Rapid snake venom poisoning detection method based on ELISA technology

Abstract

The invention belongs to the technical field of snake venom poisoning detection, and provides a snake venom poisoning rapid detection method based on an ELISA technology, and the method comprises the following steps: purifying horse anti-snake venom polyclonal antibody serum of common poisonous snakes by using Protein A and Protein G affinity chromatography and an octanoic acid-ammonium sulfate precipitation method; the purified horse anti-snake venom polyclonal antibody is used for preparing a double-antibody sandwich ELISA detection kit aiming at common snake venom, and detection conditions are optimized; the detection method and the detection kit are applied to human body sample detection of forensic identification cases of suspected snake venom poisoning death; by purifying the horse anti-snake venom polyclonal antibody and preparing the double-antibody sandwich ELISA detection kit, high-sensitivity and high-specificity detection of common snake venom is realized, and the kit is suitable for fresh and decayed biological samples, is successfully applied to forensic identification cases, provides an effective means for rapid and accurate detection of snake venom poisoning, and has a wide application prospect. And the method has important social and economic values.

Description

Technical Field

[0001] The invention belongs to the technical field of snake venom poisoning detection, in particular to a rapid snake venom poisoning detection method based on ELISA technology. Background Art

[0002] In forensic practice, cases of suspected death from snake venom poisoning are not uncommon. However, since some corpses do not have obvious snake tooth marks and pathological histological examinations show no characteristic changes, forensic identification of death from snake venom poisoning is extremely difficult and challenging.

[0003] At present, the existing detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, mass spectrometry, polymerase chain reaction, fluorescent immunoassay, biosensor detection, agglutination test, immunoelectrophoresis, optical immunoassay, fluorescent immunoassay, etc. However, the above detection methods still have problems such as insufficient specificity, low sensitivity or complicated operation.

[0004] Therefore, those skilled in the art have proposed a rapid detection method for snake venom poisoning based on ELISA technology to solve the problems raised by the background technology. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a rapid detection method for snake venom poisoning based on ELISA technology to solve the problems of insufficient specificity, low sensitivity or complex operation in the detection methods in the prior art.

[0006] A rapid detection method for snake venom poisoning based on ELISA technology, comprising:

[0007] Step 1: Purify horse anti-snake venom polyclonal antibody serum of common venomous snakes in China (Bunga, Agkistrodon acutus, Agkistrodon natans and Naja cobra) by using Protein A and Protein G affinity chromatography and caprylic acid-ammonium sulfate precipitation method;

[0008] Step 2: using the purified horse anti-snake venom polyclonal antibody, prepare a double antibody sandwich ELISA detection kit for common snake venoms, and optimize the detection conditions;

[0009] Step 3: Use the optimized detection method and the constructed detection kit to verify the biological samples of rats poisoned by snake venom;

[0010] Step 4: Apply the detection method and detection kit to the detection of human samples in forensic identification cases of suspected snake venom poisoning.

[0011] Preferably, the titers of the purified horse anti-Bungaroca polyclonal antibodies, horse anti-Agkistrodon polyclonal antibodies, horse anti-Agamoebae polyclonal antibodies, and horse anti-Naja polyclonal antibodies all reach 1:64000.

[0012] Preferably, the quantitative detection limit of the double antibody sandwich ELISA detection kit for common snake venoms is: 0.08 ng / mL for Bungarus cobra venom; 1.6 ng / mL for Agkistrodon acutus venom; 1.6 ng / mL for Agkistrodon acutus venom; and 1.6 ng / mL for Cobra venom.

[0013] Preferably, the detection method and detection kit can effectively detect snake venom in fresh and corrupt biological samples of rats poisoned by snake venom, and the kidney is the best detection sample for Bungarus cobra venom and Cobra venom, and the injection site tissue is the best detection sample for Acuta aegypti venom.

[0014] Preferably, the detection method and detection kit were successfully applied to 3 forensic identification cases of suspected snake venom poisoning, and were able to determine whether snake venom poisoning occurred and identify the species of the venomous snake.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention achieves high sensitivity and high specificity detection of common snake venoms (Bunga, Agkistrodon acutus, Agkistrodon natans, and Cobra) by purifying horse anti-snake venom polyclonal antibodies and preparing a double antibody sandwich ELISA detection kit, with a detection limit as low as nanogram level.

[0017] 2. This method is not only applicable to the detection of fresh biological samples, but also to the detection of corrupt biological samples, solving the problem of corrupt sample detection often encountered in forensic practice.

[0018] 3. The present invention has been successfully applied to forensic identification cases of suspected snake venom poisoning. It can accurately determine whether it is snake venom poisoning and determine the species of the venomous snake. It provides an important basis for the forensic identification of common snake venom poisoning, helps to build a complete chain of evidence, and assists in case detection.

[0019] 4. The horse anti-polyclonal antibody serum used in the present invention has low production cost and is easy to produce on a large scale. It is expected to be widely used in clinical diagnosis and forensic identification, and has important social and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the direct ELISA detection method of the present invention;

[0021] Figure 2 It is a schematic diagram of the indirect ELISA detection method of the present invention;

[0022] Figure 3 This is a diagram of the double antibody sandwich ELISA detection model of the present invention;

[0023] Figure 4 A schematic diagram of a suspected snake bite mark on the back of the right foot of a corpse of the present invention;

[0024] Figure 5 A schematic diagram of a suspected snake bite mark on the back of the right hand of a corpse of the present invention;

[0025] Figure 6 A schematic diagram of a suspected snake bite on the back of the right hand of a corpse of the present invention;

[0026] Figure 7 It is a schematic diagram of the potency of the horse anti-snake venom polyclonal antibody after purification of the present invention;

[0027] Figure 8 This is an example diagram of the sandwich ELISA test kit of the present invention; wherein, A: Bungarus cobra venom test area; B: Agkistrodon acutus venom test area; C: Agkistrodon acutus venom test area; D: Cobra venom test area; TEST: test area; PC: positive control area; NC: negative control area;

[0028] Fig. 9 Schematic diagram of determining the critical value of the ELISA kit of the present invention; wherein, A. Bungarus group; B. Agkistrodon group; C. Agkistrodon group; D. Naja group;

[0029] Fig.10 The figure is a schematic diagram of the standard curve for snake venom detection of the ELISA kit of the present invention, wherein A. Bungarus group; B. Agkistrodon group; C. Agkistrodon group; D. Naja group;

[0030] Fig.11 The schematic diagram of the specificity test of the Agkistrodon agkistrodon group and the Agkistrodon acutus group in the ELISA kit of the present invention; wherein, A. Agkistrodon agkistrodon group; B. Agkistrodon acutus group;

[0031] Fig.12 This is a schematic diagram of the ELISA kit for testing rats poisoned by Bungarus cobra of the present invention;

[0032] Fig.13 The figure is a schematic diagram of the ELISA kit for detecting rats poisoned by Agkistrodon agkistrodon of the present invention;

[0033] Fig.14 The schematic diagram of the ELISA kit for detecting rats poisoned by Agkistrodon acutus of the present invention;

[0034] Fig.15 The schematic diagram of the ELISA kit for detecting rats poisoned by cobra of the present invention;

[0035] Fig.16 This is a schematic diagram of the ELISA kit for detecting human samples in Case 1 of the present invention; wherein, A: Bungarus corytatus detection area; TEST: detection area; NC: negative control area; 1, 2 experimental groups; 3, 4 control groups;

[0036] Fig.17This is a schematic diagram of the ELISA kit for human sample detection in Case 2 of the present invention; wherein, C: Snake detection area; TEST: detection area; NC: negative control area; 1, 2 experimental groups; 3, 4 control groups;

[0037] Fig.18 This is a schematic diagram of the ELISA kit detection of human samples in Case 3 of the present invention; wherein A: Bungarus cobra detection area; B: Agkistrodon acutus detection area; TEST: detection area; NC: negative control area; 1, 2 experimental groups; 3, 4 control groups. DETAILED DESCRIPTION

[0038] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] Embodiment: The present invention provides a method for rapid detection of snake venom based on ELISA technology, comprising:

[0040] 1. Experimental Preparation

[0041] 1. Experimental Materials and Reagents

[0042] Horse anti-snake venom polyclonal antibody serum: horse anti-bungaroves snake venom polyclonal antibody serum, horse anti-agkistrodon acutus snake venom polyclonal antibody serum, horse anti-agropyron snake venom polyclonal antibody serum, horse anti-cobra snake venom polyclonal antibody serum, all provided by Shanghai Seron Biotechnology Co., Ltd.;

[0043] Snake venom freeze-dried powder: Bungarus venom freeze-dried powder, Agkistrodon acutus venom freeze-dried powder, Agkistrodon acutus venom freeze-dried powder, Cobra venom freeze-dried powder, provided by Kunming Institute of Zoology, Chinese Academy of Sciences;

[0044] Reagents: including pH 3.0 citrate buffer, saturated ammonium sulfate solution, sodium azide, pH 4.0 acetate buffer, caprylic acid, coating buffer, Block blocking solution, sample diluent, enzyme marker diluent, washing solution, colorimetric solution, stop solution, etc., all provided by Wuhan Keyuan Anbo Biotechnology Co., Ltd.;

[0045] Other materials: Biosun bs-0308R rabbit anti-horse IgG H&L was purchased from Wuhan Beiyinlai Biotechnology Co., Ltd.

[0046] Rabbit Anti-HorseIgG H&L HRP labeled with Biosun bs-0308R-HRP horseradish peroxidase was purchased from Wuhan Beiyinlai Biotechnology Co., Ltd.

[0047] RIPA lysis buffer G2002-100ML was purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0048] Protease inhibitor G2008-1ML was purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0049] Other reagents and materials not otherwise specified were provided by Wuhan Keyuan Anbo Biotechnology Co., Ltd.

[0050] 2. Experimental Instruments

[0051] Precision electronic analytical balance (Sartorius, Germany)

[0052] Single channel pipette (Eppendorf, Germany)

[0053] Magnetic stirrer (Shengwei, China)

[0054] Low temperature refrigerator (Meiling, China)

[0055] Ultra-low temperature refrigerator (Eppendorf, Germany)

[0056] Low temperature high speed centrifuge (Eppendorf, Germany)

[0057] Constant temperature water bath (Guohua, China)

[0058] Multifunctional microplate reader (Thermo, USA)

[0059] Horizontal shaker (Kylin-Bell, China)

[0060] Ultrapure water filtration system (Merk, USA)

[0061] Small animal anesthesia machine (Rayward, USA)

[0062] Liquid nitrogen tank (Thermo Fisher, USA)

[0063] Digital constant temperature HH-6 water bath (Guohua Group, China).

[0064] 3. Experimental animals: The experimental animals were adult male Sprague-Dawly rats, weighing 280 g. They were randomly divided into a group poisoned by Bungarus serpentis, agkistrodon acutus, agkistrodon quinquefasciatus, cobra venom and a control group. There were 3 rats in each group. They were kept in the same breeding environment for one week before the start of the experiment, with 4 to 5 rats in each cage. The rats were kept strictly according to the 12-h light cycle, and the humidity and temperature were kept constant, with frequent ventilation. The animal experiments in this study were strictly carried out in accordance with the national "Guidelines for the Care and Use of Laboratory Animals". All experimental animals were purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology.

[0065] 4. Human samples of snake venom poisoning: The human samples used in this experiment were all from the Hubei Tongji Forensic Medicine Judicial Appraisal Center. By referring to whether the bodies had hole-like defects (i.e., suspected snake bite marks), 3 suspected snake venom poisoning cases were screened out. The case information is shown in the table:

[0066] Human specimen information sheet

[0067]

[0068] 2. Experimental Methods

[0069] 1. Purification of horse antivenom polyclonal antibody serum

[0070] (1) Purification of horse anti-bungarotoxin polyclonal antibody serum using Protein A affinity chromatography

[0071] ① Take serum, add a saturated ammonium sulfate solution of the same volume as the serum while stirring, stir at room temperature for 20 minutes, and then place in a 4℃ refrigerator overnight;

[0072] ② The next day, centrifuge and discard the supernatant, add 45% saturated ammonium sulfate solution at a volume ratio of 1:2 to dissolve the precipitate; centrifuge and discard the supernatant, add an appropriate volume of PBS solution until the precipitate is completely dissolved, and place it in PBS solution for dialyzation in a 4°C refrigerator overnight, changing the dialysate every 4 hours, and change the sample 3 times;

[0073] ③ Load the sample with PBS solution of pH 8.0 to buffer the Protein A chromatography equilibrium column until the pH value of the effluent is 8.0; load the serum pretreated as above onto the chromatography column, and when the value rises, i.e. when the elution peak appears, start collecting the sample, add PBS solution of pH 8.0, and stop collecting the sample when the value drops to a stable value, i.e. when the elution peak returns to the baseline;

[0074] ④ Load the sample with citrate buffer at pH 3.0, rinse the Protein A chromatography equilibrium column, wash away the impurities, add PBS solution at pH 8.0, buffer the chromatography equilibrium column until it is neutral, add sodium azide, and store in a refrigerator at 4°C.

[0075] (2) Purification of horse anti-agkistrodon acutus venom polyclonal antibody serum, horse anti-five-step snake venom polyclonal antibody serum, and horse anti-cobra venom polyclonal antibody serum using Protein G affinity chromatography

[0076] Same steps as above.

[0077] (3) Purify the purified antibody using caprylic acid-ammonium sulfate precipitation method

[0078] ① Use a UV spectrophotometer to measure the concentration of the antibody after purification by Protein A and Protein G, and dilute the antibody with PBS solution to a concentration between 7 and 8 mg / mL;

[0079] ② Place the four antibodies in a beaker, add 2 times the volume of 60mM pH 4.0 acetate buffer dropwise while stirring, stir evenly to make it acidic;

[0080] ③ Add 0.15 times the total volume of octanoic acid in the previous step, stir at room temperature for 20 minutes at 450 rpm; after stirring evenly, centrifuge at 4°C for 20 minutes at 4500 rpm, filter with lens paper and take the supernatant;

[0081] ④Add solid ammonium sulfate and stir evenly;

[0082] ⑤ After dissolution, add ammonia water to adjust the pH value of the solution to neutral while stirring at 260rpm; centrifuge at 4℃ for 20min, 4500rpm; discard the supernatant;

[0083] ⑥ Resuspend the precipitate with PBS solution and dissolve it, put it into a dialysis bag, and place it in PBS solution in a 4°C refrigerator for overnight dialysis. Change the dialysis solution every 4 hours, and change the sample 3 times;

[0084] ⑦ Measure the antibody concentration using a UV spectrophotometer, keep half of it for sample, and add sodium azide to the other half and store it at -20℃ for later use.

[0085] (4) HRP labeling of purified antibodies

[0086] ① One night in advance, extract 5 mg of the four antibodies purified in the previous step without adding sodium azide and dialyze them in PBS solution overnight to remove impurity ions;

[0087] ② Weigh 30 mg of HRP into a brown vial and add 1 mL of pure water to dissolve; weigh 30 mg of NaIO4 into an EP tube and add 1 mL of pure water to dissolve;

[0088] ③ After both solutions are fully dissolved, add NaIO 4 The solution was added to the HRP solution and mixed at 4°C in the dark for 30 min at 110 rpm;

[0089] ④ Pipette 30 μL of ethylene glycol into an EP tube, add 1 mL of pure water to dissolve, and after fully dissolved, add to the mixed solution in (3) where the reaction is completed, and mix at room temperature in the dark for 30 minutes at 110 rpm;

[0090] ⑤ Add the liquid in step 4 to the four dialyzed antibodies respectively, place in coating buffer and react overnight in a 4°C refrigerator, replace the coating buffer every 4 hours, and change the sample 3 times;

[0091] ⑥ Weigh 4 mg of NaBH 4 In the EP tube, add 0.4 mL of pure water to dissolve; after fully dissolved, add 100 μL of the dissolving solution to each dialysis bag in the previous step, and react at 4°C in the dark for 2 h;

[0092] ⑦ Place the liquid in the previous step in PBS solution and dialyze overnight in a 4°C refrigerator at 210 rpm. Change the dialysate every 4 hours for 3 times.

[0093] ⑧ Collect samples, add equal volumes of glycerol, mix well and place in a -20℃ refrigerator for later use.

[0094] (5) Method for determining the titer of purified antibodies

[0095] Direct ELISA was used to detect the working concentration of HRP-labeled rabbit anti-horse polyclonal antibody in the ELISA plate of snake venom lyophilized powder dilution. The ELISA reaction mode was as follows: Figure 1 As shown:

[0096] ① Use coating buffer to dilute the freeze-dried powder of Bungarus, Agkistrodon acutus, Agkistrodon natans, and Cobra to 2 μg / mL, and coat the plate with 100 μL per well to prepare ELISA plates of four kinds of snake venom freeze-dried powder dilutions, and incubate at room temperature overnight;

[0097] ② The next day, discard the solution, wash the plate twice with the washing solution, and pat the plate dry; add 125 μL of blocking solution to each well and leave it at room temperature for 4 hours;

[0098] ③Discard the liquid and dry it in a drying oven at 37℃ overnight;

[0099] ④ Collect the ELISA plate and store it at 4℃ for later use;

[0100] ⑤ Before the experiment, take out the four ELISA plates and equilibrate them to room temperature;

[0101] ⑥ Dilute the HRP-labeled rabbit anti-horse polyclonal antibody to be tested with enzyme diluent at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, and add them to the first 11 wells of the corresponding ELISA plate in sequence, and add the sample diluent to the 12th well, with 100 μL per well;

[0102] ⑦ Place in a 37℃ water bath and incubate for 30 min, discard the solution, wash the plate 5 times with washing solution, and pat the plate dry;

[0103] ⑧ Add 50 μL of ELISA colorimetric solution A and solution B to each well, colorimetric for 10 min in a dark environment, add 50 μL of stop solution, and read the colorimetric solution at dual wavelengths (OD 450 -OD 630 ), select the antibody dilution concentration when the OD reading is less than 0.05 and no longer decreases as its working concentration.

[0104] (6) Determination of antibody titer after purification

[0105] The indirect ELISA method was used to evaluate the titer of the purified horse anti-snake venom polyclonal antibody. The ELISA reaction pattern was as follows Figure 2 As shown;

[0106] ① Before the experiment, take out the ELISA plates of the four kinds of snake venom freeze-dried powder dilutions prepared in the previous step and equilibrate them to room temperature;

[0107] ② Dilute the four purified antibodies to be tested with sample diluent at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and 1:1024000, and add them to the first 11 wells of the corresponding ELISA plate in sequence. Add sample diluent (negative control) to the 12th well, 100 μL per well;

[0108] ③ Place in a 37℃ water bath and incubate for 40 min, discard the solution, wash the plate 5 times with washing solution, and pat the plate dry;

[0109] ④ Each well was incubated in a 37°C water bath for 30 min with 100 μL of HRP-labeled rabbit anti-horse polyclonal antibody (the working concentration is known from the previous step), then the plate was discarded, washed 5 times with washing solution, and the plate was patted and dried;

[0110] ⑤ Add 50 μL of ELISA colorimetric solution A and solution B to each well, colorimetric for 10 min in a dark environment, add 50 μL of stop solution, and read the results with a dual wavelength microplate reader (OD 450 -OD 630 ).

[0111] 2. Preparation of double antibody sandwich ELISA test kit

[0112] (1) Assembly and operation process of double antibody sandwich ELISA kit

[0113] Purified horse anti-snake venom polyclonal antibodies of Bungarus, Agkistrodon acutus, Agkistrodon avianus, and Cobra were used as capture antibodies, and the corresponding HRP-labeled polyclonal antibodies were used as enzyme-labeled antibodies to establish a double antibody sandwich ELISA detection method for Bungarus, Agkistrodon acutus, Agkistrodon avianus, and Cobra. The reaction mode is as follows: Figure 3 The experimental operation process is as follows:

[0114] S1. Coating: The purified antibody is used as the capture antibody, diluted with coating buffer and added to the ELISA plate, 100 μL per well, and coated overnight at room temperature;

[0115] S2, plate washing: the next day, discard the solution, wash the plate with washing solution, repeat twice, and tap the plate to dry;

[0116] S3, blocking: add 125 μL of blocking solution to each well and leave to block at room temperature for 4 h;

[0117] S4, drying: discard the liquid and dry it in a drying oven at 37°C overnight;

[0118] S5, assay incubation: 100 μL per well, incubate in a 37°C water bath for 40 min;

[0119] S6, plate washing: wash the plate with washing solution, repeat 5 times, and pat the plate to dry;

[0120] S7, enzyme-labeled antibody incubation: The HRP-labeled antibody was used as the enzyme-labeled antibody and incubated, 100 μL per well, in a 37°C water bath for 30 min;

[0121] S8, plate washing: wash the plate with washing solution, repeat 5 times, and pat the plate to dry;

[0122] S9, color development: add 50 μL of TMB color development solution A and solution B to each well, and color development for 10 min at room temperature in the dark;

[0123] S10, Reading: Add 50 μL of stop solution to each well and read the double wavelength (OD 450 -OD 630 ).

[0124] (2) Optimization of experimental conditions of double antibody sandwich ELISA kit

[0125] ① Determination of the working concentration of enzyme-labeled antibody

[0126] Before the experiment, the kit prepared in the previous step was taken out and equilibrated to room temperature; the detection source was incubated with the sample diluent; the enzyme-labeled antibody was diluted with the enzyme-labeled antibody diluent at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400, and added to the first 11 wells of the ELISA plate in sequence, and the enzyme-labeled antibody diluent (negative control) was added to the 12th well; the rest of the experimental procedures were carried out according to the operating procedures of the above-mentioned kit; the antibody dilution concentration with a dual-wavelength reading OD value of about 0.04 was selected as its working concentration;

[0127] ②Determination of the optimal concentration of capture antibody

[0128] The capture antibody was diluted to 2 μg / mL, 5 μg / mL, and 10 μg / mL with sample diluent for incubation; positive samples and negative samples were tested; the remaining working conditions were selected according to the above optimized conditions, and the experimental procedures were carried out according to the operating procedures of the above kit; the dual wavelength reading OD value was recorded and the most suitable concentration of the capture antibody was selected according to the P / N value;

[0129] ③ Determination of the most suitable incubation time for the detection source

[0130] Three detection source incubation time groups of 10min, 40min, and 90min were set; each group tested the gradient dilution of snake venom dry powder, and a negative control group was set up at the same time; the other working conditions were selected according to the above optimized conditions, and the experimental procedures were carried out according to the operating procedures of the above kit; the dual-wavelength reading OD value was recorded and the most suitable incubation time of the detection source was selected according to the sensitivity;

[0131] ④ Determination of the most suitable blocking solution

[0132] 15% FCS, 5% BSA and Block solution were set as blocking solutions to block the test kit respectively; positive samples and negative samples were incubated on the test kits blocked with three different blocking solutions respectively; the remaining working conditions were selected according to the above optimized conditions, and the experimental procedures were carried out according to the operating procedures of the above test kits; the dual-wavelength reading OD values ​​were recorded and the most suitable type of blocking solution was selected according to the P / N value.

[0133] (3) Performance test of double antibody sandwich ELISA kit

[0134] ①Determination of critical value

[0135] In order to determine the detection critical value of the constructed double antibody sandwich ELISA kit, 24 sample dilutions were tested under the optimized experimental conditions, and the double wavelength reading OD value was ≥3SD+X, which was considered positive; SD was the standard deviation of the OD value of the 24 sample dilutions tested, and X was the average OD value of the 24 sample dilutions tested;

[0136] ②Sensitivity test and drawing standard curve

[0137] In order to evaluate the sensitivity of the constructed double antibody sandwich ELISA kit, the gradient dilution of snake venom powder was detected under the optimized experimental conditions. The venom powders of Bungarus, Agkistrodon, Agkistrodon acutus, and Cobra were gradiently diluted with sample diluent starting from 10 μg / mL. The sample diluent was used as the negative control in the 12th well. The OD value of the dual-wavelength reading was recorded. According to the critical value established in the previous step, the minimum detection line concentration of each group, i.e., its sensitivity, was obtained. The appropriate dilution interval was selected in combination with the dual-wavelength reading OD value, and the standard curve test was carried out. Three replicate wells were set for each concentration. The standard curve was established with different snake venom antigen concentrations and the corresponding dual-wavelength reading OD values, and the curve was fitted.

[0138] ③Specificity test

[0139] In order to evaluate the specificity of the constructed double antibody sandwich ELISA kit, the antibody group was captured by Bungarus cobra, Agkistrodon acutus, and Cobra venom lyophilized powder dilutions were tested, and the sample dilutions were used for gradient dilution from 1 μg / mL, and repeated 2 wells. The sample dilution was used as a negative control in the 12th well.

[0140] Using the pit viper capture antibody group, test the dilution of the lyophilized powder of Bungarus, Agkistrodon acutus, and Cobra venom, and perform gradient dilution with the sample diluent starting from 1 μg / mL, repeat 2 wells, and use the sample diluent in the 12th well as a negative control;

[0141] Using the Acutus acutus capture antibody group, test the dilutions of the lyophilized powder of Agkistrodon acutus, Bungarus cobra and Cobra venom, and perform gradient dilutions with the sample diluent starting from 1 μg / mL, repeat 2 wells, and use the sample diluent in the 12th well as a negative control;

[0142] The cobra capture antibody group was used to test the lyophilized powder dilutions of Agkistrodon acutus, Agkistrodon quinquefasciatus, and Bungarus cobra venom. The sample diluent was used for gradient dilution starting from 1 μg / mL, and repeated for 2 wells. The sample dilution was used as a negative control in the 12th well. The specificity of the kit was evaluated by analyzing the obtained dual-wavelength reading OD values.

[0143] 4. Repeatability test

[0144] In order to evaluate the repeatability and stability of the constructed double antibody sandwich ELISA kit, the same concentration of snake venom dry powder dilution was detected with ELISA plates coated with the same batch. Three replicate wells were made for each concentration of snake venom dry powder dilution. The mean and standard deviation of the dual-wavelength OD values ​​corresponding to each concentration were calculated, and the intra-batch repeatability of the kit was evaluated by the coefficient of variation.

[0145] Three groups of ELISA plates coated with different batches were used to detect the same concentration of snake venom dry powder dilutions. The mean and standard deviation of the dual-wavelength OD values ​​corresponding to each concentration were calculated, and the intra-batch repeatability of the kit was evaluated by the coefficient of variation.

[0146] 3. Detection of rat samples poisoned by snake venom

[0147] 1) Using the optimized method and constructed product, fresh samples of rats poisoned by snake venom were tested to verify that the detection method and product are suitable for snake venom detection in biological samples;

[0148] 2) Using the optimized method and constructed product, the spoiled samples of rats poisoned by snake venom were tested to verify that the detection method and product are also applicable to the detection of snake venom in spoiled biological samples.

[0149] 1. Preparation and collection of fresh samples from rats poisoned by snake venom

[0150] The rats were divided into 5 groups, namely, cobra venom group, Agkistrodon acutus venom group, Bungarus cobra venom group, Agkistrodon acutus venom group and control group, with 3 rats in each group; the rats were fasted and kept overnight the night before the experiment; the rats in each snake venom group were intraperitoneally injected with the corresponding snake venom according to the median lethal dose of various snake venoms, as shown in Table 1, and the rats in the control group were intraperitoneally injected with 0.4 mL of normal saline; the behavioral state of the rats after administration was observed.

[0151] Table 1 Characteristics of venom of four venomous snakes

[0152]

[0153] After 4 hours, observe whether there are any changes in the moribund state, induce anesthesia, collect orbital blood (preparation is successful if changes in the moribund state and snake venom is detected in the serum), and perfuse, separate, and grind the tissues and organs for later use; the specific process is as follows:

[0154] ① After inducing breathing in rats with isoflurane, place them in a supine position on a foam board;

[0155] ②Expose the rat's chest and abdominal cavity, and perform cardiac perfusion with normal saline (about 150 mL) until the liver turns grayish white, indicating that blood replacement is complete, and then stop perfusion;

[0156] ③ Rapidly extract the heart, liver, spleen, lung, kidney, and skin tissues at the injection site, place them on ice, rinse them with physiological saline, put them into cryopreservation tubes, and store them in a -80℃ refrigerator for later use;

[0157] ④ When using, extract 70 mg of each tissue into an EP tube and cut into pieces;

[0158] ⑤ Add 1.4 mL of pre-cooled RIPA lysis buffer and 14 μL of PMSF solution, and add 2 magnetic beads to each EP tube, and grind using a grinder (60 Hz, 60 s, 2 times);

[0159] ⑥Pipette the supernatant into a centrifuge tube and centrifuge at 4°C, 10,000 rpm for 10 min;

[0160] ⑦ The supernatant was stored in a -80℃ refrigerator for later use and recorded as the fresh group of rats poisoned by snake venom.

[0161] 2. Preparation and collection of samples from the putrefaction group of rats poisoned by snake venom

[0162] In order to prove that the optimized method and constructed product are applicable to the field of forensic practice (the specimens in forensic snake bite death cases are mostly corrupt samples), 70 mL of each biological sample ground in the above steps was extracted into an EP tube, the tube cap was opened, and placed outdoors at 35°C in a flowing and ventilated place for two weeks, which was recorded as the snake venom poisoning rat corrupt group.

[0163] 3. Testing methods and product samples in rats

[0164] The double antibody sandwich ELISA test kit constructed in this study was used to test the fresh group and the corrupted group of biological samples of snake venom poisoned rats prepared and collected, so as to evaluate the application scope of the test method and product;

[0165] ① The heart, liver, spleen, lung and kidney tissue homogenates of the fresh group and the corrupted group of rats poisoned by Bungarus cobra were selected for testing. Three replicate wells with 3 wells were set for the same sample. At the same time, the tissue homogenate of the control group of rats was set as a negative control. The sandwich ELISA test was performed under the optimized experimental conditions, and the dual-wavelength reading OD value was recorded.

[0166] ② The heart, liver, spleen, lung, kidney and skin tissue homogenates of the fresh group and the corrupt group of rats poisoned by agkistrodon agkistrodon were selected for testing. Three replicate wells with 3 wells were set for the same sample. At the same time, the tissue homogenate of the control group of rats was set as a negative control. The sandwich ELISA test was performed under the optimized experimental conditions, and the dual-wavelength reading OD value was recorded.

[0167] ③ The heart, liver, spleen, lung, kidney and skin tissue homogenates of the fresh group and the corrupt group of rats poisoned by A. acutus were selected for testing. Three replicate wells with 3 wells were set for the same sample. At the same time, the tissue homogenate of the control group of rats was set as a negative control. The sandwich ELISA test was carried out under the optimized experimental conditions, and the dual-wavelength reading OD value was recorded.

[0168] ④ The heart, liver, spleen, lung and kidney tissue homogenates of the fresh group and the corrupt group of rats poisoned by cobra were selected for testing. Three replicate wells with 3 wells were set for the same sample. At the same time, the tissue homogenate of the control group rats was set as the negative control. The sandwich ELISA test was carried out under the optimized experimental conditions, and the dual-wavelength reading OD value was recorded.

[0169] 4. Testing of human samples suspected of snake venom poisoning

[0170] Case 1

[0171] A 46-year-old woman went up the mountain to dig bamboo shoots and disappeared. Her body was found in the mountain one day later. A forensic autopsy was performed to determine the cause of death and whether it was a venomous snake bite or snake venom poisoning. The autopsy showed multiple pore-like injuries on the skin of the right foot and left wrist of the body, arranged in parallel, with edema of the skin, dark red around, and subcutaneous hemorrhage on the cut surface, which was suspected to be snake bites. Figure 4 As shown;

[0172] Two pieces of skin around the suspected snake bite were selected and ground into tissue homogenate, which was tested using a double antibody sandwich ELISA test kit. At the same time, a tissue homogenate sample from a corpse without snake venom bite was set as a negative control, and the dual-wavelength reading OD value was recorded.

[0173] Case 2

[0174] A 52-year-old man was bitten by a snake at the base of his right hand. He treated himself with Chinese medicine and bloodletting, but was found dead in bed one day later. Due to a medical dispute, he requested a forensic autopsy to determine the cause of death. The right upper limb (right palm, forearm and upper arm), right chest and right back of the corpse were highly bruised and swollen, with bloody blisters of varying sizes. The epidermis was easily peeled off, exposing the dermis, which felt like twisting hair when touched. Figure 5 As shown;

[0175] Human samples from the case were selected, ground into tissue homogenates, and tested using a double antibody sandwich ELISA test kit. At the same time, tissue homogenate samples from corpses that were not bitten by snake venom were set as negative controls, and the dual-wavelength reading OD values ​​were recorded.

[0176] Case 3

[0177] A 24-year-old man was found dead on a hillside in the garden lawn after leaving the accommodation area for one day. A forensic autopsy was performed to determine whether the cause of death was snake venom poisoning. The autopsy revealed that there were hole-like defects on the back of the right wrist, the middle of the right forearm, and the middle of the left upper arm, with black leather-like surroundings, which were suspected to be snake bites. Figure 6 As shown;

[0178] Two pieces of skin around the suspected snake bite marks in the case were selected and ground into tissue homogenate, which were tested using a double antibody sandwich ELISA test kit. At the same time, a tissue homogenate sample from a corpse without snake venom bites was set as a negative control, and the dual-wavelength reading OD value was recorded.

[0179] V. Experimental Results

[0180] 1. Optimization of snake venom detection methods and product construction

[0181] (1) Purification of horse antivenom polyclonal antibody serum

[0182] Direct ELISA method was used to measure the working concentration of rabbit anti-Madock antibody in the four snake venom ELISA plates, which was 1:2000;

[0183] The indirect ELISA method was used to detect the titer of the purified antibody. Figure 7 As shown, the titers of the four purified antibodies can all reach 1:64000;

[0184] The concentration of the purified horse anti-bungarotoxin polyclonal antibody was calculated to be 7.33 mg / mL, the concentration of the horse anti-agkistrodon polyclonal antibody was 2.15 mg / mL, the concentration of the horse anti-agara venom polyclonal antibody was 2.02 mg / mL, and the concentration of the horse anti-cobra venom polyclonal antibody was 2.44 mg / mL;

[0185] The antibody concentration and titer can fully meet the requirements of the preparation of sandwich ELISA kits and colloidal gold immunochromatography detection methods.

[0186] (2) Preparation of double antibody sandwich ELISA detection kit

[0187] 1) Kit assembly and operation procedures

[0188] The schematic diagram of the sandwich ELISA detection kit finally constructed is as follows Figure 8 As shown; the venom of Bungarus serpentis, Agkistrodon acutus, Agkistrodon quinquefasciatus and Cobra venom can be tested simultaneously in different areas in the same experiment. According to the drawn standard curve, the OD value of the test sample is substituted into the equation to obtain the specific venom concentration in the corresponding sample; the yellower the color (far left), the higher the OD value, and the higher the corresponding venom concentration.

[0189] 2) Optimization of ELISA kit test conditions

[0190] ① Determination of the working concentration of enzyme-labeled antibody

[0191] According to the above steps, the OD value results are shown in the table. After comparison, it was finally determined that the working concentration of the enzyme-labeled antibody in the Bungarus group was 1:800; the working concentration of the enzyme-labeled antibody in the Agkistrodon group was 1:2000; the working concentration of the enzyme-labeled antibody in the Agkistrodon group was 1:3000; and the working concentration of the enzyme-labeled antibody in the Cobra group was 1:1600, as shown in Table 2;

[0192] Table 2 Determination of the working concentration of enzyme-labeled antibodies in ELISA kits

[0193]

[0194] ②Determination of the optimal concentration of capture antibody

[0195] According to the above steps and conditions, the reaction results are as shown in the table. The optimum concentration of the capture antibody of the Bungarus group is 10 μg / mL, and the P / N value is 11.22 at this time; the optimum concentration of the capture antibody of the Agkistrodon group is 5 μg / mL, and the P / N value is 17.81 at this time, but considering the detection sensitivity, 10 μg / mL is finally selected as the most suitable coating antibody concentration; the optimum concentration of the capture antibody of the Acuta group is 10 μg / mL, and the P / N value is 17.02 at this time; the optimum concentration of the capture antibody of the Cobra group is 10 μg / mL, and the P / N value is 21.35 at this time, as shown in Table 3;

[0196] Table 3 Determination of the optimal concentration of capture antibody in ELISA kit

[0197]

[0198] ③ Determination of the most suitable incubation time for the detection source

[0199] According to the above steps and conditions, the reaction results are shown in the table, where the detection sensitivity of the Bungarus 40-min incubation group is better than that of the 10-min incubation group, while the 90-min incubation group will produce false positive results, indicating that the most suitable incubation time for the detection source in the Bungarus group is 40 minutes, as shown in Table 4;

[0200] Table 4 Determination of the most suitable incubation time for the detection of Bungarus group in ELISA kit

[0201]

[0202]

[0203] The sensitivity of the 40-min incubation group and the 90-min incubation group of the pit viper was better than that of the 10-min incubation group. In view of the time saving, it was finally confirmed that the most suitable incubation time for the detection in the pit viper group was 40 min, as shown in Table 5;

[0204] Table 5 Determination of the most suitable incubation time for the detection of Agkistrodon agkistrodon in ELISA kit

[0205]

[0206] The detection sensitivity of the 40-min incubation group of A. quinquefasciatus was better than that of the 10-min incubation group and the 90-min incubation group, indicating that the most suitable incubation time for the detection of A. quinquefasciatus group was 40 min, as shown in Table 6;

[0207] Table 6 Determination of the most suitable incubation time for the detection of the five-step snake group in the ELISA kit

[0208]

[0209] The detection sensitivity of the 40-min incubation group of Cobra was better than that of the 10-min incubation group, while the 90-min incubation group showed false positive results, indicating that the most suitable incubation time for the Cobra group detection was 40 min, as shown in Table 7;

[0210] Table 7 Determination of the most suitable incubation time for the cobra group detection in the ELISA kit

[0211]

[0212] ④ Determination of the most suitable blocking solution

[0213] According to the above steps and conditions, the reaction results are shown in the table, indicating that the most suitable type of blocking solution for the Bungarus group is Block solution, and the P / N value is 42.50 at this time; the Block group of the Agkistrodon group is similar to the 5% BSA group, and the Block group is selected for uniformity, and the P / N value is 24.60 at this time; the most suitable type of blocking solution for the Agropyron group is Block solution, and the P / N value is 24.07 at this time; the Block group of the Cobra group is similar to the 5% BSA group, and the Block group is selected for uniformity, and the P / N value is 27.34 at this time, as shown in Table 8;

[0214] Table 8 Determination of the most suitable blocking solution type in ELISA kit

[0215]

[0216]

[0217] 3) ELISA kit performance test

[0218] ①Determination of critical value

[0219] According to the above steps and conditions, the OD values ​​of the 24 sample dilutions are obtained, such as Fig. 9 As shown;

[0220] The calculated OD mean (X) of the Bungarus group was 0.0526, and the standard deviation (SD) was 0.0095. The positive and negative critical value of the Bungarus group was determined to be 0.081 by the formula X+3SD, that is, when the OD value ≥ 0.081, it was judged to be positive and contained Bungarus venom;

[0221] The average OD value (X) of the pit viper group was 0.0571, and the standard deviation (SD) was 0.0057. The positive and negative critical value of the pit viper group was determined to be 0.074 by the formula X+3SD, that is, when the OD value ≥ 0.074, it was judged to be positive and contained pit viper venom;

[0222] The OD mean value (X) of the Agkistrodon acutus group was 0.0601, and the standard deviation (SD) was 0.0051. The positive and negative critical value of the Agkistrodon acutus group was determined to be 0.075 by the formula X+3SD, that is, when the OD value ≥ 0.075, it was judged to be positive and contained Agkistrodon acutus venom;

[0223] The average OD value (X) of the cobra group was 0.0914, and the standard deviation (SD) was 0.033. The positive and negative critical value of the cobra group was determined to be 0.191 through the formula X+3SD, that is, when the OD value ≥ 0.191, it was judged to be positive and contained cobra venom.

[0224] ②Sensitivity test and drawing standard curve

[0225] According to the results obtained in the above steps and conditions, the snake venom detection standard curve of the ELISA kit was established as follows: Fig.10 As shown;

[0226] The calculated limit of detection (LOD) of the venom of Bungarus cobra was 80pg / mL, and the coefficient of determination (R2) was 0.9975, indicating that the actual measured value had a high degree of fit with the curve, and the calculated results of the content of Bungarus cobra venom were reliable;

[0227] The detection limit of Agkistrodon acutus venom was calculated to be 1.6 ng / mL, and the determination coefficient was 0.9955, indicating that the actual measured value had a high degree of fit with the curve, and the calculation result of the content of Agkistrodon acutus venom was reliable;

[0228] The calculated detection limit of the five-step snake venom was 1.6 ng / mL, and the determination coefficient was 0.9906, indicating that the actual measured value had a high degree of fit with the curve, and the calculation result of the content of the five-step snake venom was reliable;

[0229] The detection limit of cobra venom was calculated to be 1.6 ng / mL, and the determination coefficient was 0.9902, indicating that the actual measured value had a high degree of fit with the curve and the calculation result of the cobra venom content was reliable.

[0230] ③Specificity detection

[0231] The results obtained according to the above steps and conditions indicate that when the Bungarus capture antibody group is used to detect the venom of Agkistrodon acutus and Agkistrodon acutus, a cross reaction will occur when the venom concentration reaches 250 ng / mL or more, and when the venom of Cobra venom is detected, a cross reaction will occur when the concentration reaches 100 ng / mL or more, as shown in Table 9;

[0232] Table 9 Specific detection of Bungarus group in ELISA kit

[0233]

[0234] When the cobra capture antibody group was used to detect the venom of Bungarus serpentis, Agkistrodon acutus and Agkistrodon acutus, cross reactions occurred when the venom concentration reached 250 ng / mL or above, as shown in Table 10;

[0235] Table 10 Specificity detection of cobra group in ELISA kit

[0236]

[0237] When the pit viper capture antibody group is used to detect the venom of Bungarus cobra, no cross reaction will occur; when the venom concentration of cobra is detected, cross reaction will occur when it reaches 500ng / mL or above; when the venom of Acutus aviculare is detected, cross reaction is easy to occur; as shown in the figure, the dual wavelength reading OD value of four venom dilutions with a detection concentration of 32ng / mL is used. The results show that although the cross reaction of the pit viper capture antibody group to the detection of Acutus aviculare venom cannot be eliminated, it can be analyzed and distinguished according to its OD value at the same concentration, such as Fig.11 As shown in A;

[0238] When the A. serpentis capture antibody group was used to detect Bungarus cobra and Cobra, cross-reactions occurred when the venom concentration reached 1μg / mL or above; cross-reactions were still more likely to occur when detecting Pit Viper venom. The figure shows the dual-wavelength OD values ​​of four snake venom dilutions with a detection concentration of 50ng / mL. The results show that although the A. serpentis capture antibody group cannot eliminate the cross-reaction of Pit Viper venom, they can be distinguished based on their OD values ​​at the same concentration, such as Fig.11 As shown in B;

[0239] ④Repeatability test

[0240] According to the above steps and conditions, the results obtained are shown in Table 11;

[0241] Table 11 Repeatability test of ELISA kit

[0242]

[0243]

[0244] The average intra-batch coefficient of variation of the Bungarus group was calculated to be 3.75%, and the average inter-batch coefficient of variation was 6.68%;

[0245] The average intra-assay coefficient of variation of the pit viper group was calculated to be 2.76%, and the average inter-assay coefficient of variation was 2.94%;

[0246] The average intra-batch coefficient of variation of the five-step snake group was calculated to be 6.60%, and the average inter-batch coefficient of variation was 5.01%;

[0247] The average intra-assay coefficient of variation of the cobra group was calculated to be 6.54%, and the average inter-assay coefficient of variation was 4.31%;

[0248] According to the above data, the average value of the coefficient of variation of each group can be maintained below 10%, which shows that the detection method and product of the ELISA kit constructed in this study have good repeatability and stability.

[0249] 2. Detection of rat samples poisoned by snake venom

[0250] (1) Symptoms of snake venom poisoning in rats

[0251] The behavioral and postmortem changes of rats poisoned by each snake venom are shown below;

[0252] Bungarus poisoning group: about 1 hour later, the animals became irritable and aggressive, about 2 hours later, they began to breathe abdominally, the breathing rate and amplitude increased significantly, and urinary incontinence occurred; about 3.5 hours later, they began to twitch, opisthotonos, spasms and other near-death symptoms; after being killed, the injection site and various tissues and organs were examined, and no obvious abnormal changes were found;

[0253] Agkistrodon poisoning group: about 30 minutes later, the injection site was swollen, the rats were listless, and the activity was significantly reduced; after killing, the injection site and various tissues and organs were examined, and subcutaneous hematoma was found at the injection site, and muscle bleeding and necrosis were obvious; after opening the abdominal cavity, abdominal bleeding was found, the blood was not easy to coagulate, the spleen was enlarged, and there were bleeding spots on the kidney surface;

[0254] Five-step snake poisoning group: about 30 minutes later, the injection site was swollen, the rats were listless, and the activity was significantly reduced; blood flow did not stop when blood was taken from the eye sockets; after killing, the injection site and various tissues and organs were examined, and subcutaneous hematoma was found at the injection site, and muscle bleeding and necrosis were obvious; when the abdominal cavity was opened, abdominal bleeding, enlarged spleen, and bleeding spots on the kidney surface were found;

[0255] Cobra poisoning group: The rats became irritable and aggressive after about 30 minutes. After about 2 hours, they began to breathe abdominally, and the breathing rate and amplitude increased significantly. Convulsions and other near-death symptoms occurred after about 3.5 hours. After sacrifice, the injection site and various tissues and organs were examined, and no obvious abnormalities were found except for subcutaneous hemorrhage at the injection site.

[0256] (2) Testing methods and product samples in rats

[0257] 1) Double antibody sandwich ELISA test kit rat sample test results

[0258] ①Test results of the rat group poisoned by Bungarus krait

[0259] The results of the fresh group of rats poisoned by Bungarus krait showed that the OD values ​​of the heart, liver, spleen, lung, and kidney were all greater than 0.081, that is, they all showed positive results and Bungarus krait venom was detected; among them, the OD value of the kidney was the highest, that is, the corresponding Bungarus krait venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for Bungarus krait venom detection in biological samples, and the kidney is a better detection organ for this detection method and product in Bungarus krait poisoned rats;

[0260] The results of the corruption group of rats poisoned by Bungarus fasciatus showed that the OD values ​​of the heart, liver, spleen, lung, and kidney were all greater than 0.081, that is, they all showed positive results and Bungarus fasciatus venom was detected; among them, the OD value of the kidney was still the highest, that is, the corresponding Bungarus fasciatus venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of Bungarus fasciatus venom in corrupt biological samples, that is, it is suitable for corrupt samples commonly encountered in forensic practice, such as Fig.12 As shown;

[0261] ②Test results of rats poisoned by Agkistrodon acutus

[0262] The results of the fresh group of rats poisoned by agkistrodon showed that the OD values ​​of the heart, liver, spleen, lung, kidney, and skin at the injection site were all greater than 0.074, that is, they all showed positive results and agkistrodon venom was detected; among them, the OD value of the kidney was the highest, that is, the corresponding agkistrodon venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of agkistrodon venom in biological samples, and the kidney is a better detection organ for this detection method and product in rats poisoned by agkistrodon;

[0263] The results of the rat corruption group poisoned by Agkistrodon acutus showed that the OD values ​​of the heart, liver, spleen, lung, kidney, and skin at the injection site were all greater than 0.074, that is, they all showed positive results and Agkistrodon acutus venom was detected; among them, the OD value of the spleen was the highest, that is, the corresponding Agkistrodon acutus venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of Agkistrodon acutus venom in corrupted biological samples, that is, it is suitable for corrupted samples commonly encountered in forensic practice, such as Fig.13 As shown;

[0264] ③Test results of the rat group poisoned by Agkistrodon acutus

[0265] The results of the fresh group of rats poisoned by A. acutus showed that the OD values ​​of the heart, liver, spleen, lung, kidney and skin at the injection site were all greater than 0.075, that is, they all showed positive results and A. acutus venom was detected; among them, the OD value of the skin at the injection site was the highest, that is, the corresponding A. acutus venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of A. acutus venom in biological samples, and the skin at the injection site is a better detection organ for this detection method and product in rats poisoned by A. acutus;

[0266] The results of the spoilage group of rats poisoned by A. acutus showed that the OD values ​​of the heart, liver, spleen, lung, kidney, and skin at the injection site were all greater than 0.075, that is, they all showed positive results and A. acutus venom was detected; among them, the OD value of the skin at the injection site was still the highest, that is, the corresponding A. acutus venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of A. acutus venom in spoiled biological samples, that is, it is suitable for the spoilage samples commonly encountered in forensic practice, such as Fig.14 As shown;

[0267] ④Test results of rat cobra poisoning group

[0268] The results of the fresh group of rats poisoned by cobra showed that the OD values ​​of the heart, liver, spleen, lung, and kidney were all greater than 0.19, that is, they all showed positive results and cobra venom was detected; among them, the OD value of the kidney was the highest, that is, the corresponding cobra venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of cobra venom in biological samples, and the kidney is a better detection organ for the detection method and product in rats poisoned by cobra;

[0269] The results of the cobra poisoning rat corruption group showed that the OD values ​​of the heart, liver, spleen, lung, and kidney were all greater than 0.19, that is, they all showed positive results and cobra venom was detected; among them, the OD value of the heart was the highest, that is, the corresponding cobra venom concentration was the highest; this result suggests that the double antibody sandwich ELISA detection kit constructed in this study is suitable for the detection of cobra venom in corrupt biological samples, that is, it is suitable for corrupt samples commonly encountered in forensic practice, such as Fig.15 shown.

[0270] 3. Testing of human samples suspected of snake venom poisoning

[0271] (1) Case 1

[0272] According to the above steps and conditions, the results are as follows Fig.16 As shown in Table 12;

[0273] Table 12 Case 1 Human sample ELISA kit test

[0274]

[0275] *Note: A1 and A2 are experimental groups in the Bungarus cobra test area; A3 and A4 are control groups in the Bungarus cobra test area

[0276] The results showed that the human samples in this case were positive in the detection area of ​​the double antibody sandwich ELISA test kit for Bungarus coeruleus, indicating that the human samples in Case 1 contained Bungarus coeruleus venom, proving that the victim had been bitten by Bungarus coeruleus before his death; comprehensive analysis and investigation later confirmed that the case was caused by Bungarus coeruleus poisoning; this was consistent with the detection results of this experiment, indicating that the double antibody sandwich ELISA test kit constructed in this study has an excellent detection rate in human samples poisoned by Bungarus coeruleus venom, and can assist forensic practice in detecting cases.

[0277] (2) Case 2

[0278] According to the above steps and conditions, the results are as follows Fig.17 , as shown in Table 13;

[0279] Table 13 Case 2 Human Sample ELISA Kit Test

[0280]

[0281]

[0282] *Note: C1 and C2 are the experimental groups in the five-step snake detection area; C3 and C4 are the control groups in the five-step snake detection area

[0283] The results showed that the human samples in this case were positive in the Agkistrodon acutus detection area of ​​the double antibody sandwich ELISA test kit, indicating that the human samples in Case 2 contained Agkistrodon acutus venom, proving that the victim had been bitten by Agkistrodon acutus before his death; comprehensive analysis and investigation confirmed that the case was caused by Agkistrodon acutus poisoning, which was consistent with the detection results of this experiment, indicating that the double antibody sandwich ELISA test kit constructed in this study has an excellent detection rate for human samples poisoned by Agkistrodon acutus venom, and can assist forensic practice in case detection.

[0284] (3) Case 3

[0285] According to the above steps and conditions, the results are as follows Fig.18 , as shown in Table 14;

[0286] Table 14 Case 3 Human Sample ELISA Kit Detection

[0287]

[0288] *Note: A1 and A2 are experimental groups in the Bungarus test area; A3 and A4 are control groups in the Bungarus test area; B1 and B2 are experimental groups in the Agkistrodon test area; B3 and B4 are control groups in the Agkistrodon test area

[0289] The results showed that the human samples in this case were negative in the Bungarus serpentis area and the Agkistrodon acutus area of ​​the double antibody sandwich ELISA detection kit, indicating that the human samples in Case 3 did not contain Bungarus serpentis venom or Agkistrodon acutus venom, proving that the victim had not been bitten by Bungarus serpentis or Agkistrodon acutus before his death; after comprehensive analysis and investigation, it was found that the case was caused by nitrite poisoning, which was consistent with the results of this experiment; indicating that the double antibody sandwich ELISA detection kit constructed in this study can be used to exclude non-snake venom poisoning cases and assist forensic practice in case detection.

[0290] As can be seen from the above, the sensitivity and specificity of the double antibody sandwich ELISA detection kit are affected by many factors, including the incubation time of the test solution, the concentration of the coated antibody, the selection of the type of blocking solution, etc.; therefore, it is necessary to optimize the ratio of each reaction condition, and determine the most suitable conditions for the sandwich ELISA kit of this study through the P / N value and sensitivity; the conditions are: the coating antibody concentration is 10μg / mL, the Block solution is the blocking solution, the detection original incubation time is 40min, and the most suitable working concentrations of the enzyme-labeled antibody are 1:800; 1:2000; 1:3000; 1:1600; the dual-wavelength reading of the microplate reader can eliminate certain background errors compared with the single wavelength; the intra-batch repeat and batch The average values ​​of the coefficient of variation of the repeated tests were all lower than 10%, indicating that the detection method and product had good repeatability and stability. In this experiment, the critical value of the determination of the venom of the Bungarus cobra was 0.081, and it was judged to be positive when the OD value was ≥0.081. The critical value of the determination of the venom of the pit viper was 0.074, and it was judged to be positive when the OD value was ≥0.074. The critical value of the determination of the venom of the five-step snake was 0.075, and it was judged to be positive when the OD value was ≥0.075. The critical value of the determination of the venom of the cobra was 0.19, and it was judged to be positive when the OD value was ≥0.19. The R2 of the standard curves determined in each detection interval was above 0.99, with a high degree of fit. The concentration calculated according to this detection method and product is accurate and reliable.

[0291] The present invention successfully constructed a rapid snake venom poisoning detection method based on ELISA technology, and verified its effectiveness and reliability in the detection of snake venom poisoning rats and human samples.

[0292] In summary: This rapid detection method for snake venom poisoning based on ELISA technology, by purifying horse anti-snake venom polyclonal antibodies and preparing a double antibody sandwich ELISA detection kit, achieves high sensitivity and high specificity detection of common snake venoms, and is suitable for fresh and corrupt biological samples. It has been successfully applied to forensic identification cases, providing an effective means for the rapid and accurate detection of snake venom poisoning, and has important social and economic value.

[0293] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A rapid detection method for snake venom based on ELISA technology, characterized in that: include: Step 1: Purify horse anti-snake venom polyclonal antibody serum of common venomous snakes by using Protein A and Protein G affinity chromatography and caprylic acid-ammonium sulfate precipitation method; Step 2: using the purified horse anti-snake venom polyclonal antibody, prepare a double antibody sandwich ELISA detection kit for common snake venoms, and optimize the detection conditions; Step 3: Use the optimized detection method and the constructed detection kit to verify the biological samples of rats poisoned by snake venom; Step 4: Apply the detection method and detection kit to the detection of human samples in forensic identification cases of suspected snake venom poisoning.

2. A method for rapid detection of snake venom based on ELISA technology as claimed in claim 1, characterized in that: The titers of the purified horse anti-Bungaroca polyclonal antibody, horse anti-Agkistrodon acutus polyclonal antibody, horse anti-Agamoebae polyclonal antibody and horse anti-Naja naja polyclonal antibody all reach 1:64000.

3. A method for rapid detection of snake venom based on ELISA technology as claimed in claim 1, characterized in that: The quantitative detection limits of the double antibody sandwich ELISA detection kit for common snake venoms are: 0.08 ng / mL for Bungarus cobra venom; 1.6 ng / mL for Agkistrodon acutus venom; 1.6 ng / mL for Agkistrodon acutus venom; and 1.6 ng / mL for Cobra venom.

4. The method for rapid detection of snake venom based on ELISA technology as claimed in claim 1, characterized in that: The detection method and detection kit can effectively detect snake venom in fresh and corrupt biological samples of rats poisoned by snake venom, and the kidney is the best detection sample for poisoning by Bungarus cobra and Cobra, and the injection site tissue is the best detection sample for poisoning by Acuta quinquefasciatus.

5. A method for rapid detection of snake venom based on ELISA technology as claimed in claim 1, characterized in that: The detection method and detection kit were successfully applied to three forensic identification cases of suspected snake venom poisoning, and were able to determine whether snake venom poisoning occurred and identify the species of the venomous snake.

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