Recombinant rabbit monoclonal antibody against saxitoxin and application thereof

Abstract

The present application relates to a kind of anti-saxitoxin recombinant rabbit monoclonal antibody and its application, the amino acid sequence of the anti-saxitoxin recombinant rabbit monoclonal antibody includes heavy chain variable region and light chain variable region, respectively as shown in SEQ ID NO:1 And 10 shown in formula.The high sensitivity of it, the lowest saxitoxin can reach 7.3 pg mL ‑1 IC 50 , linear range is 0.0019 ng L ‑1 0.0282 ng L ‑1 ;Meanwhile, it also has high specificity, and other paralytic shellfish toxins and pufferfish toxin are not cross-reacted.The anti-saxitoxin recombinant rabbit monoclonal antibody prepared in the present application, compared with the anti-saxitoxin mouse monoclonal antibody on sale, the antibody provided in the present application is high in detection sensitivity, high in specificity, good in affinity, high in accuracy;It is suitable for immunological analysis kit, immunological test strip, immunoblotting and other clinical and scientific research applications.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a recombinant rabbit monoclonal antibody against scleroderma toxin and its application. Background Technology

[0002] Saxitoxin (STX) is a paralytic shellfish toxin (PSP) and one of the most toxic marine biotoxins, capable of causing numbness of organs, muscle paralysis, respiratory distress, and even death in humans. STX is biosynthesized by toxic dinoflagellates and accumulates in shellfish through the food chain. Given its high toxicity, widespread distribution, food chain accumulation, and potential for secondary contamination, STX is a mandatory testing item for aquatic product safety in countries worldwide. The permissible limit for STX in shellfish products is 80 μg / 100g (equivalent to 400 MU / 100g; 1 MU represents the average amount of toxin that kills a 20g mouse within 15 minutes), and the health warning line for STX and its analogues in drinking water is 3 μg / L. -1 .

[0003] Currently, STX detection methods mainly include reliable and widely used biological assays and highly accurate and sensitive instrumental assays, but obtaining standards for instrumental assays is relatively difficult. In contrast, immunoassays based on antigen-antibody reactions are highly specific, rapid, sensitive, and have high throughput, making them very suitable for widespread application in testing institutions at all levels. Monoclonal antibodies are the core reagents in immunoassays and are key to determining the sensitivity, analysis time, accuracy, and precision of immunoassay methods. Currently, most reported STX immunoassay methods use rabbit polyclonal antibodies and mouse monoclonal antibodies; high-affinity recombinant rabbit monoclonal antibodies have not yet been reported. Common mouse monoclonal antibodies are usually obtained by using the traditional formaldehyde condensation method to conjugate STX with carrier proteins such as bovine serum albumin, ovalbumin, and hemocyanin as immunogens, followed by screening using hybridoma technology. However, possibly due to the unique molecular structure of STX toxin, the immunogenicity of mice immunized with STX and its analogues is usually weak, requiring a large number of mice to be immunized to obtain the most potent antibodies for fusion with hybridoma cells. In summary, current immunoassays for detecting schizotoxins from marine organisms face challenges due to the diverse range of marine biotoxins, and existing antibodies often fall short of meeting practical application requirements in terms of specificity and sensitivity. Rabbit monoclonal antibodies, as recombinant antibodies, offer several advantages, including high batch-to-batch consistency, excellent specificity, ease of mass production, long-term supply, and high-throughput in vitro production, and hold promise for overcoming this bottleneck.

[0004] Regarding recombinant rabbit monoclonal antibodies against salicornin, Chinese patent applications disclose artificial antigens and antibodies against salicornin, their preparation methods, and applications, and the artificial antigens are used to prepare polyclonal antibodies. One application discloses a mouse monoclonal antibody against salicornin and its preparation method, generated using hybridoma technology, but does not disclose the rabbit monoclonal antibody or its CDR sequence. Another Chinese patent application discloses a competitive enzyme-linked immunosorbent assay (ELISA) kit for the detection of paralytic shellfish venom, which includes mouse monoclonal antibodies but does not involve rabbit monoclonal antibodies.

[0005] Regarding methods for detecting salivarius toxin, Chinese patent applications all involve colloidal gold test strips for rapid detection of paralytic shellfish venom and their preparation methods; Chinese patent applications disclose a method for rapid screening of unknown chemical hazard residues in aquatic products based on a high-resolution mass spectrometry database; Chinese patent applications disclose detection methods and applications for salivarius toxin; Chinese patent applications disclose microfluidic chips for rapid detection of salivarius toxin and their preparation methods; Chinese patent applications disclose a method for preparing and applying molecularly imprinted salivarius toxin microspheres; Chinese patent applications disclose a high-throughput detection method for marine biotoxins in aquatic products; and Chinese patent applications disclose a multi-color visualization method for rapid detection of salivarius toxin in seafood, etc., but none of them involve recombinant rabbit monoclonal antibodies against salivarius toxin. Summary of the Invention

[0006] To address the problems existing in obtaining antibodies using current technologies, this invention provides a recombinant rabbit monoclonal antibody against scleroderma toxin and its applications, which is easy to prepare, has high production capacity, and high sensitivity.

[0007] The first objective of this invention is to provide a recombinant rabbit monoclonal antibody against sclerotoxin.

[0008] The second objective of this invention is to provide a gene encoding a recombinant rabbit monoclonal antibody against sclerotoxin.

[0009] A third objective of this invention is to provide a recombinant vector.

[0010] The fourth objective of this invention is to provide a kit for detecting salicornin.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0012] A recombinant rabbit monoclonal antibody against scleroderma toxin includes a variable region and a constant region. The variable region includes a heavy chain variable region (VH) and a light chain variable region (VL). Each of the heavy chain variable region and the light chain variable region includes a frame region (FR) and a complementarity-determining region (CDR).

[0013] The CDRs of the heavy chain variable region include CDR-H1, CDR-H2, and CDR-H3.

[0014] The amino acid sequence of CDR-H1 is shown in SEQ ID NO:2;

[0015] The amino acid sequence of CDR-H2 is shown in SEQ ID NO:3;

[0016] The amino acid sequence of CDR-H3 is shown in SEQ ID NO:4;

[0017] The CDRs of the light chain variable region include CDR-L1, CDR-L2, and CDR-L3.

[0018] The amino acid sequence of CDR-L1 is shown in SEQ ID NO:11;

[0019] The amino acid sequence of CDR-L2 is EAS;

[0020] The amino acid sequence of CDR-L3 is shown in SEQ ID NO:12.

[0021] Preferably, the FRs in the heavy chain variable region include FR-H1, FR-H2, FR-H3, and FR-H4.

[0022] The amino acid sequence of FR-H1 is shown in SEQ ID NO:5;

[0023] The amino acid sequence of FR-H2 is shown in SEQ ID NO:6;

[0024] The amino acid sequence of FR-H3 is shown in SEQ ID NO:7;

[0025] The amino acid sequence of FR-H4 is shown in SEQ ID NO:8;

[0026] The FRs in the light chain variable region include FR-L1, FR-L2, FR-L3, and FR-L4.

[0027] The amino acid sequence of FR-L1 is shown in SEQ ID NO:13;

[0028] The amino acid sequence of FR-L2 is shown in SEQ ID NO:14;

[0029] The amino acid sequence of FR-L3 is shown in SEQ ID NO:15;

[0030] The amino acid sequence of FR-L4 is shown in SEQ ID NO:16.

[0031] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1.

[0032] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:10.

[0033] The present invention also claims protection for a gene encoding a recombinant rabbit monoclonal antibody against sclerotoxin, encoding any of the aforementioned recombinant rabbit monoclonal antibodies against sclerotoxin.

[0034] The present invention also claims a recombinant vector recombinantly containing the coding gene or expressing any of the recombinant rabbit monoclonal antibodies against sclerotoxin.

[0035] Preferably, the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:9.

[0036] Preferably, the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:17.

[0037] The present invention also claims a kit for detecting salicornin, containing any of the described recombinant rabbit monoclonal antibodies against salicornin.

[0038] Preferably, it further comprises a solid-phase carrier coated with a coating agent, wherein the coating agent is Gyrotoxin (GTX) conjugated with bovine serum albumin.

[0039] The genus *Gymnoxin* is an equimolar mixture of GTX 1, GTX 2, GTX 3, and GTX 4 in a 1:1:1:1 ratio.

[0040] Preferably, it also contains one or more of the following: enzyme-labeled secondary antibody, salicornin standard, TMB chromogenic solution, stop solution, washing solution, and PBST washing solution.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] A recombinant rabbit monoclonal antibody against sclerotoxin was prepared for the first time. The antibody prepared by this invention can achieve a sensitivity of pg / mL for detecting STX. -1 Furthermore, it exhibits no significant cross-reactivity with other paralytic shellfish toxins, tetrodotoxins, etc., demonstrating excellent specificity. Compared to commercially available mouse monoclonal antibodies against salicornin, the recombinant rabbit monoclonal antibody prepared in this invention offers advantages in high detection sensitivity, strong specificity, good affinity, and high accuracy; it is suitable for clinical and research applications such as immunoassay kits, immunoassay strips, and immunoblotting. In addition, as a recombinant rabbit monoclonal antibody, it exhibits high batch-to-batch consistency, high-throughput in vitro expression, and high sensitivity, enabling the establishment of a sensitive and stable STX immunoassay method. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the STX immunogen and coating agent in Embodiment 1 of the present invention; where A is the STX immunogen and B is the STX coating agent;

[0044] Figure 2 The following are the ultraviolet spectra of the STX immunogen and the coated immunogen in Example 1 of the present invention; wherein A is the ultraviolet spectrum of the STX immunogen and B is the ultraviolet spectrum of the STX coated immunogen.

[0045] Figure 3 This is a flow cytometry screening result of specific antibody-secreting cells in Example 1 of the present invention;

[0046] Figure 4 Agarose gel electrophoresis image of purified anti-STX recombinant rabbit monoclonal antibody;

[0047] Figure 5 The anti-STX recombinant rabbit monoclonal antibody IC provided in Example 1 of this invention 50 Result image. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Preparation of a high-affinity rabbit monoclonal antibody against STX

[0050] I. Preparation of STX Immunogen and Coating Genome

[0051] 1. Experimental Methods

[0052] STX Immunogen Preparation

[0053] 200 μg STX and 2 mg ovalbumin (OVA) were dissolved separately in 2 mL PBS (pH = 7.4, 0.01 M), mixed thoroughly, and then 80 μL of formaldehyde aqueous solution (by volume, formaldehyde:water = 2.5:100) was added. The mixture was incubated at 25 °C for 24 h, and then dialyzed in pH = 7.4, 0.01 M PBS solution for 72 h. The resulting product was designated as STX-OVA and stored at -20 °C for later use.

[0054] STX-coated original preparation

[0055] 200 μg of GTX (a 1:1:1:1 equimass mixture of GTX 1, GTX 2, GTX 3 and GTX 4, with CAS values ​​of 60748-39-2, 60508-89-6, 60537-65-7 and 64296-26-0) and 2 mg of bovine serum albumin (BSA) were dissolved in 2 mL of PBS (pH = 7.4, 0.01 M), mixed thoroughly, and then 80 μL of formaldehyde solution (formaldehyde:water = 2.5:100 by volume) was added. The mixture was incubated at 25 °C for 24 h, and then dialyzed in 0.01 M PBS solution at pH = 7.4 for 72 h. The resulting product was designated GTX-BSA and stored at -20 °C for later use.

[0056] 2. Experimental Results

[0057] The structures of the prepared STX immunogen STX-OVA and coated immunogen GTX-BSA are as follows: Figure 1 As shown; UV spectral results Figure 2 As shown, the UV spectral curves before and after protein coupling are significantly different, which proves that the STX immunogen STX-OVA and the coating antigen GTX-BSA were successfully prepared.

[0058] II. Immunization of New Zealand White Rabbits with STX Immunogen

[0059] 1. Experimental Methods

[0060] (1) Animal Immunization

[0061] To obtain recombinant rabbit monoclonal antibodies that recognize STX, 0.25 mg / mL STX-OVA immunogen diluted with PBS (pH=7.4, 0.01 M) solution was mixed with adjuvant and used to immunize two New Zealand white rabbits, numbered rabbit#1 and rabbit#2, respectively.

[0062] For each rabbit, the initial immunization involved mixing 500 μL of 0.25 mg / mL STX-OVA immunogen with an equal volume (500 μL) of complete Freund's adjuvant emulsifier to prepare a 1 mL immunization solution 1. Subsequently, 500 μL of 0.25 mg / mL STX-OVA immunogen was mixed with an equal volume (500 μL) of incomplete Freund's adjuvant emulsifier to prepare a 1 mL immunization solution 2. This process was repeated 14 days later for 2, 3, and booster immunizations.

[0063] The immunization method was to inject immunization at multiple points on the neck and back, followed by two booster immunizations. After three immunizations, the serum titer was measured using the icELISA method. Rabbits with high serum titers were selected and then injected subcutaneously at multiple points with 1 mL of immunization solution for a booster immunization. The spleen was harvested three days later.

[0064] (2) Affinity determination of rabbit antiserum

[0065] Serum was collected from New Zealand white rabbits one week after the third immunization, and the titer and sensitivity of the obtained antiserum were determined using icELISA.

[0066] Specifically:

[0067] Plate coating: The STX-coating precursor GTX-BSA prepared in Example 1 was diluted to 1 μg / mL with a coating buffer prepared by adding 0.375 g Na2CO3 and 0.7325 g NaHCO3 to 250 mL of water. 100 μL of the solution was added to each well of the microplate. After standing at 4 °C for 12 h, the plate was washed twice with PBST washing buffer diluted 20 times (PBS, pH = 7.4, 0.01 M, containing 0.05% Tween 20, the same below). 150 μL of 1% BSA-PBS (w / v) solution was added to each well and the plate was stood at 37 °C for 2 h. The liquid in the wells was poured out, the plate was patted dry on absorbent paper, and then dried at 37 °C for 1 h to obtain the coated icELISA plate. The plate was stored at 4 °C for later use.

[0068] Reaction: Add 50 μL of STX PBS solution of different concentrations to the coated icELISA plate, then add 50 μL of 64K antiserum solution diluted with PBS (pH=7.4, 0.01M), incubate at 37℃ for 30 min, then wash 3 times with PBST washing buffer and pat dry; label with Anti-Rabbit HRP enzyme-labeled secondary antibody (diluted 5000 times with PBST before use), incubate at 37℃ for 30 min, wash 3 times with PBST washing buffer, pat dry, develop with 3,3',5,5'-tetramethylbenzidine chromogenic solution (TMB chromogenic solution, chromogenic solution A and solution B mixed in a 1:1 volume ratio, the same below) for 15 min; terminate the reaction with 2% concentrated sulfuric acid (11.09 mL of 98% concentrated sulfuric acid dissolved in water and brought to 1000 mL).

[0069] Detection: Using a multi-functional microplate reader at a wavelength of 450 nm, the absorbance values ​​of the antiserum from rabbit#1 and rabbit#2 after immunization with different concentrations of STX were measured. Data processing was performed using Excel.

[0070] 2. Experimental Results

[0071] The results are shown in Table 1 below, with the IC50 of rabbit#2 antiserum being... 50 The minimum is 0.085 ng / mL. -1 Therefore, Rabbit#2 was selected for the preparation of spleen lymphocytes.

[0072] Table 1. Serum performance evaluation of immunized New Zealand white rabbits.

[0073]

[0074] III. Separation of spleen cells.

[0075] Three days after booster immunization, rabbit #2 had its spleen harvested under aseptic conditions and immersed in a centrifuge tube containing 50 mL of RPMI 1640 medium for 5 min. Excess fat and fibrous tissue were then removed from the spleen, which was minced and placed on a 45 μm sterile filter. 1 mL of erythrocyte lysis buffer was added, and the spleen was ground using a sterile glass pestle. 2 mL of RPMI 1640 medium was added to the sterile filter to rinse it. The resulting ground and filtered spleen cells were centrifuged in a 50 mL centrifuge tube at 1000 rpm for 10 min. The cell pellet was resuspended in 4 mL of RPMI 1640 medium and then added dropwise to the separation medium at a 1:1 volume ratio. After centrifugation at 400 g for 20 min, four cell layers were obtained from top to bottom: dilution medium, lymphocyte layer, separation medium layer, and erythrocyte layer. Collect the lymphocyte layer into 5 mL of PBS and mix thoroughly. Centrifuge at 400 g for 15 min. Wash the lymphocyte pellet twice with culture medium. Resuspend the cell pellet in RPMI 1640 medium containing 5% serum and adjust the cell concentration to 102. 5 mL -1 Spread it evenly in a cell culture dish and culture it for use in subsequent experiments.

[0076] IV. B Lymphocyte Sorting

[0077] 1. Experimental Methods

[0078] For specific methods of sorting and culturing B lymphocytes, please refer to the method disclosed in Chinese authorized patent CN110016462A, "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".

[0079] 2. Experimental Results

[0080] The results are as follows Figure 3 As shown in the figure, the circled area represents the sorted B lymphocytes, which are then cultured in single-well 96-well cell culture plates.

[0081] V. Cloning of the gene encoding rabbit monoclonal antibody

[0082] 1. Experimental Methods

[0083] The supernatant of the screened B lymphocytes was coated with the original coated icELISA. The specific operation was the same as the antiserum titer test described above. B lymphocytes with an absorbance greater than 1 in the absence of the drug and an absorbance close to 0 in the presence of the drug were considered as B lymphocyte positive clones.

[0084] Cells from B lymphocyte-positive clones were collected, lysed at 70°C for 10 min, and then 1 U / μL was added to the PCR tube. -1 Mix 5 μL of DNase I and 1 μL of DNase I buffer, centrifuge, and incubate at room temperature for 5 min. Add 1.2 μL of 25 mM EDTA, invert the tube, centrifuge, and incubate at 70°C for 5 min to obtain cellular RNA from B lymphocyte-positive clones. Reverse transcribe the extracted RNA into cDNA. Add 2 μL of Oligo(dT) to the PCR tube containing the cellular RNA from the B lymphocyte-positive clones. 20 (50mM) and 1μL of 10mM dNTP Mix were mixed, centrifuged, and then incubated at 70°C for 5 min. After incubation on ice for 2 min, 6μL of 5×RT Buffer and 1μL of LNaseOUT™ (40UμL) were added sequentially. -1 ), 1μL SuperScriptTMIII RT (200UμL -1 Add 1 μL of 0.1 M DTT, mix well and centrifuge, then incubate in a PCR instrument at 50℃ for 50 min, 85℃ for 5 min, followed by adding 1 μL of RNase H (2 U μL). -1 ), let stand at 37℃ for 20 minutes, and the product is the reverse transcribed cDNA.

[0085] Using reverse-transcribed cDNA as a template, PCR denaturation, annealing, and extension were performed at 95℃ for 3 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, for 30 cycles, followed by 72℃ for 10 min. After these steps, plasmid and ligase were added and mixed. The mixture was then briefly centrifuged for 5 s and incubated at room temperature for 5 min before being transferred to ice for subsequent transformation reactions. A portion of the ligation product was added to 50 μL of DH5α competent cells for transfection. 350 μL of preheated LB medium (containing 1 g Tryptone, 0.5 g yeast extract, and 1 g NaCl dissolved in 100 mL of water, antibiotic-free) was added and incubated at 37°C with shaking at 250 rpm for 60 min. Then, 200 μL of the incubated medium was added to an agar plate containing ampicillin and spread evenly. The plate was inverted and incubated at 37°C for 12–16 h. A single colony was picked and inoculated into 10 mL of LB medium (containing ampicillin at a final concentration of 0.1%) and incubated overnight at 37°C with shaking. The bacterial culture was then sent for sequencing to confirm the sequence.

[0086] 2. Experimental Results

[0087] Sequencing results showed that the sequence of the recombinant rabbit monoclonal antibody against scutellarin is as follows:

[0088] Heavy chain variable region amino acid sequence:

[0089] MNHLWVRLLLVAAPGWGLALVMEESGGRLVTPPTPLTLTCQASGIDLSSYAMSWVRQAPGKGLEWIGIISTANSTYYASWAKGRFTISKASTTVDLKIISPTTEDAATYFCARSLNLDDDSGDYYFNLWGPGTLVTVSS(SEQID NO:1),

[0090] This includes three CDR areas and four FR areas:

[0091] CDR-H1: GIDLSSYA (SEQ ID NO:2),

[0092] CDR-H2:ISTANST(SEQ ID NO:3),

[0093] CDR-H3:ARSLNL(SEQ ID NO:4),

[0094] FR-H1:ALVMEESGGRLVTPPTPLTLTCQAS(SEQ ID NO:5),

[0095] FR-H2: MSWVRQAPGKGLEWIGI(SEQ ID NO:6),

[0096] FR-H3:YYASWAKGRFTISKASTTVDLKIISPTTEDAATYFC(SEQ ID NO:7),

[0097] FR-H4:WGPGTLVTVSS(SEQ ID NO:8);

[0098] Nucleotide sequence of the gene encoding the heavy chain variable region:

[0099] ATGATACACCTGTGGGTCAGGCTGCTGCTGGTGGCCGCTCCTTCTTGGTCTCTGTGACTGGTCATGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGGGGCCCCTGACATCACCTGCACAGCTCTGAGATTCTCCAAAGGTAGATGAGCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTAGTAGCATGAATACAGCCTCGACGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCCTCCACGGTGGATCTGAAAATCACCACCACGACAACCGAGGACACGGCCTCCTATTTCTGTGCCAGATCCCTCTATAATAGCTGATGACTCTGGTGATTACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA(SEQ ID NO:9)

[0100] Amino acid sequence of the light chain variable region:

[0101] MVLQTQVFISLLLWISGAYGAAVLEETPSVTPGTPGGTVTINCTASQSVYNNNNLSWKQGQYQQPPKLLIYEASKLASGVSSRFSGSGSGTQFTLTISDVQCDDAATYYCLGGYKRADCNAFGGGTELEIK(SEQ ID NO:10),

[0102] Which includes three CDR regions and four FR regions:

[0103] CDR-L1: QSVYNNNN(SEQ ID NO:11);

[0104] CDR-L2: EAS;

[0105] CDR-L3: LGGYKRADCNA(SEQ ID NO:12);

[0106] FR-L1: AAVLEETPSVTPGTPGGTVTINCTAS(SEQ ID NO:13);

[0107] FR-L2: LSWKQGQYQQPPKLLIY(SEQ ID NO:14);

[0108] FR-L3: KLASGVSSRFSGSGSGTQFTLTISDVQCDDAATYYC (SEQ ID NO: 15);

[0109] FR-L4: FGGGTELEIK (SEQ ID NO: 16);

[0110] The nucleotide sequence encoding the variable region of the light chain:

[0111] ATGGTGCTGCAGACCCAGGTGTTCATCTCTCTGCTGCTGTGGATCTCCGGC

[0112] GCCTACGGCGCAGCCGTGCTGGAGGAGACACCATCAGTGACACCTGGGAC

[0113] CCCTGGAGGCACAGTCACCATCAATTGCACAGCCAGTCAGAGTGTTTATA

[0114] ATAACAACAACTTATCCTGGTATCAGCAGAAAAGGCCAGCAGCCTCCCAAG

[0115] CTCCTGATCTACAGGGCATCCAAACTGGCATCTGGGGTCTCATCACGGTTC

[0116] AGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGAGTGCA

[0117] GTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATAAACGGGCCGGATGCATAGCTTTCGGCGGAGGGACCGAGCTGGAGATCAAA(SEQ ID NO:17)

[0118] Example 2: Production and purification of recombinant rabbit monoclonal antibody against STX

[0119] I. Experimental Methods

[0120] Preparation and purification of monoclonal antibodies: In order to obtain multiple recombinant rabbit monoclonal antibodies with high affinity for recognizing STX, this invention optimizes the codons of the heavy chain variable region and light chain variable region obtained by sequencing in Example 1 according to the preferences of mammalian cell expression systems, and amplifies and synthesizes the full gene of the variable region, which is then loaded into a mammalian cell expression vector containing the constant region of rabbit monoclonal antibodies.

[0121] Specifically, the obtained heavy chain variable region gene and light chain variable region gene were homologously recombined with the RGFc-PCMV3 and RCL-PCMV3 vectors, which were digested with KasI and BamHI, respectively, to obtain expression vectors containing rabbit monoclonal antibody sequences. After the recombinant monoclonal antibody plasmid was transformed into competent DH5α cells, 350 μL of preheated LB medium at 37°C was added, and the cells were cultured with shaking at 250 rpm and 37°C for 60 min. Then, 200 μL was added to ampicillin-containing agar medium, spread evenly, and incubated at 37°C for 12–16 h. Positive clones were selected for sequencing and plasmid extraction. The extracted plasmids from positive clones were transfected into HEK293 cells, and after 48–130 hours of transfection, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing STX.

[0122] Collect the cell suspension, centrifuge to obtain the supernatant, and purify the antibody using affinity chromatography. Determine the concentration of the purified monoclonal antibody using the BCA method, then aliquot and lyophilize.

[0123] II. Experimental Results

[0124] The harvested antibodies were validated using SDS-PAGE, such as... Figure 4 As shown, the results indicate that the obtained anti-STX recombinant rabbit monoclonal antibody (wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10) shows a clear band around 150 kDa, and no impurity bands are shown, indicating that the obtained anti-STX recombinant rabbit monoclonal antibody has good purity and the band size is correct, which is the target protein obtained in this invention.

[0125] Example 3: Establishment of the standard curve for the enzyme-linked immunosorbent assay (ELISA) method for detecting scutellarin.

[0126] I. Experimental Methods

[0127] Binding and inhibition properties were analyzed using icELISA.

[0128] The specific steps are as follows:

[0129] The plate coating procedure is the same as described in Example 1. Add 50 μL of STX PBS solution of different concentrations to the coated icELISA plate, then add 50 μL of purified 6.7 μg / L anti-STX recombinant rabbit monoclonal antibody prepared in PBS (pH = 7.4, 0.01 M) (wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10). Incubate at 37°C for 30 min, then wash three times with PBST buffer and pat dry. Label with Anti-Rabbit HRP enzyme-labeled secondary antibody and incubate at 37°C for 30 min, wash three times with PBST buffer, pat dry, and develop with 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution for 15 min. Terminate the reaction with 2% concentrated sulfuric acid (11.09 mL of 98% concentrated sulfuric acid dissolved in water and brought to a final volume of 1000 mL).

[0130] The absorbance values ​​of the anti-STX recombinant rabbit monoclonal antibody after reaction with different concentrations of STX were obtained by using a multi-functional microplate reader at a wavelength of 450 nm and terminating with 2% concentrated sulfuric acid. The data were processed using Excel.

[0131] II. Experimental Results

[0132] The results are as follows Figure 5 As shown, the IC50 of the purified anti-STX recombinant rabbit monoclonal antibody obtained in Example 2 for detecting STX was... 50 It is 0.0073 ng / mL -1 (7.3 pg mL) -1 The detection range is 0.0019~0.0282ng / L. -1 This indicates that the established icELISA immunoassay method can detect STX with ultrasensitivity, with a detection minimum of pg / mL. -1 The order of magnitude.

[0133] Example 4: Specificity of the enzyme-linked immunosorbent assay (ELISA) method for detecting scutellarin.

[0134] I. Experimental Methods

[0135] The steps for wrapping the plate are the same as in Example 1. Add 50 μL of different concentrations of small molecule competitors to the coated icELISA plate, including GTX1 & 4 (a 1:1 mass mixture of GTX1 and GTX4, CAS 60748-39-2 and 64296-26-0), GTX2 & 3 (a 1:1 mass mixture of GTX2 and GTX3, CAS 60508-89-6 and 60537-65-7), N-sulfocarbamoyl GTX1 & 2 (C1 & C2, a 1:1 mass mixture of C1 and C2, CAS 80173-30-4 and 80226-62-6), neosalicytoxin (NEO), desaminoglycantoxin (dcSTX, CAS 58911-04-9), and tetrodotoxin (TTX, CAS 58911-04-9). 4368-28-9) These paralytic shellfish toxin structural analogs were then added to 50 μL of purified 6.7 μg / L anti-STX recombinant rabbit monoclonal antibody (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10) prepared in PBS (pH=7.4, 0.01M). The mixture was incubated at 37°C for 30 min, then washed three times with PBST buffer and patted dry. 100 μL of Anti-Rabbit HRP enzyme-labeled secondary antibody diluted 5000 times with PBST was added and incubated at 37°C for 30 min. The mixture was washed three times with PBST buffer, patted dry, and developed with 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution for 15 min. The reaction was terminated with 2% concentrated sulfuric acid (11.09 mL of 98% concentrated sulfuric acid dissolved in water and brought to a final volume of 1000 mL).

[0136] The absorbance of the reaction solution after termination with 2% concentrated sulfuric acid was detected at 450 nm using a multi-functional microplate reader. The absorbance values ​​obtained after the anti-STX recombinant rabbit monoclonal antibody reacted with different concentrations of small molecule competitors, including GTX1&4, GTX2&3, C1&C2, NEO, dcSTX, and TTX—paralytic shellfish toxin structural analogs—were obtained. The IC50 values ​​were obtained using Excel data processing. 50 value.

[0137] Antibody specificity was assessed by analyzing cross-reactivity with other paralytic shellfish toxins, etc. The cross-reactivity ratio (CR) was calculated using the following formula:

[0138] CR = IC 50(STX) / IC 50(其他麻痹性贝类毒素和 / 或河豚毒素)

[0139] II. Experimental Results

[0140] The results are shown in Table 2. The results show that the anti-STX recombinant rabbit monoclonal antibody purified in Example 2 has almost no cross-reactivity with other paralytic shellfish toxins and / or tetrodotoxins.

[0141] Table 2 Cross-reactivity rates of recombinant rabbit monoclonal antibodies against STX:

[0142]

[0143]

[0144] Example 5: A test kit for safflower toxin

[0145] I. Components of the Reagent Kit

[0146] The pre-wrapped board, the steps of which are as follows;

[0147] The STX-coated precursor GTX-BSA prepared in Example 1 was diluted to 1 μg / mL with a coating buffer made of 0.375 g Na2CO3 and 0.7325 g NaHCO3 in 250 mL of water. 100 μL of this buffer was added to each well of an ELISA plate. After standing at 4°C for 12 h, the plate was washed twice with PBST (pH 7.4, 0.01 M, containing 0.05% Tween 20, hereinafter the same) diluted 20 times. Then, 150 μL of 1% BSA-PBS (w / v) solution was added to each well and the plate was incubated at 37°C for 2 h. The liquid in the wells was poured out, the plate was patted dry on absorbent paper, and then dried at 37°C for 1 h to obtain the coated icELISA plate. The plate was stored at 4°C for later use.

[0148] Enzyme-labeled secondary antibody: Anti-Rabbit HRP secondary antibody enzyme-labeled reagent, which needs to be diluted 5000 times with PBST before use;

[0149] STX standard solutions of different concentrations;

[0150] Detection antibody: The anti-STX recombinant rabbit monoclonal antibody purified in Example 2, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0151] Antibody and sample dilution solvent PBS (pH=7.4, 0.01M);

[0152] Diluent for labeled antibody (Anti-Rabbit HRP secondary antibody enzyme-labeled reagent): PBST (PBS, pH=7.4, 0.01M, containing 0.05% Tween 20);

[0153] TMB colorimetric solution; colorimetric solution A and solution B are mixed in a 1:1 volume ratio;

[0154] Termination solution: 2% concentrated sulfuric acid solution, 11.09 mL of 98% concentrated sulfuric acid dissolved in water and diluted to 1000 mL;

[0155] Concentrated washing buffer: PBST (PBS, pH=7.4, 0.01M, containing 0.05% Tween 20), diluted 20 times before use for washing plates.

[0156] II. Instructions for using the reagent kit

[0157] Pre-number and label the standard blank wells (B0), standard wells, and sample wells, and perform a 3-well replicate test. Add 50 μL of PBS to B0, add 50 μL of standard solution of different concentrations to each standard well, and add 50 μL of diluted sample solution to each sample well. Dilute the detection antibody to a working concentration of 6.7 μg / L with 0.01M PBS (pH 7.4), add 50 μL of the diluted antibody solution to all the wells, and incubate at 37°C for 30 min. Wash the microwells 5 times with PBST diluted 20 times, pat dry on absorbent paper, and add 100 μL of enzyme-labeled secondary antibody to each well. Incubate at 37°C for 30 min, then wash the microwells 5 times with PBST washing solution diluted 20 times. After patting dry on absorbent paper, add 100 μL of pre-developed TMB chromogenic solution to each well and incubate at 37°C in the dark for 10 min. Add 50 μL of 2% concentrated sulfuric acid stop solution to each well and read the value at 450 nm wavelength on a microplate reader within 5 min.

[0158] III. Result Interpretation

[0159] The average value of the standard blank at 450 nm wavelength is recorded as B0, and the average value of the values ​​at 450 nm wavelength for different STX drug concentrations and the average value of the sample test wells is recorded as B0. x Use Excel (or software capable of processing icELISA data) to calculate the B-values ​​of different STX drug concentrations or sample wells. x The / B0 ratio and the standard deviation of each set of parallel data. The logarithm of the standard concentration is plotted on the x-axis, B... x Using the / B0 ratio as the ordinate, plot a standard curve. Based on the average absorbance value of the sample wells, the abscissa of the corresponding point on the curve can be obtained; this is the logarithm of the STX concentration. The antilogarithm is the STX concentration in the assay solution. Since the sample has been pre-diluted, the sample concentration obtained from the standard curve must be multiplied by the dilution factor.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant rabbit monoclonal antibody against scleroderma toxin, characterized in that, It includes a heavy chain variable region and a light chain variable region, each of which includes a frame region (FR) and a complementarity-determining region (CDR). The complement-determining regions (CDRs) of the heavy chain variable region include CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO:2; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:3; The amino acid sequence of CDR-H3 is shown in SEQ ID NO:4; The complementary determinant regions (CDRs) of the light chain variable regions include CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO:11; The amino acid sequence of CDR-L2 is EAS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

12.

2. The recombinant rabbit monoclonal antibody against *Scleroderma vesiculosus* toxin according to claim 1, characterized in that, The frame region FR of the heavy chain variable region includes FR-H1, FR-H2, FR-H3, and FR-H4. The amino acid sequence of FR-H1 is shown in SEQ ID NO:5; The amino acid sequence of FR-H2 is shown in SEQ ID NO:6; The amino acid sequence of FR-H3 is shown in SEQ ID NO:7; The amino acid sequence of FR-H4 is shown in SEQ ID NO:8; The framework region FR of the light chain variable region includes FR-L1, FR-L2, FR-L3, and FR-L4. The amino acid sequence of FR-L1 is shown in SEQ ID NO:13; The amino acid sequence of FR-L2 is shown in SEQ ID NO:14; The amino acid sequence of FR-L3 is shown in SEQ ID NO:15; The amino acid sequence of FR-L4 is shown in SEQ ID NO:

16.

3. The recombinant rabbit monoclonal antibody against *Scleroderma vesiculosus* toxin according to claim 1 or 2, characterized in that, The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:

1.

4. The recombinant rabbit monoclonal antibody against *Scleroderma vesiculosus* toxin according to claim 1 or 2, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

10.

5. A gene encoding a recombinant rabbit monoclonal antibody against scleroderma toxin, characterized in that, The recombinant rabbit monoclonal antibody against sclerotoxin as described in any one of claims 1 to 4 is encoded.

6. The encoding gene according to claim 5, characterized in that, The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:

9.

7. The encoding gene according to claim 6, characterized in that, The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:

17.

8. A recombinant vector, characterized in that, The recombinant vector is recombined with the encoding gene of any one of claims 5 to 7, or expresses the recombinant rabbit monoclonal antibody against sclerotoxin of any one of claims 1 to 4.

9. A kit for detecting schizoaflavins, characterized in that, It contains the recombinant rabbit monoclonal antibody against sclerotoxin as described in any one of claims 1 to 4.

10. The reagent kit according to claim 9, characterized in that, It also contains a solid-phase carrier coated with a coating agent, wherein the coating agent is a genistein coupled with bovine serum albumin.

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