A method for detecting ophophagus and viverid snake venom and application thereof

Abstract

The application relates to the field of biological medicine, in particular to a detection method and application of ophidiotoxin and vivero toad venom. (1) Antibodies obtained by immunizing a mixture of SVSP chimeras and Disintegrin chimeras and two kinds of antigens of 3FT chimeras can effectively identify ophidiotoxin and vivero toad venom; (2) Antibodies obtained by immunizing two kinds of antigens of multicinctus 3FT chimeras and Atra 3FT chimeras can effectively identify ophidiotoxin and silver ring snake venom; (3) The cation exchange chromatography method is used to enrich the snake venom in the sample, and the sensitivity of the to-be-detected snake venom in blood and tissue fluid is improved; (4) Standard samples of cobra and silver ring snake venom are established for the first time. Compared with the prior art, the method has the effects of identifying vivero toad venom and ophidiotoxin by searching for difference proteins, predicting linear antigen epitopes, removing the same and highly similar linear antigen epitopes and serially recombinantly expressing the linear antigen epitopes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and application for detecting venom from snakes of the Spectacularidae and Viperidae families. Background Technology

[0002] There are more than 210 species of snakes in my country, belonging to 9 families and 66 genera, including more than 60 venomous snakes, of which more than 10 are highly venomous (2018 Chinese Expert Consensus on Snakebite Treatment, Chinese Expert Consensus on Snakebite Treatment Expert Group). Common venomous snakes in my country include: (1) Elapidae: Cobra (Naja atra, abbreviated N. atra), King Cobra (Ophiophagus Hannah), Banded Krait (Bungarus fasciatus, abbreviated B. fasciatus), and Silver Krait (Bungarus multicinctus, abbreviated B. multicinctus); (2) Viperidae: Viper (Daboia russelii siamensis, abbreviated D. siamensis), Sharp-nosed Viper (Deinagkistrodon), Pit Viper (Trimeresurus mucrosquamatus, abbreviated T. mucrosquamatus), Viper (Agkistrodon halys), and Bamboo Viper (Trimeresurus stejnegeri, abbreviated T. stejnegeri); (3) Sea Snakes: 16 species including the Blue-ringed Sea Snake (Hydrophis cyanocinctus) (Chinese Venomous Snakes and Snakebite Treatment, 2008 Blue Ocean).

[0003] Snakebites are acute injuries with rapid onset and progression. Some snake venoms can be fatal within 2-3 hours, leaving patients little time for rescue. Even if not fatal, snake venom can cause complications, severely damaging organ function, making treatment for snakebite victims much more difficult.

[0004] Currently, the diagnosis and differentiation of snakebites in clinical practice includes the following three main aspects: (1) Differentiation between venomous and non-venomous snake bites relies on the shape of the fang marks and the condition of the wound. (2) Differentiation between snakebites from various venomous snakes relies on the doctor's experience to judge based on local and systemic symptoms. (3) The severity of snakebites is judged by the Simple Assessment Scale for Clinical Severity of Snakebites or the Snakebite Severity Rating Scale (2018 Chinese Expert Consensus on Snakebite Treatment, Expert Group on Chinese Expert Consensus on Snakebite Treatment). Farmers working in the fields or outdoor workers, after being bitten by a snake, cannot identify the specific snake species due to the lack of convenient testing methods, and cannot determine whether they have been bitten by a common venomous snake. Similarly, there are no specific snake venom testing methods in hospitals, and only routine laboratory tests can be performed, including tests for thrombin, blood cells, and neuropeptides. These tests are not only time-consuming and prone to missing the best treatment window, but also cannot identify the species and type of snake venom that caused the snakebite. Therefore, the diagnosis and differentiation of snakebites heavily rely on the doctor's personal experience and skills. Primary care physicians may lack diagnostic experience, leading to patient deaths. Therefore, there is an urgent need to develop a rapid detection method for field and clinical use to identify whether a person has been bitten by a venomous snake and even what kind of venomous snake it is.

[0005] Currently, snake venom detection methods are mainly divided into the following three categories (Li Zhangyong et al., Research Progress in In Vivo Snake Venom Detection, 2015). ⑴ Enzyme-linked immunosorbent assay (ELISA). The ELISA method for detecting snake venom and its antibodies was first proposed and used by Theakston et al. in 1983. It can accurately detect snake venom antigens within 1-5 ng / mL (Ann Trop Med Parasitol, 1983, 77(3); 311-314). After many years of research, the results show that the ELISA method has the advantages of accuracy, sensitivity, and high specificity, and is suitable for the detection of snake venom in vivo. Its disadvantage is that it is not suitable for immediate detection during field operations; ⑵ Chemiluminescence detection method. Its sensitivity is higher than that of enzyme-linked immunosorbent assay (ELISA). Its disadvantage is that it needs to be performed in the laboratory and requires special instruments and equipment. (3) Colloidal gold test card. This method is suitable for immediate detection and screening needs during field operations and can complement the ELISA method used in hospitals. Because snake venom is composed of various related toxins, cross-reactions are unavoidable when using antibodies obtained from immunization with these natural snake venoms as detection reagents. For example, Labrousse et al. used an ELISA method to detect viper venom in infected rabbit serum samples. This method typically takes about 20 minutes and requires about 200 μL of blood. However, its specificity is poor, and cross-reactions are frequent. Another example is Chandler et al., who extracted antisera from six highly toxic snake venoms from Australia, conjugated them to six glass tubes, and then connected the tubes to a syringe to create an immunodiagnostic kit. This kit can diagnose snakebites within 30 minutes, and while it has high sensitivity, non-specific reactions cause significant interference.

[0006] With the development of proteomics, the venom of various venomous snakes has been isolated, purified, and its components determined. Viper venom uniquely contains three-finger toxin (3FT), while viper venom uniquely contains snake venom serine proteases (SVSP), CTL / SNACLEC (C-type lectins and C-type lectin like), and disintegrin. Snake venom from Spectralidae snakes mainly contains snake venom metalloprotease (SVMP), phospholipase A2 (PLA2), and three-fingered toxin protein (3FT). However, the content of these components varies among different types of venom from Spectralidae snakes. For example, the venom of the banded krait contains 16% PLA2, 4% SVMP, 75% 3FT, and other proteins; the venom of the cobra contains 12% PLA2, 1.6% SVMP, 84% 3FT, and other proteins; while the venom of the king cobra contains 2.8% PLA2, 11.9% SVMP, 64.5% 3FT, and other proteins (a review and database of snake venom proteomes, 2017, Theo Tasoulis and Geoffrey K. Isbister). Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention provides a method and application for detecting venoms from spectacled snakes and viper venoms. The method involves identifying differentially expressed proteins from the complex protein families of viper venoms and spectacled snake venoms, predicting their linear antigenic epitopes, removing identical and highly similar linear antigenic epitopes, and then tandemly recombinating and expressing them to obtain specific antigens. This method is effective in identifying viper venoms and spectacled snake venoms.

[0008] To achieve the above objectives, a method for detecting venom from eagle-like snakes and viper-like snakes is designed, characterized by:

[0009] (1) Antibodies obtained by immunization with a mixture of SVSP chimera and Disintegrin chimera and two antigens of 3FT chimera can effectively identify venoms of spectacled snakes and viper venoms;

[0010] (2) Antibodies obtained by immunization with two antigens, multicinctus 3FT chimera and Atra 3FT chimera, can effectively identify venom of spectacled snakes and krait venom.

[0011] (3) The cation exchange chromatography method was used to enrich the snake venom in the sample, which improved the sensitivity of the snake venom to be tested in blood and tissue fluid.

[0012] (4) Standard samples of cobra and banded krait venom were established for the first time.

[0013] The detection method described is applicable to ELISA, colloidal gold test card method, chemiluminescence method and fluorescence method.

[0014] The specific procedures for preparing the SVSP chimera, Disintegrin chimera, 3FT chimera, multicinctus 3FT chimera, and Atra 3FT chimera are as follows:

[0015] S1. Based on the venom of four viper species and four eagle species, phylogenetic trees and linear epitope predictions were performed to obtain recombinant antigens.

[0016] S2 involves transfecting recombinant antigens into *E. coli* BL bacteria via tandem linking with polypeptides, gene synthesis, and conversion. 21 In (DE)3, SVSP chimeras, Disintegrin chimeras, 3FT chimeras, multicinctus 3FT chimeras, and Atra 3FT chimeras were prepared.

[0017] S3, prepare four antibodies from the five chimeras in step S2.

[0018] The specific process of step S1 is as follows:

[0019] S1-1, using SVSP as the keyword, several protein sequences of venom from four viper species were obtained from the UniProtKB library;

[0020] S1-2, the sequence of S1-1 was analyzed using the phylogenetic tree tool on the UniProtKB website;

[0021] S1-3, select 8 sequences from them, perform antigen prediction in the IDEB database, and select those with a threshold greater than 0.5 and a length greater than 5aa as the selected SVSP sequences and predicted linear antigen epitopes;

[0022] S1-4, remove identical or highly similar sequences from the linear antigenic epitopes in S1-3 to obtain a summary of predicted linear antigenic epitopes, and obtain the amino acid sequences as shown in SEQ ID NO:1~SEQ ID NO:38;

[0023] S1-5, using disintegrin as the keyword, several protein sequences of the venoms of four viper species were obtained from the UniProtKB library;

[0024] S1-6, repeat steps S1-2 to S1-4 to obtain the amino acid sequences as shown in SEQ ID NO:39 to SEQ ID NO:93;

[0025] S1-7, the venoms of four eagle species were used to obtain several protein sequences in the UniProtKB library using three-finger toxin as the keyword;

[0026] S1-8, repeat steps S1-2 to S1-4 to obtain the amino acid sequences shown in SEQ ID NO:94 to SEQ ID NO:135.

[0027] The specific process of step S2 is as follows:

[0028] S2-1, SEQ ID NO: 1-38 is concatenated using GG, GGG, GGGS, and KK to obtain sequence SEQ ID NO: 136, whose nucleotide optimized sequence is SEQ ID NO: 137;

[0029] S2-2, Sequence SEQ ID NO: 136 and the polypeptide were tandemly linked using GG, GGG, GGGS, and KK to obtain sequence SEQ ID NO: 138, whose nucleotide optimized sequence is SEQ ID NO: 139; and sequence SEQ ID NO: 138 was defined as an SVSP chimera;

[0030] S2-3, the sequence SEQ ID NO: 39-93 and the polypeptide are tandemly linked using GG, GGG, GGGS, and KK to obtain the sequence SEQ ID NO: 140, whose nucleotide optimized sequence is SEQ ID NO: 141;

[0031] S2-4, the sequence SEQ ID NO: 140 was synthesized and transformed into Escherichia coli BL21(DE)3 to obtain the Disintegrin chimera;

[0032] S2-5, the sequence SEQ ID NO: 94-135 and the polypeptide are tandemly linked using GG, GGG, GGGS, and KK to obtain the sequence SEQ ID NO: 142, whose nucleotide optimized sequence is SEQ ID NO: 143;

[0033] S2-6, the sequence SEQ ID NO: 142 was synthesized and transformed into Escherichia coli BL21(DE)3 to obtain a 3FT chimera;

[0034] S2-7, Sequences SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 97 were tandemly linked using GG to obtain sequence SEQ ID NO: 144; The polypeptide sequence of sequence SEQ ID NO: 144 was named multicinctus 3FT chimera;

[0035] S2-8, Sequences SEQ ID NO: 98-101, SEQ ID NO: 103-105 and the polypeptide were tandemly linked using GG and GGGS to obtain sequence SEQ ID NO: 145, whose nucleotide-optimized sequence is SEQ ID NO: 146; S2-9, Sequence SEQ ID NO: 145 was used for gene synthesis and transformed into E. coli BL. 21 In (DE)3, the Atra 3FT chimera is obtained.

[0036] The polypeptide is an HLA-DR-restricted epitope of CD4+T in upper tetanus.

[0037] The specific process of step S3 is as follows:

[0038] S3-1: Select 12 New Zealand White rabbits and randomly divide them into four groups;

[0039] S3-2, immunized separately with: (1) SVSP chimera + Disintegrin chimera, mixed at a concentration of 1:1; (2) 3FT chimera; (3) multicinctus 3FT chimera; (4) Atra 3FT chimera;

[0040] S3-3, First immunization: Use Freund's complete adjuvant 0.5ml + 0.5ml antigen (2mg / ml) for subcutaneous immunization at 3-5 sites, and perform booster immunization every 21 days;

[0041] S3-4, Second immunization: 0.5ml Freund's incomplete adjuvant + 0.5ml antigen (2mg / ml) were used for subcutaneous immunization at 3-5 sites, and a total of 5 booster immunizations were performed. Blood was then collected to obtain 4 types of antibodies.

[0042] S3-5, the four antibodies from step S3-4 are purified using the octanoic acid precipitation method.

[0043] The SVSP chimera, disintegrin chimera, 3FT chimera, multicinctus 3FT chimera, and Atra 3FT chimera are shown in sequences SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, and SEQ ID NO:145, respectively.

[0044] The amino acid sequences shown in SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144 and SEQ ID NO: 145 have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology.

[0045] Antiserum from rabbits immunized with SVSP chimera and disintegrin chimera mixture, 3FT chimera, multicinctus 3FT and Atra3FT can be purified by protein G, immunoaffinity chromatography or caprylic acid precipitation, preferably caprylic acid precipitation combined with ultrafiltration purification.

[0046] Using the principle of cation exchange chromatography, the venom of cobra and banded krait in buffer solution, tissue fluid and blood was enriched. The enrichment conditions were: washing buffer was 20 mM PB (pH 6.8 ± 0.1) containing 0.15-0.2 M NaCl, and elution conditions were 20 mM PB (pH 6.8 ± 0.1) containing 0.3-0.35 M NaCl.

[0047] The standards for cobra venom and banded krait venom were prepared by dissolving a large batch of freeze-dried venom powder in purified water, mixing, centrifuging, and then lyophilizing the supernatant. Protein quantification was performed using the Kjeldahl method or ultraviolet spectrophotometer, and component analysis was performed using SDS-PAGE or mass spectrometry.

[0048] The preparation method of the snake venom standard is as follows: Weigh the freeze-dried snake venom powder, dissolve it in purified water, shake to mix, and centrifuge at high speed (10,000 rpm, 10 min). Take the supernatant, dispense it into vials, and freeze-dry. Perform ultraviolet spectroscopy analysis, setting the ultraviolet wavelength of the ultraviolet-visible spectrophotometer to 200-400 nm and scanning at medium speed. Perform SDS-PAGE electrophoresis analysis, using a 15% protein separating gel.

[0049] The specific operation of the ELISA method is as follows: Antibodies produced by the mixture of SVSP chimera and disintegrin chimera, and 3FT chimera antigens can effectively identify the venom of spectacled and viper species. Antibodies produced by the two antigens multicinctus 3FT and Atra 3FT can effectively distinguish the venom of cobra and banded krait, with detection limits reaching 1 ng / ml for cobra venom and 2 ng / ml for banded krait venom.

[0050] The specific operation of the colloidal gold detection card method is as follows: Four antibodies obtained by immunization with a mixture of SVSP chimera and disintegrin chimera, 3FT chimera, multicinctus 3FT and Atra 3FT, enriched samples and standards are used. It can effectively detect the venom of snakes in the families Spectacleidae and Viperidae, as well as the venom of cobras and kraits, with the detection limit reaching 10 ng / ml for cobra venom and 50 ng / ml for krait venom.

[0051] An application of a method for detecting venom from spectacled snakes and viper venom, characterized in that:

[0052] (1) Effectively identifies the venom of Spectacular snakes and Viperidae snakes;

[0053] (2) Effectively distinguishes between cobra and banded krait venom;

[0054] (3) It can be used in hospitals to test patient samples, including but not limited to blood, tissue fluid from wounds, and rinsing fluid from bite sites, and to guide the dosage of anti-Bungarus krait and cobra venom.

[0055] Compared with the prior art, the present invention provides a method and application for detecting venoms of spectacled snakes and viper venoms. It searches for differentially expressed proteins among members of the complex protein family of viper venoms and spectacled snake venoms, predicts their linear antigenic epitopes, removes identical and highly similar linear antigenic epitopes, and expresses them in tandem recombination to obtain specific antigens, which can be used to identify viper venoms and spectacled snake venoms.

[0056] This method provides comprehensive coverage of differentially expressed proteins in both snake families while reducing the size of the expressed proteins. Similarly, by comparing the differentially expressed linear epitopes of high-abundance 3FT proteins in the venoms of four spectacled snakes, including cobra and krait venoms, antibodies were prepared that can distinguish the venoms of these four spectacled snakes. Attached Figure Description

[0057] Figure 1 Evolutionary tree of SVSP proteins in four viper species.

[0058] Figure 2 Evolutionary tree of Disintegrin protein in four viper species.

[0059] Figure 3 Evolutionary tree of 3FT proteins in four species of spectacled venomous snakes.

[0060] Figure 4SDS-PAGE assay for recombinant protein expression. 1 represents Atra 3FT; 2 represents the 3FT chimera; 3 represents the Disintegrin chimera; 4 represents the SVSP chimera; 5 represents the expression product of SEQ ID NO: 136; M represents the protein marker (purchased from Bio-Rad #161-0377), with molecular weights from top to bottom of 250, 150, 100, 75, 50, 37, 25, and 10 KD.

[0061] Figure 5 HPLC was used to purify equine anticobra antiserum. In the figure, (1) shows the purification of protein G; (2) shows the precipitation of octanoic acid.

[0062] Figure 6 This is a chromatogram of cobra venom purified by cation exchange chromatography. In the figure, 1 and 2 represent the pH and conductivity of the buffer solution, respectively; A, B, and C are the protein elution peaks detected by OD280, where A is the loading flow-through solution, B is the first elution peak during linear elution (NaCl concentration 180-250 mM), and C is the second elution peak during linear elution (NaCl concentration 280-350 mM).

[0063] Figure 7 SDS-PAGE of cobra and banded krait venom standards. 1 represents cobra venom (10ug); 2 represents banded krait venom (10ug); 3 represents cobra venom (20ug); 4 represents banded krait venom (20ug); 5 represents cobra venom (40ug); 6 represents banded krait venom (40ug); M represents protein markers (purchased from Bio-Rad #161-0377), with molecular weights from top to bottom as follows: 250, 150, 100, 75, 50, 37, 25, 15, and 10 KD.

[0064] Figure 8 For the detection of banded krait venom and cobra venom using colloidal gold test cards: (A) detection with equine anticobra antiserum; (B) detection with equine antibanded krait antiserum; (C) detection with Atra 3FT and multicinctus 3FT antibodies; (D) detection with Atra 3FT and multicinctus 3FT antibodies for specificity. Detailed Implementation

[0065] The present invention will now be further described with reference to the accompanying drawings.

[0066] 1. Screening for immune antigens.

[0067] 1.1 Inactivation of natural snake venom from cobras and kraits and equine immunization:

[0068] Cobra and banded krait venoms were inactivated, and blood plasma was obtained by immunizing horses with these two types of blood plasma, which were then used as raw materials.

[0069] 1.2 Preparation of recombinant antigen:

[0070] 1.2.1 Linear epitope expression of proteins unique to the venom of four viper species:

[0071] 1.2.1.1 Phylogenetic tree and linear epitope prediction of SVSP in the venom of four viper species:

[0072] In the UniProtKB library (www.uniprot.org), 6 protein sequences were obtained using the keywords "SVSP siamensis AND reviewed: yes", 6 sequences using "SVSP deinagkistrodon AND reviewed: yes", 7 sequences using "SVSP mucrosquamatus AND reviewed: yes", and 25 sequences using "SVSP stejnegeri AND reviewed: yes". These sequences were analyzed using the phylogenetic tree tool on the UniProtKB website. The results of the phylogenetic tree analysis are shown below. Figure 1 As shown.

[0073] Figure 1 This indicates that in the evolutionary tree, branches A and B are vipers, branches C and D are mainly pit vipers, branch E is a viper, branches G, H and I are bamboo pit vipers, and the sequences in J, K, M and N are present in all four snake species.

[0074] Eight sequences were selected and antigen prediction was performed in the IDEB database (http: / / tools.immuneepitope.org / bcell / ). Sequences with a threshold greater than 0.5 and a length greater than 5 aa were selected as the included SVSP sequences and predicted linear antigenic epitopes, as shown in Table 1. Identical or highly similar sequences were removed from these predicted linear epitopes in Table 1, resulting in a summary of predicted linear antigenic epitopes, as shown in Table 2.

[0075] Table 1

[0076]

[0077] Table 2

[0078] Serial Number amino acid sequence Serial Number amino acid sequence SEQ ID NO: 1 SKKVLNEDEQTRN SEQ ID NO: 20 SSTKDILPDVP SEQ ID NO: 2 KNNNEVLDK SEQ ID NO: 21 ECVAPYP SEQ ID NO: 3 PVKETFP SEQ ID NO: 22 GGIDTCHQDSG SEQ ID NO: 4 GGKDTCGGDS SEQ ID NO: 23 YPCAQPR SEQ ID NO: 5 HSCGQGP SEQ ID NO: 24 AQRTSEL SEQ ID NO: 6 TDWIQRN SEQ ID NO: 25 SVEFDYEETRY SEQ ID NO: 7 HAVCQ SEQ ID NO: 26 SKNYTKWD SEQ ID NO: 8 ECDIN SEQ ID NO: 27 SPNETFPD SEQ ID NO: 9 KYVKFDDEQGRE SEQ ID NO: 28 TCARPR SEQ ID NO: 10 YFYNCSNNLTTRD SEQ ID NO: 29 KKLLNEDEQIRN SEQ ID NO: 11 PSRDVLPD SEQ ID NO: 30 PIEVTYP SEQ ID NO: 12 PCAQPNM SEQ ID NO: 31 PCGQSLK SEQ ID NO: 13 AQKSSEL SEQ ID NO: 32 QKSPELV SEQ ID NO: 14 KKVLNEDEQTRN SEQ ID NO: 33 PTKVTYP SEQ ID NO: 15 PVKETFPD SEQ ID NO: 34 LKVLNKDALRRFPKEKYFCLNIRNDTIWD SEQ ID NO: 16 YPELLA SEQ ID NO: 35 SPNATLP SEQ ID NO: 17 HPCGQGP SEQ ID NO: 36 YAVCQA SEQ ID NO: 18 SIQFDDEQR SEQ ID NO: 37 NPCAQPRKP SEQ ID NO: 19 YYRCDEKLTKWE SEQ ID NO: 38 YTDWIQSIISGNTDA

[0079] 1.2.1.2 Phylogenetic tree and linear epitope prediction of disintegrin in four viper toxins:

[0080] The search keyword SVSP in section 1.2.1.1 was replaced with disintegrin, and the same operations (including phylogenetic tree analysis, linear epitope prediction, and removal of identical or highly similar predicted linear epitopes) were performed. A total of 13 protein sequences were found. The phylogenetic tree analysis results are as follows: Figure 2 As shown in the phylogenetic tree, the disintegrin protein in each type of snake venom is essentially a separate lineage with relatively distant phylogenetic relationships. Therefore, one member from each of the four venomous snakes was selected for linear epitope prediction. The selected disintegrin sequences and predicted linear antigenic epitopes are shown in Figure 3. After removing identical or highly similar sequences, a summary of the predicted linear antigenic epitopes is obtained, as shown in Table 4.

[0081] Table 3

[0082]

[0083] Table 4

[0084] Serial Number amino acid sequence Serial Number amino acid sequence SEQ ID NO: 39 SSIILESGNVNDYE; SEQ ID NO: 67 SNDDEHKGMVLPGTKCAD SEQ ID NO: 40 KVTALPKGAVQQPEQKYED; SEQ ID NO: 68 SSIILESGNVDDY; SEQ ID NO: 41 YYPDGREITTNPPVE; SEQ ID NO: 69 PRRVSALPKGAVQPKYED; SEQ ID NO: 42 PLKLSNSEA; SEQ ID NO: 70 YSPDGREITTYPSVED; SEQ ID NO: 43 ENIEKEDEIPKMCGVTQTNWESDKPIKKASQLVSTSAQFNK; SEQ ID NO: 71 PLKLPDSEA; SEQ ID NO: 44 KKCNSTA; SEQ ID NO: 72 IYDINLCRYDYSANGMVAQGTKCA SEQ ID NO: 45 CDRDLINVTSS; SEQ ID NO: 73 YIKYNRDSNKI; SEQ ID NO: 46 EWRASDLMTRKS; SEQ ID NO: 74 SQNDLITVQPASSIT; SEQ ID NO: 47 MYHDGKNCIC; SEQ ID NO: 75 GNWRKTVLLNQQNH; SEQ ID NO: 48 LSDQPSKL; SEQ ID NO: 76 DEDKNEKKCK; SEQ ID NO: 49 IHDYQRYLTRYKPKCIFNPPLRKDIVSPPVCGNEIWEEGEECDCGSPANCQN; SEQ ID NO: 77 SDPPAQLFSDC; SEQ ID NO: 50 CDAATCKLKPGAE; SEQ ID NO: 78 LNAPLRTDTVSTPVSGNEFLEAGEECDCGSPENPCC; SEQ ID NO: 51 CCYQCKIKTAGTVCRRARDECDVPEHCTGQSAECPRDQLQQNGKPCQN; SEQ ID NO: 79 AATCKLRPGAQCA; SEQ ID NO: 52 DCPIMRNQCISLFGSRANVAKDSCFQENLKGSYYGYCRKENGRKIPCAP; SEQ ID NO: 80 QCRFKKKRTICRRARGDNPDDRCTGQSADCPRNG SEQ ID NO: 53 NSPRNKNPC; SEQ ID NO: 81 RKVTAVPKGAVQQKYED; SEQ ID NO: 54 SCMDQHKGMVDPGTKCEDGKVCNNKRQCV; SEQ ID NO: 82 YSPDGREITTYPSVED; SEQ ID NO: 55 VNTAYQSTTG SEQ ID NO: 83 LKLPDSEA; SEQ ID NO: 56 SPDGREITINPPVED; SEQ ID NO: 84 YENVEKEEEAPKMCGVTETNWKSDEPIKKASQLVVTAEQQRFPR; SEQ ID NO: 57 EPMKLSDSEA; SEQ ID NO: 85 YTKHKKNL; SEQ ID NO: 58 ENVEKEDEAPKMCGVTQKWKSYEPIKKISQLNLPEQQIYDP; SEQ ID NO: 86 WKKNDKITAQSASN; SEQ ID NO: 59 KYNGDLD; SEQ ID NO: 87 NWRETVLLKRKR; SEQ ID NO: 60 EDKITVKPD; SEQ ID NO: 88 DHDGNQCN; SEQ ID NO: 61 EWRKTYLLAEKK; SEQ ID NO: 89 INNPPSERFSGCSMG; SEQ ID NO: 62 SPEPSK; SEQ ID NO: 90 LTAYNPQCILNALSKRDIITPPVCGNELLEEGEECDCGSPENCQYQCCNA; SEQ ID NO: 63 DFIMNHNPECIDNEPLGTDIISPPLCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQC; SEQ ID NO: 91 TCKLHSWVECE; SEQ ID NO: 64 CCEQCKFRTSGTECRASMSECDPAEHCTGQSSECPADVFHKNGEPCLD; SEQ ID NO: 92 ECCEQCRFKKAGAVCRAARTECDIPENCTDQSADCPTDSFHRNGQPCLY; SEQ ID NO: 65 NCPIMYHQCYALFGADIYEAEDSCFESNKKGNYYGYCRKENGKKIPCASEDV; SEQ ID NO: 93 CPVMHYQCYGLFGPNATVGQDGCFDANDRGDEYFYCRKENEKYIPCAQEDV; SEQ ID NO: 66 KDDSPGQNN;

[0085] 1.2.1.3 Predicting linear antigenic epitopes using tandem expression of SVSP protein sequences:

[0086] By chaining together SEQ ID NO: 1-38 in Table 2 using GG, GGG, GGGS, KK, etc., a chimera SEQ ID NO: 136 is formed, and its optimized nucleotide sequence is SEQ ID NO: 137.

[0087] To enhance its ability to induce antibody expression, the HLA-DR restriction epitopes of tetanus CD4+T, namely the peptides (PITINNFRYSDPVNNDTIIM and YCKGLDIYYKAFKIT), were tandemly added to SEQ ID NO: 136, thus forming the sequence SEQ ID NO: 138, whose nucleotide optimized sequence is SEQ ID NO: 139.

[0088] The sequences SEQ ID NO: 137 and SEQ ID NO: 139 were sent to Shanghai Bioengineering Co., Ltd. or Shanghai Jierui Bioengineering Co., Ltd. for gene synthesis (hereinafter referred to as gene synthesis), and then transformed into Escherichia coli BL. 21 The protein was prepared in (DE)3. The expressed protein was detected by SDS-PAGE, as shown below. Figure 4 As shown.

[0089] 1.2.1.4 Immunize mice with the expression products of SEQ ID NO: 138 and SEQ ID NO: 136:

[0090] Six adult BALB / c mice were randomly divided into two groups and injected with the chimeras expressed in SEQ ID NO: 136 and SEQ ID NO: 138, respectively. For the initial immunization, each mouse received 20 μg of chimeric protein mixed with an appropriate amount of Freund's adjuvant, emulsified, and injected using a sterile syringe. Two weeks later, a second immunization was performed using incomplete Freund's adjuvant instead of complete Freund's adjuvant, with the chimeric protein dose remaining at 20 μg per mouse. This second immunization was repeated four times (two weeks apart), i.e., one initial immunization and four second immunizations. Blood was then collected intravenously, and serum from three mice in each group was mixed in equal volumes for comparison of the immunogenicity of the two chimeras using the following ELISA method.

[0091] 1.2.1.5 Comparison of the effects of SEQ ID NO: 138 and SEQ ID NO: 136 on mouse immunization using ELISA:

[0092] Add 100 μl of 0.05 mol / L pH 9.6 carbonate buffer containing 5 μg of uninactivated viper venom to each well of a 96-well plate and coat overnight at 4°C. Block with 250 μl of blocking buffer [2% BSA (0.05% Tween-20 and 5% lactose)] at 37°C for 2 h. Dilute positive serum 100, 200, 400, 800, and 1600 times with phosphate buffer at pH 7.4, and dilute negative serum (serum from mice not immunized with the antigen) to the same dilution as positive serum. Add 100 μl to each well, and repeat each dilution in 3 wells. Measure the average value and select the reaction group with the largest positive serum to negative serum A450 ratio (P / N value), which is the best antigen.

[0093] Results: After 800-fold dilution of serum, the OD450 readings of SEQ ID NO: 138 and SEQ ID NO: 136 were 1.6 and 0.9, respectively, indicating that the SEQ ID NO: 138 chimera possesses high immunogenicity. Therefore, the peptides (PITINNFRYSDPVNNDTIIM and YCKGLDIYYKAFKIT) were used for fusion expression of other antigens for immunization and antibody preparation. The expression product of SEQ ID NO: 138 was named the SVSP chimera.

[0094] 1.2.1.6 Disintegrin chimera expression:

[0095] Using the same strategy and method as in 1.2.1.3, SEQ ID NO: 39-93 and the polypeptides (PITINNFRYSDPVNNDTIIM and YCKGLDIYYKAFKIT) in Table 4 were tandemly linked using GG, GGG, GGGS, KK, etc., to obtain SEQ ID NO: 140, whose optimized nucleotide sequence is SEQ ID NO: 141. The gene was synthesized and transformed into *E. coli* BL21(DE)3, ultimately obtaining the expression product, such as... Figure 4 As shown. The product expressed by SEQ ID NO: 140 is named Disintegrin chimera.

[0096] 1.2.2 Specific proteins in the venom of four species of spectacled snakes:

[0097] 1.2.2.1 Determination of high-abundance venom proteins in the venom of four cobra species:

[0098] A search on PubMed (http: / / PubMed.ncbi.nlm.nih.gov) using the statement "((naja naja)[Title / Abstract]OR(Bungarus multicinctus)[Title / Abstract]OR(Bungarusfasciatus)[Title / Abstract])AND(proteomics[Title / Abstract]OR proteomic[Title / Abstract])" yielded 59 articles as of June 30, 2021. Further analysis revealed three articles studying the proteomes of cobra, banded krait, and golden krait venom in China. Two of these articles contained 3FT abundance data, showing the 3FT sequences and their abundance (greater than 1%) in cobra and banded krait venom, as shown in Table 5.

[0099] Table 5

[0100]

[0101] As shown in Table 5, P60615 and P60616 account for 47% of the total venom content of the banded krait, while the contents of the three proteins Q9PTT0, Q9W6W9 and Q9DEQ3 account for 30% of the total venom content of the cobra. They are the main components and can be considered as detection targets.

[0102] 1.2.2.2 3FT phylogenetic tree analysis and linear antigenic epitope prediction:

[0103] A search of the UniProtKB database (www.uniprot.org) using the keywords "three-finger toxin""Bungarus fasciatus" AND reviewed: yes", "three-finger toxin""Bungarus multicinctus" AND reviewed: yes", "three-finger toxin""naja atra" AND reviewed: yes", and "three-finger toxin""ophiophagus hannah" AND reviewed: yes" yielded a total of 122 protein sequences. The phylogenetic analysis results are as follows... Figure 3 As shown.

[0104] from Figure 3 From the phylogenetic tree, the 3FTs of each venomous snake are clustered separately, indicating relatively distant phylogenetic relationships. To ensure the detection of 3FTs in each venomous snake, high-abundance 3FTs were selected from cobra and banded krait venoms, while one member from each clade was selected from king cobra and krait venoms. Linear antigenic epitope prediction was then performed, and the selected 3FT protein sequences and their predicted linear antigenic epitopes are shown in Table 6.

[0105] Table 6

[0106]

[0107] Table 7

[0108] Serial number Amino acid sequence Serial number Amino acid sequence SEQ ID NO: 94 TATSPISAVT SEQ ID NO: 115 PQPKKEW SEQ ID NO: 95 YRKMWCDVFCSSRGKV SEQ ID NO: 116 KTGDRIISEACPPGQDL SEQ ID NO: 96 PSKKPYE SEQ ID NO: 117 TWCDVFCGTRGRVI SEQ ID NO: 97 CNPHPKQ SEQ ID NO: 118 PTVKPHEQ SEQ ID NO: 98 CHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRT SEQ ID NO:119 NCNPHPK SEQ ID NO:99 PSVKNGIE SEQ ID NO:120 DVTSQTCPDG SEQ ID NO:100 RKCNKLVPLFYKTCPA SEQ ID NO:121 TWCDAWCGSRGKRV SEQ ID NO:101 NLCYKMFMVSNLTVPV; SEQ ID NO:122 PKVNPGV SEQ ID NO:102 DVCPKSSLLVK SEQ ID NO:123 DNCNPFP SEQ ID NO:103 YNHLSRTPETTEICPDS SEQ ID NO:124 CPKVKPGVD SEQ ID NO:104 LADGNDVRI SEQ ID NO:125 LNTPLPLIYKTCPIG SEQ ID NO:105 CPELRPTGK SEQ ID NO:126 KMTIKKLPSKYDV SEQ ID NO:106 YSSSMNKDSKTCQKWET SEQ ID NO:127 DICPKSSAD SEQ ID NO:107 QEAFKPHSSGMII SEQ ID NO:128 LKQEPFQPETTTT SEQ ID NO:108 KAQFPNIETQCK SEQ ID NO:129 NLFWSDHSEIK SEQ ID NO:109 QRDVKPHPS SEQ ID NO:130 CHRVHGLQTCEPDQKFCFRKTTMFFPNHPV SEQ ID NO:110 SSCGKGAMCNGPS SEQ ID NO:131 EEYCKRIHTCRDGEN SEQ ID NO:111 LNQQSSEPQTTETCPNG SEQ ID NO:132 KGFYEGKQLGKQFR SEQ ID NO:112 YNKTWNTHRGSRT SEQ ID NO:133 PEGKPNE SEQ ID NO:113 KVKPGI SEQ ID NO:134 HESLFFETTETCSDGQNLCYAKWFAVFPGARR SEQ ID NO:114 CLTKYSQDNESSKTCPSGQKLCFKKWETGKLLGT SEQ ID NO:135

[0109] 1.2.2.3 3FT chimeric recombination expression:

[0110] Using the same strategy and method as in 1.2.1.5, SEQ ID NO: 94-135 and peptides (PITINNFRYSDPVNNDTIIM and YCKGLDIYYKAFKIT) from Table 7 (summary of predicted linear antigenic epitopes after removing highly similar or identical ones) were concatenated using GG, GGG, GGGS, KK, etc., to obtain SEQ ID NO: 142, whose nucleotide-optimized sequence is SEQ ID NO: 143. The gene was synthesized and transformed into *E. coli* BL... 21 In (DE)3, the final expression product is obtained, such as PDKVPLE As shown. The expression product of SEQ ID NO: 142 is named the 3FT chimera.

[0111] 1.3 Differential Immunogens between Cobras and Banded Krait:

[0112] Table 6 shows that the predicted linear antigenic epitopes of cobra P60615 and P60616 are basically the same, consisting of four sequences SEQ ID NO: 94-97. The predicted linear antigenic epitope of cobra SEQ ID NO: 102 is basically the same as that of krait SEQ ID NO: 95. If they are used, there will be cross-reactivity. Therefore, SEQ ID NO: 102 and SEQ ID NO: 95 are removed.

[0113] The banded krait sequence SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO: 97 were selected and tandemly linked using GG to obtain sequence SEQ ID NO: 144. The polypeptide sequence of SEQ ID NO: 144 is: TATSPISAVTGG PSKKPYEGGCNPHPKQ. This sequence was named the multicinctus 3FT chimera. This sequence was sent to Gir Biochemical for synthesis and linked with keyhole limpet hemocyanin (KLH). Because SEQ ID NO: 144 has a very small molecular weight, it is not easy to produce a good immune effect. Generally, it is coupled with a large molecule such as BSA or KLH to enhance the immune effect.

[0114] Cobra selected SEQ ID NO: 98-101 and SEQ ID NO: 103-105, and linked them with the polypeptides (PITINNFRYSDPVNNDTIIM and YCKGLDIYYKAFKIT) using GG and GGGS, then tandemly copied them to obtain sequence SEQ ID NO: 145, whose nucleotide-optimized sequence is SEQ ID NO: 146. The gene was synthesized and transformed into *E. coli* BL21(DE)3, ultimately obtaining the expression product, as shown below. Figure 4 As shown. The expression product of SEQ ID NO: 145 is named the Atra3FT chimera.

[0115] 2. Antibody preparation.

[0116] 2.1 Purification of equine anti-cobra polyclonal antibodies:

[0117] 2.1.1 Protein G purification of equine anti-cobra polyclonal antibody:

[0118] 1. Column packing and equilibration: Pack 100 ml of Protein G (purchased from BorgL (Shanghai) Biotechnology Co., Ltd., catalog number: AA104307) into a 26x40 mm chromatography column (purchased from GE Healthcare); Equilibrate the column with 20 mM PB (pH 7.3±0.1) 3CV equilibration buffer.

[0119] 2. Centrifuge the plasma at 8000 rpm for 20 min and collect the supernatant. After filtration and centrifugation, dilute the plasma 1:2 with PB (pH 7.3 ± 0.1) buffer.

[0120] 3. Sample loading: After baseline equilibration, load 1 mL of plasma for every 2 mL of packing material, and load 150 mL of plasma diluent for every 100 mL of packing material. Repeat the loading 2-3 times.

[0121] 4. Wash with 20mM PB (pH 7.3±0.1) 3CV buffer.

[0122] 5. Elution: Elute with Gly-hydrochloric acid buffer (pH 2.8 ± 0.2) for 3-5 CV, and collect the eluted sample. Immediately adjust the pH of the eluent to neutral.

[0123] 6. Rinse the column to neutral pH using 20mM PB buffer (pH 7.3±0.1), and then preserve the column using 20% ​​ethanol.

[0124] 7. Perform HPLC according to Example 6.2 to detect the collected products.

[0125] 2.1.2 Purification by octanoic acid precipitation:

[0126] 1. Take 50ml of plasma stock solution, add 100ml of ultrapure water, slowly add n-octanoic acid stock solution dropwise to make the final concentration 5%, and adjust the pH value to maintain it between 5 and 6. Stir well at room temperature and let stand for 1 hour.

[0127] 2. Centrifuge the supernatant at 4℃ for 20 minutes at 12000 rpm / min and collect the supernatant.

[0128] 3. Tandem Ultrafiltration: The supernatant of the filtered protein solution was ultrafiltered three times using a 100 kDa ultrafiltration membrane, and the filtrate was collected. Then, the filtrate was concentrated using a 30 kDa ultrafiltration membrane, and the concentrate was collected, replacing the filtrate with PBS buffer. The purified concentrate was analyzed by HPLC.

[0129] 2.1.3 Comparison of purification effects of Protein G and caprylic acid precipitation:

[0130] 2.1.3.1 HPLC detection:

[0131] from Figure 4 It is evident that protein G, compared to octanoic acid precipitation for purifying madocoplastin, resulted in lower purity. The octanoic acid precipitation method yielded madocoplastin with a purity as high as 95%, which can be used for subsequent product development. In fact, other proteins in plasma can also non-specifically bind to protein G.

[0132] 2.1.3.2 Comparison of purification effects using ELISA detection method:

[0133] Referring to the ElISA method in 1.2.1.5, the main differences are as follows:

[0134] 1. Add 100 μl of 0.05 mol / L pH 9.6 carbonate buffer containing 10 μg / ml of uninactivated cobra venom to each well of a 96-well plate and coat overnight at 4°C.

[0135] 2. The polyclonal antibody obtained by purifying and precipitating protein G with caprylic acid was determined. Its protein concentration and purity were determined according to HPLC. It was then adjusted to be consistent with the starting amount of IgG (IgG content 1 mg / ml). It was diluted 2000, 4000, 8000, 16000, 32000, 64000 and 128000 times with phosphate buffer at pH 7.4 as primary antibody.

[0136] 3. Rabbit anti-horse IgG (FC specific, purchased from Sigma, catalog number: SAB3700145-2mg) labeled with HRP was diluted 1:4000 as a secondary antibody. When the OD450 reading was between 1.0 and 1.5, the ratio of the two was considered appropriate.

[0137] Results: When the eluent obtained from protein G purification was diluted 64,000 times, the OD450 was approximately 1.0, while the polyclonal antibody obtained from octanoic acid precipitation combined with ultrafiltration, after being diluted 128,000 times, had an OD450 of approximately 1.4. Therefore, the binding affinity of protein G to equine polyclonal antibodies purified by octanoic acid precipitation was lower, possibly due to the extreme elution conditions of protein G (pH 2.8) and the differences in binding affinity between protein G and different IgG subtypes in horses, leading to the loss of some IgG or loss of activity. Similarly, conjugating snake venom to a medium and purifying it using immunoaffinity chromatography also presents the same problem. Therefore, this invention uses octanoic acid precipitation to purify equine antiserum against cobras and banded kraits to obtain polyclonal antibodies for subsequent experiments.

[0138] 2.2 Preparation of rabbit polyclonal antibodies:

[0139] 2.2.1 Rabbit polyantibody immunization:

[0140] Twelve New Zealand white rabbits were randomly divided into four groups. They were immunized with: (1) SVSP chimera + Disintegrin chimera (mixed at a 1:1 concentration); (2) 3FT chimera; (3) multicinctus 3FT; and (4) Atra 3FT. The first immunization used 0.5 ml of Freund's complete adjuvant + 0.5 ml of antigen (2 mg / ml) subcutaneously at 3-5 sites. A booster immunization was performed every 21 days using 0.5 ml of Freund's incomplete adjuvant + 0.5 ml of antigen (2 mg / ml) subcutaneously at 3-5 sites, for a total of 5 booster immunizations. Blood was then collected.

[0141] 2.2.2 Purification of rabbit polyclonal antibodies by octanoic acid precipitation:

[0142] The four rabbit polyclonal antibodies described in 2.2.1 were prepared according to the method in 2.1.2.

[0143] 3. Enrichment of samples by cation chromatography.

[0144] 3.1 Purification of cobra venom by cation chromatography:

[0145] 3.1.1 SP column gradient purification of cobra venom:

[0146] 1. Column packing and equilibration: Pack 20 ml of SP Sepharose 4FF into a 16x20 mm chromatography column (purchased from GE Healthcare); equilibrate the column with 20 mM PB (pH 6.8 ± 0.1) 3CV buffer.

[0147] 2. Sample loading: Prepare a 5 mg / ml solution of cobra venom in 20 mM PB (pH 6.8 ± 0.1), load 20 ml of the solution at 2 ml / min, and collect the flow-through.

[0148] 3. Washing: Wash with 20mM PB (pH 6.8±0.1) 3CV buffer.

[0149] 4. Linear elution: Two pumps were set up: pump A pumped 20 mM PB (pH 6.8 ± 0.1), and pump B pumped 20 mM PB (pH 6.8 ± 0.1) containing 0.7 M NaCl. The entire process took 70 min to complete. The entire process was monitored with OD280 and the protein of each peak was collected.

[0150] 5. Column cleaning and storage: Clean the medium with 0.5M NaOH and 1M NaCl solution, and then store it with 20% ethanol.

[0151] 6. ELISA was used to detect the purification effect of cation chromatography.

[0152] Results: From the tomography diagram (e.g.) Figure 5 As shown in the figure, there are three main peak bands: A is the sample flow through the solution; B is the first peak during linear elution (NaCl concentration 180-250 mM); and C is the second peak during linear elution (NaCl concentration 280-350 mM).

[0153] 3.1.2 Indirect ELISA method for detecting purification effect:

[0154] Referring to the ElISA method in 1.2.1.5, the main differences are as follows:

[0155] 1. Add 100 μl of 0.05 mol / L pH 9.6 carbonate buffer containing 10 μg of uninactivated cobra venom (as a positive control) and three peak samples A, B and C (as test samples) to each well of a 96-well plate, and coat overnight at 4°C.

[0156] 2. Primary antibody: Antibody obtained by immunizing rabbits with Atra 3FT chimeric antibody and then precipitated with caprylic acid.

[0157] Results: The reaction with the primary antibody Atra 3FT was mainly concentrated in the C peak sample. Therefore, the cationic chromatography purification conditions were optimized as follows: the loading buffer / washing buffer was 20 mM PB (pH 6.8 ± 0.1) containing 150–200 mM NaCl, and the elution buffer was 20 mM PB (pH 6.8 ± 0.1) containing 0.3–0.35 M NaCl.

[0158] Similarly, the venom of the banded krait can also be purified by cation chromatography, under the same purification conditions as that of the cobra venom.

[0159] 3.2 Sample enrichment using a simplified cation chromatography processor:

[0160] 3.2.1 Simple cation chromatography processor:

[0161] Create a baffle at the tip of a syringe and add 50 μL of SP Sepharose 4FF. Separate the gel into two layers with paper. Before use, cap the tip and fill the entire tube with 20% ethanol.

[0162] To use, open the cap and remove the plunger. Add 200 μL of 20 mM PB (pH 6.8 ± 0.1) buffer containing 0.15–0.2 M NaCl to wash the gel. Then add the sample and allow it to flow through. Finally, add 100 μL of 20 mM PB (pH 6.8 ± 0.1) containing 0.3–0.35 M NaCl to elute the sample. Collect the sample in a 200 μL centrifuge tube and perform the analysis.

[0163] 3.2.2 Enrichment of rabbit blood samples using a simplified cation exchange chromatography processor:

[0164] Take 1 ml of unimmunized rabbit serum and add 20 ng of uninactivated cobra and banded krait venom, respectively. Then, use a simple cation exchange column with 20 mM PB (pH 6.8 ± 0.1) and 20 mM PB (pH 6.8 ± 0.1) containing 0.1–0.3 M NaCl. The concentration limit is then determined according to the ELISA method in 3.1.2.

[0165] The results showed that cobra and banded krait venom could accumulate in rabbit blood, with an accumulation factor of up to 3 times, which is obviously beneficial for increasing the venom content of patients with snake bites.

[0166] 3.2.3 Enrichment of tissue fluid samples using a simplified cation exchange chromatography processor:

[0167] Take 1 ml of tissue fluid from the wound of an unimmunized rabbit, and add 100 ng of uninactivated cobra and banded krait venom respectively. Then, use a simple cation exchange column with 20 mM PB (pH 6.8 ± 0.1) and 20 mM PB (pH 6.8 ± 0.1) containing 0.1-0.3 M NaCl. The concentration limit is then determined according to the ELISA method in 3.1.2.

[0168] The results showed that cobra and banded krait venom could be enriched in rabbit tissue fluid, with an enrichment factor of 2.5 times, which is obviously beneficial for increasing the venom content of patients with snakebites during testing.

[0169] 4. Application scenarios of ELISA detection method.

[0170] 4.1 Indirect ELISA to determine antibody specificity:

[0171] Referring to the ElISA method in 1.2.1.5, the main differences are as follows:

[0172] 1. Add 100 μl of 0.05 mol / L pH 9.6 carbonate buffer containing 10 μg of uninactivated snake venom to each well of a 96-well plate and coat overnight at 4°C. The coated snake venom included four species of eagles (cobra, king cobra, krait, and banded krait) and five species of vipers (viper, pit viper, pit viper, and bamboo viper), used to identify the horse anti-cobra and banded krait antiserum obtained from caprylic acid precipitation, and the four antibodies obtained in section 2.2.2.

[0173] 2. The polyclonal antibodies against cobra and banded krait venom purified with caprylic acid in 2.1.2 and the four rabbit polyclonal antibodies obtained in 2.2.2 were adjusted to have the same starting IgG amount (IgG content 1 mg / ml). They were then diluted 2000, 4000, 8000, 16000, 32000, 64000 and 128000 times with phosphate buffer at pH 7.4 to serve as primary antibodies.

[0174] 3. Use HRP-labeled mouse anti-rabbit IgG diluted at 1:4000 as a secondary antibody. If a colorimetric reaction occurs, the ratio of the two antibodies is considered appropriate when the OD450 reading is between 1.0 and 1.5.

[0175] Results: As shown in Table 8 (six antibody combinations for detecting nine snake venoms): (1) Even with overcoating (10ug / ml), the four rabbit polyclonal antibodies showed no obvious color reaction after being diluted 16,000 times, thus demonstrating that the antibodies did not cross-react. The polyclonal antibodies produced after immunizing rabbits with these four molecules have significant specificity. (2) Horse antibodies against banded krait and cobra venoms and other snake venoms showed cross-reactivity. Therefore, no further research was conducted on these two antibodies.

[0176] Table 8

[0177]

[0178] Note: "x" represents no color change; "√" represents a noticeable color change.

[0179] 4.2 Determination of the detection limits of rabbit polyclonal antibodies using multicinctus 3FT and Atra 3FT:

[0180] 4.2.1 HRP-labeled rabbit polyclonal antibodies against multicinctus 3FT and Atra 3FT using the sodium periodate method:

[0181] 1. Weigh 30 mg HRP and dissolve it in 6 ml of distilled water. Add 1.2 ml of freshly prepared 0.1 M NaIO4 solution and stir for 20 minutes at room temperature in the dark. Transfer the solution to a dialysis bag and dialyze against 1 mM pH 4.4 sodium acetate buffer at 4°C overnight.

[0182] 2. Add 120 μl of 0.2 M pH 9.5 carbonate buffer to raise the pH of the aldehyde-modified HRP to 9.0-9.5. Then immediately add 60 mg of IgG (antibody) to 6 ml of 0.01 M carbonate buffer and gently stir at room temperature in the dark for 2 hours.

[0183] 3. Add 0.6 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and incubate at 4°C for 2 hours. Transfer the solution to a dialysis bag and dialyze against 0.15 M pH 7.4 PBS overnight at 4°C.

[0184] 4. Add an equal volume of saturated ammonium sulfate dropwise while stirring, and let stand at 4°C for 1 hour.

[0185] 5. Centrifuge at 3000 rpm for half an hour and discard the supernatant. Wash the precipitate twice with semi-saturated ammonium sulfate, and finally dissolve the precipitate in a small amount of 0.15M PBS (pH 7.4).

[0186] 6. Place the above solution into a dialysis bag and dialyze it against 0.15M PB buffered saline at pH 7.4 to remove ammonium ions (detected with Naphthol's reagent). Centrifuge at 10,000 rpm for 30 minutes to remove the precipitate. The supernatant is the enzyme conjugate. Aliquot and store frozen.

[0187] 4.2.2 Evaluation of antibody detection limit using double-sandwich ELISA:

[0188] Referring to the ElISA method in 1.2.1.5, the main differences are as follows:

[0189] 1. Coating: Add 100 μl of 0.05 mol / L pH 9.6 carbonate buffer containing 10 μg / ml HRP-labeled rabbit multicinctus 3FT or Atra 3FT to each well of a 96-well plate and coat overnight at 4°C.

[0190] 2. After sealing and washing, add serially diluted (1 μg-0.1 ng) crude venom from the banded krait or cobra and incubate at 37°C for 90 min. Three wells without any snake venom were selected as negative controls.

[0191] 3. Primary antibody: Add 100 μL of rabbit polyclonal antibodies labeled multicinctus 3FT and Atra 3FT (1 mg / mL) diluted 1:2000 with HRP and incubate at room temperature for 45 minutes.

[0192] 4. After washing, add the colorimetric solution and the stop solution, and measure the light absorption value at 450nm in each well.

[0193] The results showed that a positive result and detectable content were defined as an OD450 greater than twice the negative control reading. The lowest level of Atra 3FT rabbit polyclonal antibody detection in cobra was 1 ng / ml, while the lowest level of multicinctus 3FT detection in banded krait was 2 ng / ml.

[0194] 4.3 Preparation of Standards:

[0195] Weigh 15g of uninactivated cobra venom lyophilized powder, dissolve it in 1L of purified water, vortex to mix, and centrifuge at high speed (10000rpm, 10min). Collect the supernatant and aliquot it into 500µl vials for lyophilization. Store the lyophilized solution at -80°C. Take 10 vials of lyophilized snake venom and reconstitute each vial with 500µl of water. After complete dissolution, determine the protein content using a UV-Vis spectrophotometer at 280nm. The average protein content of the 10 vials is taken as the target value for the standard protein. Snake venom component analysis is performed using SDS-PAGE electrophoresis with a 15% protein separating gel.

[0196] Prepare standard freeze-dried products of banded krait venom using the same steps, and perform protein content and component analysis.

[0197] The results showed that the protein concentration of the cobra venom standard was 12.5 mg / ml, and the protein concentration of the banded krait venom standard was 13.2 mg / ml, with inter-vial CVs both less than 3%. Observation of the SDS-PAGE electrophoresis images of cobra venom and banded krait venom revealed the characteristic bands at 3FT (molecular weight approximately 10 kDa), indicating successful preparation. Figure 6 As shown.

[0198] 5. Colloidal gold test card.

[0199] 5.1 Preparation method:

[0200] 5.1.1 Preparation of gold-labeled probes for colloidal gold-labeled anti-cobra venom / anti-clade krait venom specific antibodies:

[0201] Heat 100 mL of 0.01% chloroauric acid aqueous solution to boiling. While stirring, add 1.6 mL of 1% trisodium citrate aqueous solution until the liquid color stabilizes to a wine-red color, obtaining a colloidal gold solution. Adjust the pH to 9.0, add antibody to a final concentration of 30-40 μg / mL, and stir for 20 minutes. Add 5 mL of 10% BSA and stir for 20 minutes. Then add 1 mL of 10% PEG2000 and stir for 20 minutes. Centrifuge at 5000 rpm for 10 minutes, collect the supernatant, and centrifuge the supernatant again at 15000 rpm for 30 minutes. Discard the supernatant and store the precipitate in 10 mL of 0.3% sodium tetraborate solution to obtain the labeled colloidal gold probe solution.

[0202] 5.1.2 Assemble the cobra venom / clade krait venom immunochromatographic test strip:

[0203] The test strip consists of a support plate, a sample pad, a gold-labeled pad, a detection membrane, and an absorbent pad. The gold-labeled pad is made of glass fiber that adsorbs colloidal gold-labeled antibodies. The detection membrane is a nitrocellulose membrane with a blot for cobra venom detection line T and a control line C. Detection line T is an antibody blot (antibody coating concentration of 4 mg / mL), and control line C is a goat anti-horse IgG secondary antibody blot (secondary antibody coating concentration of 4 mg / mL).

[0204] 5.2 Specificity detection:

[0205] Prepare solutions of cobra venom and banded krait venom at different concentrations (100ug / mL, 50ug / mL, 10ug / mL, 1ug / mL, 100ng / mL, etc.), and test the solutions of different concentrations using the test strips prepared in 5.1.1.

[0206] The results showed that when the cobra venom immunochromatographic test strip detected krait venom and cobra venom, both the krait venom immunochromatographic test strip and the cobra venom immunochromatographic test strip showed only one red band at control line C, regardless of the venom concentration (Figure 8D). However, when using equine anti-krait and cobra venom tests, cross-reactions were observed (e.g., ...). Figure 7 (As shown in A and 8B).

[0207] 5.3 Sensitivity Testing:

[0208] The cobra venom / clade krait venom standard (the venom standard prepared in 4.3) was serially diluted with PBS buffer (20 mM PBS, 150 mM NaCl, pH 7.2) to concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 1 μg / mL and 10 μg / mL, respectively, and detected using the test strip prepared in 5.1.1.

[0209] The test results showed that when the concentration of krait venom was above 50 ng / mL, two red bands appeared: the test line (T) and the control line (C). When the concentration was below 50 ng / mL, only the control line (C) appeared as a single red band. Similarly, when the concentration of cobra venom was above 10 ng / mL, two red bands appeared: the test line (T) and the control line (C). When the concentration was below 10 ng / mL, only the control line (C) appeared as a single red band. Figure 8 Figure 8 (as shown in C).

[0210] The beneficial effects of this invention are as follows: First, by using tandem recombinant expression of specific antigenic epitopes from four venomous snakes each of the Spectacidae and Viperidae families, not only is the toxicity of naturally derived snake venom overcome, but also the differences in venom composition from different regions, snake ages, and even different seasons, ensuring the stability of the immunogenicity; moreover, the antibodies produced after immunization can effectively identify the family characteristics of snakebites. Second, by utilizing the specific antigenic epitopes of cobra and krait venom, the antibodies produced after immunization can effectively distinguish between cobra and krait venom. These antibodies are suitable for detection by ELISA, chemiluminescence, fluorescence, and colloidal gold test cards. Finally, the enrichment of toxins in the sample improves sensitivity, making the detection method clinically applicable.

[0211] Sequence information:

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Claims

1. A group of antigens for detecting venom from spectacled snakes and viper venom, characterized in that: It consists of two antigens: an SVSP chimera with an amino acid sequence as shown in SEQ ID NO:138 and a Disintegrin chimera with an amino acid sequence as shown in SEQ ID NO:140, and a 3FT chimera with an amino acid sequence as shown in SEQ ID NO:

142.

2. A group of antigens for detecting venom from spectacled snakes and viper venom, characterized in that: It consists of four antigens: a mixture of SVSP chimera (amino acid sequence as shown in SEQ ID NO:138) and Disintegrin chimera (amino acid sequence as shown in SEQ ID NO:140), a 3FT chimera (amino acid sequence as shown in SEQ ID NO:142), a multicinctus 3FT chimera (amino acid sequence as shown in SEQ ID NO:144), and an Atra 3FT chimera (amino acid sequence as shown in SEQ ID NO:145).

3. The use of the antigen according to claim 1 or 2 in the preparation of antibodies for detecting venom from spectacled snakes and viper venom, wherein the antibodies are obtained by immunizing rabbits with the antigen, respectively.

4. The application according to claim 3, characterized in that: The detection methods are applicable to ELISA, colloidal gold test card method, chemiluminescence method and fluorescence method.

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