

[0084] Example 2: Ascites fluid preparation and monoclonal antibody purification

[0085] 2.1 Ascites preparation

[0086] (1) Balb / c mice were injected intraperitoneally with 0.3-0.5 ml of phytane or liquid paraffin. After 7-15 days, the selected hybridoma cell lines were expanded and cultured and then inoculated into the peritoneum of Balb / c mice.

[0087] (2) Seven days after the injection of hybridoma cells, the mice began to swell in the abdomen. They were observed daily. When the mice had difficulty eating, difficulty walking, and dull fur, they were euthanized and the ascites was extracted once.

[0088] (3) Mice were euthanized by dislocation of the cervical vertebrae and disinfected by immersion in 75% alcohol for 5 minutes;

[0089] (4) Use surgical scissors to cut a small opening in the mouse's abdomen, peel off the surrounding skin to expose the abdominal cavity, then cut a small opening in the abdominal cavity, insert a dropper into the abdominal cavity to aspirate the accumulated fluid into a clean 15ml centrifuge tube.

[0090] (5) Centrifuge at 3000 rpm for 10 min. The colorless and transparent middle layer is the ascites.

[0091] 2.2 Antibody purification

[0092] (1) The ascites fluid was diluted with PBS pH 7.4, centrifuged and the supernatant was purified by protein G affinity chromatography.

[0093] (2) Equilibration: Equilibrate the purification column with 0.4M PB buffer (pH 7.0);

[0094] (3) Column loading: Slowly pass the diluted ascites supernatant through the column to ensure that the antibody binds better to the protein G column;

[0095] (4) Washing: Wash the column again with equilibration buffer;

[0096] (5) Elution: Elute the antibody bound to the column with 0.1M glycine buffer (pH 2.7) and add 1M Tris-HCl (pH 8.0) to neutralize the glycine, so that the pH is neutral for antibody preservation.

[0097] (6) The purified monoclonal antibody was subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown, the heavy and light chains of the antibody can be clearly seen.

[0098] Example 3 Construction and amplification of recombinant plasmids

[0099] 3.1 Nucleotide sequence sequencing

[0100] (1) Monoclonal hybridoma cells were sent to Shanghai Bio-Tech Co., Ltd. for sequencing to obtain the nucleotide sequences of the antibody heavy and light chains. Table 1 shows the sequences of important regions of the monoclonal antibody.

[0101] Table 1. Nucleotide and amino acid sequences of important regions of monoclonal antibody W14B5G5

[0102]

[0103]

[0104]

[0105] The nucleotide sequence of the heavy chain constant region of W14B5G5 is shown below (SEQ ID NO:21):

[0106] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

[0107] The amino acid sequence (SEQ ID NO:22) of the heavy chain constant region of W14B5G5 is as follows:

[0108] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0109] The nucleotide sequence of the light chain constant region of W14B5G5 is shown below (SEQ ID NO:23):

[0110] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0111] The amino acid sequence (SEQ ID NO:24) of the light chain constant region of W14B5G5 is as follows:

[0112] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0113] 3.2 Construction of expression vector

[0114] (1) Construct appropriate upstream and downstream primers for the heavy and light chains of W14B5G5 respectively, and perform PCR amplification on the target sequence (heavy or light chain of W14B5G5) to obtain PCR products.

[0115] (2) Gel recovery: The PCR products were recovered using a gel recovery kit.

[0116] (3) Double digestion of PCR products to expose restriction enzyme sites. The pcDNA3.1 vector (Shanghai Baiying Biotechnology Co., Ltd.) was double digested to obtain a linearized vector.

[0117] (4) Ligation of recombinant plasmids: Ligate the recombinant plasmids according to the reaction system in Table 2. Recombinant plasmids include heavy chain recombinant plasmids and light chain recombinant plasmids.

[0118] Table 2. Reaction System

[0119]

[0120] Note: The recombinase is from Shanghai Bio-Tech Co., Ltd.

[0121] (5) After constructing the plasmid, let it stand for 2-3 minutes to lower the temperature, and then perform transformation and bacterial coating experiments at 37°C overnight.

[0122] 3.3 Colony Screening Experiment

[0123] (1) Pick a single colony from the overnight plate.

[0124] (2) Colony PCR was performed using BI-CMV-F (SEQ ID NO:25): CGCAAATGGGCGGTAGGCGTG; BI-SEQ-R (SEQ ID NO:26): AGCGTAAAAGGAGCAACATAGT.

[0125] (3) After the colony PCR results are correct, select the corresponding colonies, add them to the culture medium, and incubate overnight.

[0126] 3.4 Plasmid extraction (using the plasmid extraction kit from Tiangen Biotech Co., Ltd.)

[0127] (1) Take 1-5 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible (if there is a lot of bacterial culture, the bacterial precipitate can be collected into a centrifuge tube by multiple centrifugations).

[0128] (2) Add 250 μL of solution I (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose, 100 μg / ml RNase A) to the centrifuge tube containing the bacterial precipitate, and vortex to completely suspend the bacterial cell precipitate.

[0129] (3) Add 250 μL of solution II (250 mM NaOH, 1% SDS (sodium dodecyl sulfate)) to the centrifuge tube and gently invert it 6-8 times to fully lyse the bacteria.

[0130] (4) Add 350 μL of Solution III (3M potassium acetate, 5M acetic acid) to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min, and carefully transfer the supernatant to another clean centrifuge tube with a pipette, trying not to aspirate the precipitate.

[0131] (5) Take the supernatant from the previous step, add 0.6 times the volume of anhydrous ethanol, and mix thoroughly (if the volume is too large, mix in two batches and then load onto the column).

[0132] (6) Add the mixture obtained in the previous step into the adsorption column (insert the adsorption column into the collection tube), place at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0133] (7) Add 750 μL of washing solution to the adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube.

[0134] (8) Add 700 μL of washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube.

[0135] (9) Centrifuge at 12000 rpm for 2 min, and place the adsorption column open at room temperature or in a 50°C incubator for several minutes to remove the residual washing solution in the adsorption column.

[0136] (10) Place the adsorption column into a clean centrifuge tube, and add 50-200 μL of elution buffer (10 mM Tris-HCl, pH = 7.5, 80% ethanol) preheated in a 65°C water bath to the center of the adsorption membrane. Let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 1 min.

[0137] (11) To increase plasmid recovery efficiency, the obtained eluent (10 mM Tris-HCl, pH = 7.5) can be added back to the adsorption column, incubated at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min. The plasmid results are as follows: Figure 3 As shown, it matches the expected size.

[0138] Example 4: Recombinant expression of monoclonal antibodies in cells

[0139] 4.1 Preparation before cell transfection

[0140] (1) Remove the required CHO (Thermo Fisher Scientific) cell tubes from liquid nitrogen and thaw them in a 37°C water bath for 1-2 minutes to allow the cells to thaw quickly. Do not completely immerse the cell tubes in water.

[0141] (2) Transfer the thawed cell suspension to a container containing 30 ml of preheated ExpiCHO. TM In the expression medium, the cells were inoculated into 125 ml polycarbonate disposable sterile, breathable conical flasks and cultured in a shaker at 37°C and 8% CO2 relative humidity ≥80%. After 3-5 days, the CHO cells were cultured to a suitable cell density and then counted.

[0142] 4.2 Cell transfection

[0143] (1) Take 150×10 6 The CHO cell suspension was centrifuged and the supernatant was discarded. The cells were then resuspended in 5 ml of transfection reagent (GenePulseer Electroporation Buffer, #1652676).

[0144] (2) After mixing, add plasmid to a final concentration of 600 ng / μl. Repeatedly pipette the suspension to ensure that the CHO cells and plasmid DNA are mixed evenly.

[0145] (3) Add the mixture to the electric shock tube and place the electric shock tube into the electric shock instrument to prepare for electric shock.

[0146] (4) Set the electrotransfer parameters: voltage 160V; pulse length 15ms; electrotransfer tube width 2mm; pulse number 1.

[0147] (5) Press the “start” button to execute the electric rotation procedure.

[0148] (6) After the electroporation is completed, the cells in the electroporation tube are dispensed into a shake flask containing culture medium and incubated for 40 minutes.

[0149] (7) After incubation, the cells are placed in a shaker at 37°C, 120 rpm, 8% CO2 and relative humidity ≥80% for 48 hours of culture. The monoclonal antibodies in the cell supernatant can be detected.

[0150] 4.3 Antibody Purification - Protein A Affinity Column Purification

[0151] (1) Equilibration chromatography column: 1xPBS, flow rate 1ml / min, 20ml

[0152] (2) Sample loading: flow rate 1 ml / min

[0153] (3) Washing: 1xPBS, flow rate 1ml / min, 20ml

[0154] (4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collected in aliquots, approximately 1000 μl per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument.

[0155] (5) Dialysis: Aspirate the high-concentration protein into a dialysis bag and place it in a beaker containing 1×PBS for dialysis. Dialyze three times.

[0156] Example 5: Preparation and Evaluation of the Detection Performance of a Rapid Test Strip for Five-Step Snake Venom

[0157] 5.1 Probe Preparation

[0158] (1) Add 5 mL of 100 mM 2-(N-morpholino)ethanesulfonic acid buffer to 0.125 mL of latex microsphere solution and mix well. Add a certain amount of N-hydroxysuccinimide (NHS) solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution. Shake at room temperature for 30 min.

[0159] (2) Add a certain amount of horse anti-five-step snake venom polyclonal antibody (Shanghai Seren Biotechnology Co., Ltd.) to the above solution, incubate at room temperature for 2-3 hours, add 1% BSA solution for blocking after incubation, and continue incubation for 2-3 hours.

[0160] (3) Centrifuge the solution at 15000 rpm for 10-30 minutes, discard the supernatant, and reconstitute the precipitate with the preservation solution.

[0161] (4) The above solution is uniformly impregnated into the polyester fiber film and then transferred to a 45°C oven for drying.

[0162] 5.2 Nitrocellulose membrane coating

[0163] (1) Nitrocellulose membranes were coated using an XYZ three-dimensional coating instrument, which was purchased from Shanghai Jinbiao Biotechnology Co., Ltd. The test line (T line) was coated with the recombinant monoclonal antibody W14B5G5 against the venom of the five-step snake, and the control line (C line) was coated with goat anti-equine secondary antibody. The goat anti-equine secondary antibody was purchased from Shanghai Solarbio Co., Ltd.

[0164] 5.3 Test strip assembly

[0165] Assemble the sample pad (glass fiber), nitrocellulose membrane (including T-line and C-line), polyester fiber membrane (including probe), and absorbent paper. Use a strip cutter to cut the large plate into 4mm wide test strips. Place the cut test strips into the lower compartment of the outer packaging casing, then place the upper cover of the casing on top, overlapping and pressing firmly. The sample pad, polyester membrane, nitrocellulose membrane, absorbent paper, and strip cutter were all purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0166] 5.4 Use and Result Interpretation of Rapid Test Strips for Five-Step Snake Venom

[0167] (1) Add 3-4 drops of sample into the well and observe the results after 15 minutes. If both the T line and the C line are visible, the result is positive. If only the C line is visible, the result is negative. The appearance of only the T line or no band is invalid.

[0168] 5.5 Evaluation of Test Strip Detection Performance

[0169] (1) Sensitivity Experiment

[0170] Five-step snake venom solutions with concentrations of 10 ng / ml, 50 ng / ml, and 100 ng / mL were prepared using physiological saline (0.9% NaCl, pH 7.2) as samples. 100 μl of sample was added to each well, and the results were observed after 15 minutes. The test results are as follows: Figure 4 As shown: a test result of 10 ng / mL is weakly positive; test results of 50 ng / mL and 100 ng / mL are moderately positive and strongly positive, respectively; the minimum detection limit of the test strip is 10 ng / mL.

[0171] (2) Cross-disciplinary experiments

[0172] Standard solutions of viper venom, cobra venom, and krait venom (all from Shanghai Seren Biotechnology Co., Ltd.) with a concentration of 500 ng / mL were prepared using physiological saline (0.9% NaCl, pH 7.2). 100 μl of each sample was added to the well, and the results were observed after 15 minutes. Figure 5 As shown, the test results for the other four types of snake venom were all negative.

[0173] Example 6: Evaluation of the protective effect of five-step snake-specific antibodies on animals

[0174] 6.1 Preparation of diluent: Weigh 8.5g of sodium chloride, 4.5g of boric acid, and 0.5g of sodium tetraborate, add water for injection to make up to 1000ml, filter, and sterilize by moist heat at 121℃ for 30 minutes.

[0175] 6.2 Dilution of snake venom

[0176] Five-step snake venom dilution: Accurately pipette the liquid glycerol five-step snake venom stock solution and dilute it with diluent so that the 2LD50 amount (100μg) does not exceed 0.8ml. When mixing with the anti-five-step snake venom monoclonal antibody, add diluent to 2ml.

[0177] 6.3 Mixing:

[0178] (1) Experimental group 2: 1 ml of 2.21 mg / ml anti-five-step snake monoclonal antibody W14B5G5 stock solution, add twice the test amount of five-step snake venom (2LD50), and add diluent to 2 ml.

[0179] (2) Negative control group 2: Take 1 ml of diluent, add 2 LD50 test dose of five-step snake venom, and add diluent to 2 ml.

[0180] (3) The two mixtures were placed in a 37°C water bath for 45 minutes and then injected into mice immediately.

[0181] 6.4 Injection: Four mice from experimental group 2 and negative control group 2 were injected intraperitoneally with 0.4 ml each.

[0182] 6.5 Observation: The experimental mice were observed once daily, and the incidence and mortality were recorded for 48-72 hours. The results showed that in the negative control group 2, all four mice died within 24 hours; in the experimental group 2, only one mouse survived after 72 hours, with a mortality rate of 75%. Compared with the negative control group, this indicates that the monoclonal antibody W14B5G5, which specifically binds to the venom of the five-step snake, has a certain neutralizing protective effect in mice.

[0183] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Modifications and improvements to the present invention are possible without exceeding the concept and scope defined by the claims. Therefore, the content of the embodiments in this specification should not be construed as a limitation of the present invention.

Claims

1. An antibody or antigen-binding fragment that binds to a Five-Paced Pit Viper venom, characterized in that, an antibody or antigen-binding fragment that binds to a Five-Paced Snake venom, c1) the amino acid sequences of three heavy chain complementarity determining regions of the heavy chain variable region, HC-CDR1, HC-CDR2, HC-CDR3, are set forth in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, respectively; and c2) the amino acid sequences of three light chain complementarity determining regions of the light chain variable region, LC-CDR1, LC-CDR2, LC-CDR3, are set forth in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, respectively; the antibody that binds to a Five-Paced Snake venom is a monoclonal antibody; the antigen-binding fragment that binds to a Five-Paced Snake venom is: d1) a single chain Fv; or d2) a disulfide-bonded Fv; or d3) a Fab fragment; or d4) a F(ab’)2 fragment; or d5) a Fab’ fragment.

2. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 1, characterized in that, the amino acid sequences of the heavy chain variable region and the light chain variable region are set forth in SEQ ID NO: 2 and SEQ ID NO: 10, respectively, or have at least 90% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

3. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 2, characterized in that, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 92% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

4. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 3, characterized in that, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 94% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

5. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 4, wherein, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 95% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

6. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 5, wherein, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 96% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

7. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 6, wherein, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 97% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

8. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 7, characterized in that, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 98% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

9. The antibody or antigen-binding fragment thereof that binds to the Five-Paced Pit Viper venom of claim 8, wherein, the amino acid sequences of the heavy chain variable region and the light chain variable region have at least 99% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 10, respectively.

10. The antibody or antigen-binding fragment that binds to the Five-Paced Pit Viper venom of claim 1, wherein, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is set forth in SEQ ID NO: 22, and the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:

24.

11. The antibody or antigen-binding fragment that binds to the Five-Paced Pit Viper venom of claim 1, wherein, is a monospecific antibody or antigen-binding fragment.

12. The antibody or antigen-binding fragment that binds to the Five-Paced Pit Viper venom of claim 1, wherein, is a bispecific antibody or antigen-binding fragment.

13. The antibody or antigen-binding fragment that binds to the Five-Paced Pit Viper venom of claim 1, wherein, is a multispecific antibody or antigen-binding fragment.

14. A nucleic acid, characterized in that, a nucleic acid fragment encoding the antibody or antigen-binding fragment that binds to a Five-Paced Snake venom of any one of claims 1-13.

15. An expression vector, characterized in that, the nucleic acid of claim 14.

16. A host cell characterized in that, the expression vector of claim 15.

17. A composition characterized in that, an antibody or antigen-binding fragment that binds to a Five-Paced Rattlesnake venom according to any one of claims 1-13.

18. A test strip, characterized in that a test strip for the detection of a Five-Paced Rattlesnake venom, the test strip comprising an antibody or antigen-binding fragment that binds to a Five-Paced Rattlesnake venom according to any one of claims 1-13 coated on a detection line;

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