CN113832132B - Solder tip SVMP protein specific short peptide, solder tip SVMP protein antibody and snake bite detection kit - Google Patents
Solder tip SVMP protein specific short peptide, solder tip SVMP protein antibody and snake bite detection kit Download PDFInfo
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Abstract
The invention provides a soldering iron SVMP protein specific short peptide, an anti-soldering iron SVMP protein antibody and a snake bite detection kit, belonging to the technical field of antibody and immunodetection. A soldering iron head SVMP protein specific short peptide, the amino acid sequence is shown in SEQ ID NO 1. The soldering iron tip SVMP protein resisting monoclonal antibody SVMP-mc18 obtained by screening the short peptide has higher specific affinity with soldering iron tip natural SVMP protein. The snake bite diagnosis kit prepared from the SVMP protein monoclonal antibody can be used for diagnosing whether a snake bite is bitten by a soldering iron head, and greatly improves the snake bite diagnosis efficiency and the result accuracy by taking the antibody-antigen immunoreaction as the detection diagnosis basis.
Description
Technical Field
The invention belongs to the technical field of antibody and immunodetection, and particularly relates to a soldering iron SVMP protein specific short peptide, an anti-soldering iron SVMP protein antibody and a snake bite detection kit.
Background
The soldering iron head snake, named as original spearhead viper (named as Trimeresurus muscovatus), is one of the most aggressive snakes in Asia, is also one of ten poisonous snakes in China, and has the characteristics of small body size and strong toxicity.
At present, the method for identifying the bite of the viper is mainly identified clinically by the main methods of family members or patients such as the identification of the viper atlas, clinical manifestations, snake tooth marks and the like, and the diagnosis error of the methods is very large. In a large number of cases, the snake species are not identified for bite, thereby delaying the valuable treatment window and leading to death. Based on the antibody-antigen immunoreaction as the detection and diagnosis basis, the snake bite diagnosis efficiency can be greatly improved. At present, the anti-snake venom serum varieties in China are few, and the anti-snake venom serum varieties cannot cover all the virulent snake varieties. The toxic components contained in snake venom are very complex and mainly comprise enzyme, neurotoxic polypeptide, bioactive factor and membrane active polypeptide. There is no report of biomarker detection for snake venom of flathead snake, which seriously blocks the rapid detection of flathead snake bite.
Disclosure of Invention
In view of the above, the present invention aims to provide a solder tip SVMP protein specific short peptide, which can be used for specific screening of SVMP protein monoclonal antibodies.
The invention also aims to provide an anti-soldering bit SVMP protein antibody and a snake bite detection kit, which realize the rapid diagnosis of soldering bit snake bite.
The invention provides a soldering iron SVMP protein specific short peptide, and the amino acid sequence of the short peptide is shown in SEQ ID NO. 1.
The invention provides a monoclonal antibody SVMP-mc18 for resisting solder tip SVMP protein obtained by screening short peptide, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO:2, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 3. as shown.
The invention provides an ELISA detection kit for soldering iron head snake bite, which comprises a solid phase carrier coated with a polyclonal antibody of anti-soldering iron head SVMP protein and an enzyme-labeled monoclonal antibody SVMP-mc18 of the anti-soldering iron head SVMP protein.
Preferably, the polyclonal antibody against the flathead SVMP protein is prepared by immunizing an animal with SVMP protein isolated and purified from flathead snake venom.
Preferably, the amino acid sequence of the solder tip SVMP protein is as set forth in SEQ ID NO:4, respectively.
Preferably, the coating concentration of the polyclonal antibody against tip SVMP protein in the solid phase carrier is 1-100 mug/ml.
Preferably, the detection kit further comprises a washing solution, a sample diluent, a substrate, a color development solution and a positive standard.
Preferably, the positive standard comprises an isolated and purified SVMP protein or a short peptide specific to the solder tip SVMP protein.
The amino acid sequence of the solder tip SVMP protein specificity short peptide provided by the invention is shown in SEQ ID NO. 1.SVMP protein exists in various snake venom, comparison shows that the snake venom SVMP proteins from different sources exist in a conserved region and a hypervariable region, the SVMP protein short peptide provided by the invention specifically exists in the snake venom of the flatiron head, and the specific detection of the flatiron head snake bite can be realized by screening the monoclonal antibody of the SVMP protein of the flatiron head snake by adopting the short peptide.
The invention provides a monoclonal antibody SVMP-mc18 for resisting solder tip SVMP protein obtained by screening short peptide, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 2. the amino acid sequence of the light chain variable region is shown as SEQ ID NO:3, respectively. The monoclonal antibody SVMP-mc18 has stronger specificity, antiserum secreted by the prepared hybridoma cell strain is subjected to immune combination reaction with five kinds of snake venom (including bamboo leaf, viper, cobra and flatiron), and only flatiron snake venom can be subjected to strong specific combination with the antiserum. And then, antiserum secreted by different hybridoma cell strains and the iron head SVMP protein specific short peptide are subjected to immune combination reaction, SVMP-mc4, SVMP-mc9 and SVMP-mc18 can be combined with SVMP short peptide antigen, and the SVMP-mc18 and the SVMP short peptide antigen have strong affinity, so that the result accuracy of the detection kit is ensured by the characteristic that the SVMP-mc18 is specifically combined with the iron head SVMP protein.
Drawings
FIG. 1 is the tip of a soldering iron SVMP (snake venous metalloprotease TM-3, access 1 kuf) NCBI blast result, boxed as the specific sequence (SEQ ID NO: 1);
FIG. 2 shows the results of a homology modeling of the SVMP proteins of the iron tip, the black regions marked as the hypervariable regions of the SVMP proteins (SVMP-pep-1, SVMP-pep-2 and SVMP-pep-3);
FIG. 3 shows the medium-pressure liquid phase (cation column) separation and purification of SVMP protein from iron tip (FIG. A) and the WB results of each peak after purification (FIG. B), wherein peak No. 11 is the peak of SVMP protein;
FIG. 4 shows SDS-PAGE results of denaturation of SVMP protein from solder tip after crude soldering iron poison and purification;
FIG. 5 is a graph showing the results of screening SVMP monoclonal antibodies using five natural snake venoms and natural SVMP proteins;
FIG. 6 is a graph showing the results of a second round of screening of SVMP polyclonal antibodies using SVMP-peptide-1;
FIG. 7 shows the results of the specificity and spatial accessibility assays of monoclonal and polyclonal antibodies against native SVMP protein antigen; wherein A is the ELISA and WB detection results of the anti-SVMP monoclonal antibody SVMP-mc18 respectively; b, respectively obtaining ELISA and WB detection results of the anti-SVMP polyclonal antibody;
FIG. 8 shows the results of five snake venom detection (ELISA) after successful pairing of anti-SVMP monoclonal antibodies and polyclonal antibodies;
FIG. 9 is a standard curve of flathead venom concentration assay showing good linearity in the range of 6.25ng/ml to 100 ng/ml.
Detailed Description
The invention provides a soldering iron SVMP protein specific short peptide, the amino acid sequence of which is shown in SEQ ID NO 1 (TKYSSNFKKI).
In the invention, SVMP protein sequences of various venomous snakes are compared to obtain a section of highly-variable amino acid sequence conserved in the flatiron snake species with other venomous snakes, and the adoption of the amino acid sequence can specifically distinguish the flatiron SVMP protein from other snake venom SVMP proteins, thereby providing a tool for the subsequent screening of anti-flatiron SVMP protein antibodies.
The invention provides a monoclonal antibody SVMP-mc18 for resisting solder tip SVMP protein obtained by screening short peptide, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO:2 (EIQLQQSGPELMKPGASVKVSCKASGYSFTDYNMYWVKQSHGKSLE WIGYIDPYNGGTSYNQKFKGKATLTVDKSSSTAFMHLNSLTSEDSAVYYCARSPLITTLGKRYFDVWGAGTTVTVSSEIQLQQSGPELMKPGASVKVSCK ASGYSFTDYNMYWVKQSHGKSLEWIGYIDPYNGGTSYNQKFKGKATLT VDKSSSTAFMHLNSLTSEDSAVYYCARSPLITTLGKRYFDVWGAGTTVTVSS), the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:3 (DIQMTQSPASLSASVGETVTITCRTSENIYIYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYGTPLTFG AGTKLELKDIQMTQSPASLSASVGETVTITCRTSENIYIYLAWYQQKQGKS PQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYGTPLTFGAGTKLELK).
In the invention, the monoclonal antibody SVMP-mc18 for resisting the solder tip SVMP protein is secreted by a hybridoma cell strain. Five natural snake venom antigens and natural flatiron SVMP antigens are respectively coated on a solid phase carrier, antiserum secreted by a plurality of screened hybridoma cell strains is subjected to indirect ELISA detection, and the result shows that only the flatiron snake venom and the natural flatiron SVMP antigens can be strongly specifically combined. And then performing indirect ELISA screening by taking the soldering iron SVMP protein specific short peptide as a coating antigen, wherein SVMP-mc4, SVMP-mc9 and SVMP-mc18 can be combined with the SVMP short peptide antigen, and the SVMP-mc18 has stronger affinity property.
The invention provides an ELISA detection kit for soldering iron head snake bite, which comprises a solid phase carrier coated with a polyclonal antibody of anti-soldering iron head SVMP protein and an enzyme-labeled monoclonal antibody SVMP-mc18 of the anti-soldering iron head SVMP protein.
In the present invention, the polyclonal antibody against the solder tip SVMP protein is preferably prepared by immunizing an animal with SVMP protein isolated and purified from the snake venom of the solder tip. The amino acid sequence of the solder tip SVMP protein is preferably as shown in SEQ ID NO:4 (EQQRFPQRYIKLAIVVDHGMYTKYSSNFKKIRKRVHQMVSNINEMCRPLNIAITLALLDVWSEKDFITVQADAPTT AGLFGDWRERVLLKKKNHDHAQLLTDTNFARNTIGWAYVGRMCDEKYSVAVVKDHSSKVFMVAVTMTHELGHNLGMEHDDKDKCKCDTCIMSAVIS DKQSKLFSDCSKDYYQTFLTNDNPQCILNAP). The number of immunizations of an animal preferably comprises 2, with an interval of 2 weeks. For the immunization, the SVMP protein is preferably injected into animals after being mixed with an adjuvant in equal volume. After immunization, antiserum was collected and the antibody titer was determined by indirect ELISA.
In the present invention, the coating concentration of the polyclonal antibody against tip SVMP protein in the solid phase carrier is preferably 1 to 100. Mu.g/ml, more preferably 5 to 50. Mu.g/ml, and most preferably 10. Mu.g/ml. The coating solvent is preferably 20mM phosphate buffer. The coating method of the present invention is not particularly limited, and a coating method known in the art may be used. After coating, it is preferably closed. The blocking solution is BSA solution with the concentration of 1-10%. The solid phase carrier preferably comprises an enzyme label plate, magnetic particles or a plastic tube and the like.
In the present invention, the enzyme in the enzyme-labeled monoclonal antibody SVMP-mc18 against tip SVMP protein is preferably horseradish peroxidase (HRP). The preparation method of the enzyme-labeled monoclonal antibody SVMP-mc18 for resisting the soldering iron tip SVMP protein is not particularly limited, and the enzyme-labeled antibody method well known in the field can be adopted.
In the present invention, the detection kit preferably further comprises a washing solution, a sample diluent, a substrate, a developing solution and a positive standard. The positive standard preferably comprises an isolated and purified SVMP protein or a short peptide specific to the solder tip SVMP protein. The washing solution, the sample diluent, the substrate, and the developing solution are not particularly limited in kind, and an ELISA reagent known in the art may be used.
The method for using the kit is not particularly limited, and a double-antibody sandwich ELISA detection method well known in the art can be adopted.
The detection kit provided by the invention is used for detecting the snake venom of the soldering iron head based on the double-antibody sandwich principle, and the monoclonal antibody and the polyclonal antibody for resisting the SVMP protein, which are provided by the invention, have high affinity and strong specificity and are capable of being combined with the SVMP protein of the soldering iron head, so that the rapid diagnosis of the soldering iron head bite is realized, and the snake bite detection efficiency and the accuracy of a detection result are greatly improved.
The tip SVMP protein-specific short peptide, the anti-tip SVMP protein antibody and the snake bite detection kit provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
The terms "anti-tip SVMP protein and tip SVMP protein antibody", "tip SVMP protein and tip SVMP protein antibody" and "antibody" are used interchangeably and refer to an antibody that specifically binds to tip SVMP protein.
The term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
The term "isolated" refers to a substance that is separated from its original environment, which is the natural environment if the substance is natural. If the polynucleotide or protein in a living cell is not isolated or purified in a natural state, the polynucleotide or protein is isolated from other substances present in the natural state.
The term "hybridoma cell" refers to a cell obtained by fusing myeloma cells and B lymphocytes in the course of producing a monoclonal antibody.
The term "ELISA" refers to a qualitative and quantitative detection method in which a soluble antigen or antibody is bound to a solid phase carrier such as polystyrene and an immunoreaction is carried out by utilizing the binding specificity of the antigen and the antibody.
The invention provides antibodies against soldering iron tip SVMP protein and soldering iron tip SVMP protein, wherein the antibodies can be specifically combined with the soldering iron tip SVMP protein and the soldering iron tip SVMP protein respectively, and the antibodies are prepared from the naturally purified soldering iron tip SVMP protein and the soldering iron tip SVMP protein respectively.
Example 1
SVMP sequences from different snake species were downloaded from NCBI website, and solder tip SVMP protein specific polypeptide sequences were obtained by NCBI blast analysis and homology modeling analysis (FIGS. 1 and 2). The sequences were found to be divided into conserved regions (white, light grey background) and hypervariable regions (grey, dark grey background). The hypervariable region of the SVMP protein is boxed and the specific sequence (SEQ ID NO:1, TKYSSNFKKI) is designated SVMP-peptide-1.SVMP-peptide-1 antigen polypeptide is synthesized by Gill Biochemical (Shanghai) Co., ltd by solid phase synthesis. Each short peptide antigen synthesized polypeptide antigen 10mg and purity over 95%.
Example 2
A method for preparing an anti-SVMP characteristic polypeptide (SVMP-peptide-1) polyclonal antibody, comprising the following steps:
(1) KLH-polypeptide Complex antigen preparation
Synthesizing polypeptide by biochemical synthesis, adding a cysteine (C) residue at C terminal, and combining with hemocyanin (KLH) to prepare KLH-polypeptide complex antigen. The specific method was carried out according to the kit (K2039-5, bioVision, USA) instructions.
(2) Immunizing animals
The natural tip KLH-polypeptide complex antigen was dissolved in physiological saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1 mg of antigen) was emulsified in equal amounts of complete Freund's adjuvant (F-5881, sigma) and injected subcutaneously along multiple sites of the back of male New Zealand white rabbits, two rabbits per antigen. After 2 weeks of intervals, booster immunizations were administered intradermally along multiple sites along the back of male New Zealand rabbits using Freund's incomplete adjuvant (F-5506, sigma) emulsified with an equal volume of antigen (0.5 ml, containing 0.5mg of antigen). Two booster immunizations were performed at 2 weeks intervals. Rabbit sera were collected 14 days after the second booster immunization. And (3) taking each antigen as a coating antigen, taking serum before immunization as negative control, and detecting the titer of the antibody by adopting an indirect ELISA method. The method comprises the following specific steps:
1) Coating: KLH-polypeptide complex antigen (5. Mu.g/ml) was diluted with the coating solution and added to a 96-well plate in an amount of 100. Mu.l per well and 100. Mu.l of the coating solution to a blank well, and coated overnight at 4 ℃.
2) Washing: the next day, the coating solution in the wells was poured off and washed three times with 250. Mu.l of wash solution per well for 3-5 minutes each time, and the wells were patted dry as much as possible.
3) And (3) sealing: the blocking solution was applied to 250. Mu.l/well for 1h at 37 ℃. Then washed three times and patted dry.
4) Mu.l of rabbit immune serum after gradient dilution is added into the positive hole, and 100. Mu.l of sealing solution is added into the negative hole and the blank hole, and the temperature is 37 ℃ for 1h. Then washed three times and patted dry.
5) 100 μ l of horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibody was added to the positive, negative and blank wells, and incubated for 1h at 37 ℃ in a dark incubator. Then washed three times and patted dry.
6) Color development: after washing, 100. Mu.l of color developing solution is added into each well, and the mixture is incubated for 15 to 30 minutes at 37 ℃ in the dark.
7) And (4) terminating: the stop solution is 100 μ l per well, and the reading is carried out by a microplate reader at 450/630nm dual wavelength.
Wherein, the formula of the solution used is as follows:
1. coating liquid: 0.05M carbonate buffer pH 9.6; sodium carbonate 1.59g + sodium bicarbonate 2.93g, dissolved in 1L deionized water.
2. Washing liquid: cells were treated with Phosphate Buffered Saline (PBS) +0.1% Tween-20. Note: cells were plated with PBS: 0.2g of potassium chloride; potassium dihydrogen phosphate, 0.2g; sodium chloride, 8g;12 g of sodium dihydrogen phosphate monohydrate, 2.16g of deionized water and 1L of deionized water.
3. Blocking solution (antibody dilution): washing solution 1g per 100ml + Bovine Serum Albumin (BSA).
4. Color development liquid: 3,3',5,5' -Tetramethylbenzidine (TMB) single-component color developing solution (Solarbio, beijing).
5. Stopping liquid: 2mol/L sulfuric acid, 178.3ml water +21.7ml concentrated sulfuric acid, slowly stirring and mixing.
Example 3
The separation and purification method of the solder tip SVMP protein comprises the following steps:
a Resource S column was mounted on an FPLC (fast protein liquid chromatography, AKTA pure) instrument, and after washing the column bed according to the specification, 50mg of crude flatiron toxin was weighed and dissolved in 2ml of A solution, and the solution was loaded onto the well-balanced Resource S column through a loading valve. Detecting with ultraviolet detector at 280nm and 215nm, collecting each peak, respectively collecting each elution peak, lyophilizing, and storing at-20 deg.C. The method comprises the following specific steps: equilibrating with solution A (flow rate 2 ml/min), loading, washing away unbound protein with solution A, performing linear elution with solution B, and collecting each peak. Wherein, the formula of the solution is as follows:
liquid A (KCl: 0.2g, KH 2 PO 4 ：0.2g，Na2HPO 4 ·12H 2 O3.47g, pH 7.2); liquid B (KCl: 0.2g, KH 2 PO 4 ：0.2g，Na 2 HPO 4 ·12H 2 O:3.47g, naCl:58g, pH 7.2) was filtered using a 0.45 μm filter (Millipore)And ultrasonic degassing is performed.
WB detection was performed using an anti-SVMP-peptide-1 polypeptide antigen antibody to confirm the peak of the target protein (see FIG. 3). Finally, the purified soldering iron tip SVMP protein is subjected to denaturing SDS-PAGE electrophoresis detection protein purification, and the result is shown in figure 4. The results showed that the native solder tip SVMP protein (SEQ ID NO: 4) was isolated and purified from snake venom.
Example 4
Preparation method of soldering iron tip SVMP protein monoclonal antibody
1) And (3) immunizing a mouse by using the natural solder tip SVMP protein antigen to obtain spleen cells of the immunized mouse.
5 Balb/c mice were immunized with the native solder tip SVMP protein prepared in example 2, respectively. Wherein Balb/c mice are white-variant laboratory mice, each mouse averagely completes at least 4 times of immunization and 3 times of blood sampling detection, and after the first, second and third blood sampling detection, the target mice with higher titer (screened by the natural antigen) are screened, in the embodiment, 2 mice are screened from 5 mice, and the splenocytes of the 2 mice are obtained.
2) Fusing splenocytes and mouse myeloma cells, and screening out hybridoma cell strains capable of being combined with the natural solder tip SVMP protein antigen.
And (3) performing cell fusion on SP2/0 mouse myeloma cells from Balb/c mice and spleen cells of 2 screened mice, culturing, observing, detecting and performing negative and positive control tests after the cell fusion to obtain a hybridoma cell strain capable of generating an anti-solder tip SVMP protein antibody, and continuously culturing and selecting the hybridoma cell strain.
3) Inoculating the hybridoma cells into the abdominal cavity of a mouse for induced culture to obtain a cell culture solution after culture, and purifying the cell culture solution to obtain the soldering iron SVMP protein monoclonal antibody capable of immunoreacting with the soldering iron SVMP protein.
Before inoculating hybridoma cells, the mice are injected with 0.5mL of liquid paraffin into the abdominal cavity, and the hybridoma cell suspension prepared by the method is injected into the abdominal cavity of the mice for 2 multiplied by 10 after one week 6 Per mL, 0.5mL hybridoma cell suspension per mouse was injected.
After about 7-10 days after the hybridoma cells are inoculated, ascites of a mouse with obviously swollen abdomen is collected under an aseptic condition, centrifugal treatment is carried out for 10min at 12000g, impurities such as lipid, cells and the like are removed, and supernatant obtained by the centrifugal treatment is taken and inactivated for 30min at 56 ℃ to obtain the SVMP protein monoclonal antibody.
4) The titer of the anti-iron tip SVMP protein antibody (the coating antigen is a natural antigen) is detected by an indirect ELISA (Enzyme Linked Immunosorbent Assay). Specifically, five natural snake venom and natural SVMP proteins are used as coating antigens to screen monoclonal antibodies, wherein the five natural snake venom antigens are shown as A in figure 5, each hole is 100 mu l, the snake venom antigens with the concentration of 10 mu g/ml are coated on a 96-well plate, BSA is blocked and washed, the monoclonal antibodies are diluted by the antibodies secreted by the prepared hybridoma cell strains to be incubated, anti-mouse HRP labeled secondary antibodies are added to be incubated after washing, and display and OD are performed after washing 450 And (6) reading. The specific results are shown in FIG. 5. Only the solder tip can generate specific affinity reaction with the monoclonal antibody, and the monoclonal antibodies secreted by different hybridoma cell strains can generate affinity reaction with the natural SVMP protein.
A second round of screening was performed on the polyclonal antibodies using SVMP-peptide-1. And (3) screening monoclonal cell strains and antibodies with high antibody titer by using the SVMP-peptide-1 polypeptide as a coating antigen. SVMP-peptide antigen (100. Mu.l per well, 10. Mu.g/ml) was coated on 96-well plates, BSA blocked and washed, diluted monoclonal antibody incubated, washed and incubated with anti-murine HRP-labeled secondary antibody, washed and displayed and OD 450 And (6) reading. The results showed that SVMP-mc4, SVMP-mc9 and SVMP-mc18 were able to bind to SVMP-peptide polypeptide antigen (FIG. 6). The monoclonal antibody SVMP-mc18 entrusts Jin Weizhi Biotechnology Inc., suzhou, to determine the light chain variable region and the heavy chain variable region, the specific sequences are shown in SEQ ID NO 2 and SEQ ID NO 3, and the results of the variable region analysis are shown in Table 1.
TABLE 1 monoclonal antibody SVMP-mc18 variable region of each region
Therefore, in the embodiment, the monoclonal antibody is prepared by an in vivo induction method of an animal inoculated in an abdominal cavity, after the hybridoma cells are cloned successfully, the hybridoma is inoculated in the abdominal cavity of a mouse to induce and generate a large amount of ascites, and simultaneously, a large amount of antibody is secreted in the ascites, so that the concentration of the antibody is high, and the antibody with high titer for resisting the soldering iron tip SVMP protein can be screened conveniently.
Example 5
The preparation method of the anti-natural solder tip SVMP protein polyclonal antibody comprises the following specific steps:
native tip SVMP protein was dissolved in saline and filtered through a 0.22 μm sterile filter. 0.5ml of antigen (1 mg of antigen) was emulsified in equal amounts of complete Freund's adjuvant (F-5881, sigma) and injected subcutaneously along multiple sites of the back of male New Zealand white rabbits, two rabbits per antigen. After 2 weeks of intervals, booster immunizations were administered intradermally along multiple sites along the back of male New Zealand rabbits using Freund's incomplete adjuvant (F-5506, sigma) emulsified with an equal volume of antigen (0.5 ml, containing 0.5mg of antigen). Two booster immunizations were given at 2 weeks intervals. Rabbit sera were collected 14 days after the second booster immunization.
Example 6
The prepared monoclonal antibody and polyclonal antibody against the solder tip SVMP protein are respectively combined with solder tip SVMP antigen and five snake venom (bamboo leaf, adder, viper, cobra and solder tip) to detect, and the specific steps are the same as the steps of detecting the antibody titer by the 'indirect ELISA method' in the embodiment 4. Meanwhile, the specificity and space accessibility of the antibody are detected by a western blotting method (WB).
The results are shown in FIG. 7. Wherein A is the result of ELISA and WB detection of an anti-SVMP monoclonal antibody SVMP-mc18. The ELISA and WB results show that the anti-SVMP monoclonal antibody SVMP-mc18 has good specificity. B is the result of ELISA and WB detection of the anti-SVMP polyclonal antibody. ELISA and WB results show that the anti-SVMP polyclonal antibody has low specificity and has obvious cross reaction with the bamboo leaf green, the viper and the viper.
Example 7
The monoclonal antibody is paired with the polyclonal antibody, namely the establishment of the double-antibody sandwich ELISA method
The specific method of antibody pairing is as follows. Coating: diluting the natural SVMP polyclonal antibody to 10 mu g/ml by using the coating solution, wherein each well is 100 mu l, and the natural SVMP polyclonal antibody is coated overnight at 4 ℃; and (3) sealing: discarding the coating solution, washing with PBST for 3 times, 5min each time, and incubating at 37 deg.C for 1h with 200 μ l of blocking solution per well; incubation of the samples: discarding the blocking solution, washing with PBST for 3 times, each time for 5min, diluting five crude toxins (bamboo leaf green, viper, cobra and iron head) with PBS (500 ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml and 7.8125 ng/ml), and adding 100 μ l per hole. Incubating at 37 ℃ for 1h; incubation of anti-SVMP monoclonal antibodies: samples were discarded, PBST washed 3 times for 5min each, and HRP-bearing monoclonal antibody was incubated at a dilution ratio of 1:3000 (the concentration of the antibody stock solution is 1 mg/ml), adding 100 mu l of the antibody stock solution into each well, and incubating for 1h at 37 ℃; and (3) developing: discarding the monoclonal antibody, washing with PBST for 3 times, adding 100 μ l TMB color development solution, and incubating at 37 deg.C in dark for 10min; and (4) terminating: mu.l of stop buffer was added to each well and the absorbance at 450nm was measured for each well.
The result shows that the pairing mode taking the polyclonal antibody as the coating antibody and the monoclonal antibody as the detection antibody is the optimal pairing mode.
Example 8
Establishment of standard curve for detecting snake venom of solder tip
Diluting an anti-tip SVMP protein polyclonal antibody (10 mu g/ml) by PBS, adding 100 mu l of a 96-well plate and 100 mu l of a coating solution into a blank well according to the amount of 100 mu l per well, and coating for 1 hour at 37 ℃; pouring out the coating liquid in the plate holes, washing three times by using washing liquid, wherein each time is 250 mu l, and each time is 5 minutes, and drying the plate holes as much as possible; sealing the solution in each hole by 250 mu l for 1 hour at 37 ℃, then washing for three times, and patting to dry; adding 100 μ l diluted snake venom into the positive well, adding 100 μ l blocking solution into the negative well and the blank well, incubating at 37 deg.C for 1 hr, washing for three times, and patting to dry; adding 100 μ l of Horse Radish Peroxidase (HRP) labeled anti-tip SVMP protein monoclonal antibody (the concentration of an antibody stock solution is 1mg/ml, and the antibody stock solution is diluted according to 1; adding a color development solution into each 100 mu l of washed wells, and incubating for 25 minutes at 37 ℃ in a dark place; the stop solution is 100 μ l per well, and the reading is carried out by a microplate reader at 450/630nm dual wavelength.
Reference is made to FIG. 9 for the flathead venom standard curve. The regression equation is y =0.00343x +0.07938, wherein R 2 =0.994. The results showed good linearity in the range of 6.25ng/ml to 100 ng/ml.
Example 9
A kit for detecting flathead venom by double-antibody sandwich ELISA comprises the following components:
a solid phase carrier coated with an anti-bit SVMP protein polyclonal antibody; the coating concentration is 10 mug/ml;
horse radish peroxidase labeled anti-solder tip SVMP protein monoclonal antibody;
the reagent comprises diluent, a chromogenic substrate, stop solution, a sealing film and a positive standard substance, wherein the positive standard substance is a soldering iron tip natural SVMP protein solution, and the preparation method comprises the following steps:
1. and (3) preparing a crude toxic solution, specifically, respectively taking out a proper amount of freeze-dried powder of the soldering iron head stored in a laboratory, and dissolving the freeze-dried powder with a proper volume of ultrapure water to obtain the crude toxic solution.
2. The crude venom solution samples were diluted 5-fold with phosphate buffered saline as described above (solution A). Then filtered by a 0.22 mu m filter membrane, loaded on a Resource S column and eluted by a linear gradient of 0 to 1mol/L NaCl solution. Then, detecting and collecting each elution peak at the wavelength of 280nm and 215nm, and freeze-drying until obtaining the natural SVMP protein standard product with the purity of more than 95%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (8)
1. A solder tip SVMP protein specificity short peptide is characterized in that the amino acid sequence of the short peptide is shown in SEQ ID NO. 1.
2. The monoclonal antibody SVMP-mc18 against solder tip SVMP protein screened from the short peptide of claim 1, wherein the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as shown in SEQ ID NO:2, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO:3, respectively.
3. An ELISA kit for detecting solder tip snake bite, which comprises a solid phase carrier coated with a polyclonal antibody against solder tip SVMP protein and an enzyme-labeled monoclonal antibody SVMP-mc18 against solder tip SVMP protein according to claim 2.
4. The test kit of claim 3, wherein the polyclonal antibody against the flatiron SVMP protein is prepared by immunizing an animal with SVMP protein isolated and purified from flatiron snake venom.
5. The assay kit of claim 4, wherein the amino acid sequence of the solder tip SVMP protein is as set forth in SEQ ID NO:4, respectively.
6. The detection kit of claim 3, wherein the coating concentration of the polyclonal antibody against tip SVMP protein in the solid phase carrier is 1-100 μ g/ml.
7. The detection kit according to any one of claims 3 to 6, wherein the detection kit further comprises a washing solution, a sample diluent, a substrate, a developing solution and a positive standard.
8. The detection kit according to claim 7, wherein the positive standard is isolated and purified solder tip SVMP protein or a solder tip SVMP protein-specific short peptide according to claim 1.
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