LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)-based method for detecting activity of abrus precatorius toxin

Abstract

The invention relates to the technical field of toxin detection. The invention provides an LC-MS / MS. The method comprises the following steps: mixing a to-be-detected sample with a reaction system A for reaction, detecting by utilizing LC-MS / MS, determining the nature of the sample according to an abscisic acid peak area, and if the to-be-detected sample is a positive sample, judging whether the to-be-detected sample is a positive sample or not, and if the to-be-detected sample is a negative sample, judging whether the to-be-detected sample is a positive sample or not. And substituting (the ratio of the peak area of the shedding adenine of the positive sample to the corresponding peak area of the internal standard acyclovir) and (the ratio of the peak area of the shedding adenine of the negative sample to the corresponding peak area of the internal standard acyclovir) into the standard curve to obtain the concentration of the abrus precatorius toxin. On the basis of detecting the peak area of reaction abscisic acid for sample qualitative determination, the concentration of the abscisic acid toxin is further determined through the standard curve established by LC-MS / MS, the purpose of detecting the toxin is achieved, and the scheme is relatively short in reaction time, has more advantages in analysis of complex matrix samples, and is better in detection effect, higher in sensitivity and stronger in specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of toxin detection, and in particular to a method for detecting the activity of abrinus toxin based on LC-MS / MS. Background Art

[0002] Abrin toxin (AT) is a plant toxin derived from the leguminous vine Abrin, with a molecular weight of about 60-65 kDa and an isoelectric point between 6 and 8. It is composed of an A chain with N-glycosidase activity and a B chain with galactose specificity, which are connected together by a disulfide bond. The B chain mediates the entry of the A chain into the cell. The A chain exerts N-glycosidase activity and specifically cuts adenine at the 4324th site of the ribosome 28rRNA, resulting in protein inactivation, thereby exerting toxic effects. AT is divided into four subtypes: a, b, c, and d, among which the a subtype has the strongest cytotoxicity and the highest content. AT can enter the human body through different infection routes and cause poisoning, including the respiratory tract, digestive tract, and intramuscular injection. There are currently many specific detection methods to detect this toxic plant protein, but few are based on toxin activity detection. Therefore, it is of great significance to explore a method that can quickly and accurately detect AT activity, which is also crucial for identifying biologically active toxins. Summary of the invention

[0003] The object of the present invention is to provide a method for detecting the activity of abrin toxin based on LC-MS / MS. On the basis of qualitatively analyzing the sample by detecting the peak area of ​​adenine shedding in the detection reaction, the concentration of abrin toxin is further determined by a standard curve established by LC-MS / MS to achieve the purpose of detecting the abrin toxin. The detection scheme has simple reaction components and relatively short reaction time, and has more advantages in the analysis of complex matrix samples, better detection effect, higher sensitivity and stronger specificity.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for detecting the activity of abrin toxin based on LC-MS / MS, which is characterized by comprising the following steps:

[0006] (1) A series of gradient diluted abrin toxin standards were mixed with reaction system A for reaction, and detected by LC-MS / MS, and then a standard curve was established with the concentration of abrin toxin as the horizontal axis and (the ratio of the peak area of ​​adenine shed from the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​adenine shed from the negative sample to the peak area corresponding to the internal standard acyclovir) as the vertical axis;

[0007] (2) The sample to be tested is mixed with reaction system A for reaction, and detected by LC-MS / MS. The sample is qualitatively analyzed according to the peak area of ​​shed adenine. If the sample to be tested is a positive sample, (the ratio of the peak area of ​​shed adenine of the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​shed adenine of the negative sample to the peak area corresponding to the internal standard acyclovir) is substituted into the standard curve described in step (1) to calculate the concentration of abrin toxin; when the ratio of the peak area of ​​shed adenine of the sample to be tested to the peak area of ​​shed adenine of the negative control group is greater than 5, it indicates that the sample is a positive sample.

[0008] Preferably, the reaction system A comprises ammonium citrate + Mg 2+ +EDTA buffer, BSA solution, oligonucleotide chain substrate and internal standard acyclovir solution.

[0009] Preferably, the liquid chromatography conditions of the LC-MS / MS are: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM ammonium formate, pH=4; organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL.

[0010] Preferably, the MRM mode parameters of the LC-MS / MS are: adenine m / z 136→m / z 92, m / z 136→m / z 119; acyclovir m / z 226.1→m / z 134.9, m / z 226.1→m / z 152.05.

[0011] Preferably, the reaction time of step (2) is 1.5-2.2 h, and the reaction temperature is 42-46°C.

[0012] Preferably, the ammonium citrate + Mg in step (2) 2+ +EDTA buffer pH 4.0-4.1, ammonium citrate + Mg 2 + The final concentration of ammonium citrate in the +EDTA buffer is 0.1-10 mM, ammonium citrate + Mg 2+ +Mg in EDTA buffer 2+ The final concentration is 0.1-5mM, ammonium citrate + Mg 2+ +The final concentration of EDTA in EDTA buffer is 0.1-10mM;

[0013] The final concentration of the BSA solution is 8-12 μg / mL;

[0014] The final concentration of the oligonucleotide chain substrate is 0.1-0.3 μM;

[0015] The final concentration of the internal standard acyclovir solution is 8-12 ng / mL.

[0016] Preferably, the sequence of the oligonucleotide chain substrate in step (2) is as shown in SEQ ID NO.11.

[0017] Preferably, the negative control group in step (2) is tested with sterile water.

[0018] By adopting the above technical solution, the present invention has the following beneficial effects:

[0019] The technical solution of the present invention uses the N-glycosidase activity of abrin toxin to specifically cut adenine on the ribosome to establish a detection method. The reaction principle is that AT exerts N-glycosidase activity to make the substrate shed adenine. The sample is qualitatively determined by detecting the peak area of ​​shed adenine in the reaction, and the concentration of abrin toxin is further determined by a standard curve established by LC-MS / MS to achieve the purpose of detecting toxins. The detection scheme has simple reaction components and relatively short reaction time. It has more advantages in the analysis of complex matrix samples, better detection effect, higher sensitivity and stronger specificity.

[0020] The experiment in the present invention also shows that the optimal reaction system is ammonium citrate + Mg at pH 4.05. 2+ +EDTA buffer, BSA solution with a final concentration of 10μg / mL, linear single-stranded DNA 20A substrate with a final concentration of 0.2μM, and internal standard acyclovir solution with a final concentration of 10ng / mL; the optimal reaction temperature is 45°C, and the optimal reaction time is 2h. The detection method of the present invention is combined with the above-mentioned optimal reaction parameters to measure the abrin toxin sample enriched with antibody-coated magnetic beads, and the detection sensitivity can reach 0.391ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The standard curve of adenine standard with internal standard ACV added;

[0022] Figure 2 The reaction diagrams for different types of substrates ( Figure 2 A is a single-stranded DNA substrate, B is a single-stranded RNA substrate, and C is a double-stranded DNA substrate);

[0023] Figure 3 The effects of different types of buffer, different pH, different substrate concentrations and different BSA concentrations on the deadenylation intensity ( Figure 3 A in the figure is different types of buffer, B is different pH, C is a line graph of different substrate concentrations, D is a bar graph of different substrate concentrations, and E is different BSA concentrations);

[0024] Figure 4 The effect of different reaction temperatures on the deadenylation strength;

[0025] Figure 5 The effect of different reaction times on the deadenylation intensity;

[0026] Figure 6 To investigate the effect of enrichment with different magnetic beads on the intensity of deadenination;

[0027] Figure 7 The sensitivity of sample enrichment detection without antibody-coated magnetic beads ( Figure 7 A in the figure is the deadenylation intensity under different concentrations of abrin toxin, and B is the linear relationship between abrin toxin and the deadenylation intensity);

[0028] Figure 8 The sensitivity of sample enrichment detection using antibody-coated magnetic beads ( Figure 8 A in the figure is the deadenylation intensity under different concentrations of abrin toxin, and B is the linear relationship between abrin toxin and the deadenylation intensity);

[0029] Fig. 9 The sensitivity of the detection of PBS sample enrichment using antibody-coated magnetic beads ( Fig. 9 A in the figure is the deadenylation intensity under different concentrations of abrin toxin, and B is the linear relationship between abrin toxin and the deadenylation intensity);

[0030] Fig.10 The sensitivity of the detection of milk sample enrichment using antibody-coated magnetic beads ( Fig.10 A in the figure is the deadenylation intensity under different concentrations of abrin toxin, and B is the linear relationship between abrin toxin and the deadenylation intensity);

[0031] Fig.11 The sensitivity of serum sample enrichment using antibody-coated magnetic beads ( Fig.11 A in the figure is the deadenylation intensity under different concentrations of abrin toxin, and B is the linear relationship between abrin toxin and the deadenylation intensity). DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting the activity of abrin toxin based on LC-MS / MS, which is characterized by comprising the following steps:

[0033] (1) A series of gradient diluted abrin toxin standards were mixed with reaction system A for reaction, and detected by LC-MS / MS, and then a standard curve was established with the concentration of abrin toxin as the horizontal axis and (the ratio of the peak area of ​​adenine shed from the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​adenine shed from the negative sample to the peak area corresponding to the internal standard acyclovir) as the vertical axis;

[0034] (2) The sample to be tested is mixed with reaction system A for reaction, and detected by LC-MS / MS. The sample is qualitatively analyzed according to the peak area of ​​shed adenine. If the sample to be tested is a positive sample, (the ratio of the peak area of ​​shed adenine of the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​shed adenine of the negative sample to the peak area corresponding to the internal standard acyclovir) is substituted into the standard curve described in step (1) to calculate the concentration of abrin toxin; when the ratio of the peak area of ​​shed adenine of the sample to be tested to the peak area of ​​shed adenine of the negative control group is greater than 5, it indicates that the sample is a positive sample.

[0035] In the present invention, the reaction principle is that the abrin toxin exerts N-glycosidase activity to cause the substrate to shed adenine. On the basis of qualitative analysis of the sample by detecting the peak area of ​​shed adenine in the reaction, the concentration of the abrin toxin is further determined by a standard curve established by LC-MS / MS to achieve the purpose of detecting the toxin.

[0036] In the present invention, the liquid chromatography conditions of the LC-MS / MS are: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM ammonium formate, pH=4; organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL.

[0037] In the present invention, the MRM mode parameters of the LC-MS / MS are: adenine m / z 136→m / z 92, m / z 136→m / z 119; acyclovir m / z 226.1→m / z 134.9, m / z 226.1→m / z 152.05.

[0038] In the present invention, the sample to be tested is preferably enriched with antibody-coated magnetic beads before use, which can significantly improve the sensitivity of abrin toxin detection. The magnetic beads in the present invention are preferably B magnetic beads (Dynabeads TMM-270Streptavidin streptavidin), the mass ratio of the antibody to the magnetic beads is preferably 1: (95-105), further preferably 1: (98-102), and further preferably 1: 100. The amount of magnetic beads used in each sample to be tested (the volume of each sample to be tested is preferably 500 μL) in the present invention is preferably 25-35 μg, further preferably 27-32 μg, and further preferably 30 μg.

[0039] In the present invention, the reaction system A comprises ammonium citrate + Mg 2+ +EDTA buffer, BSA solution, oligonucleotide chain substrate and internal standard acyclovir solution.

[0040] In the present invention, the ammonium citrate + Mg 2+ The pH of the ammonium citrate + Mg+EDTA buffer is preferably 4.0-4.1, more preferably 4.02-4.08, and even more preferably 4.05. 2+ The final concentration of ammonium citrate in the +EDTA buffer is preferably 0.1-10 mM, more preferably 0.5-5 mM, and even more preferably 1 mM; ammonium citrate + Mg 2+ +Mg in EDTA buffer 2+ The final concentration is preferably 0.1-5 mM, more preferably 0.1-1 mM, and more preferably 0.5 mM; ammonium citrate + Mg 2+ The final concentration of EDTA in the +EDTA buffer is preferably 0.1-10 mM, more preferably 0.5-5 mM, and even more preferably 1 mM.

[0041] In the present invention, the final concentration of the BSA solution is preferably 8-12 μg / mL, more preferably 9-11 μg / mL, and even more preferably 10 μg / mL.

[0042] In the present invention, the oligonucleotide chain substrate is preferably a linear single-stranded DNA 20A, and the sequence of the oligonucleotide chain substrate is shown in SEQ ID NO.11, and the specific sequence is 5'-AAAAAAAAAAAAAAAAAAAAAAA-3'. The final concentration of the oligonucleotide chain substrate of the present invention is preferably 0.1-0.3 μM, more preferably 0.15-0.25 μM, and further preferably 0.2 μM.

[0043] In the present invention, the final concentration of the internal standard acyclovir solution (ACV) in the reaction system A is preferably 8-12 ng / mL, more preferably 9-11 ng / mL, and even more preferably 10 ng / mL.

[0044] In the present invention, the reaction time is preferably 1.5-2.2 h, more preferably 1.8-2.1 h, and more preferably 2 h; the reaction temperature is preferably 42-46°C, more preferably 43-45.5°C, and more preferably 45°C.

[0045] In the present invention, the negative control group was tested with sterile water.

[0046] In the present invention, after the reaction is completed, aqueous ammonia is preferably added to terminate the reaction.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The present invention Antibody Coupling Kit (Cat. No.: 14311D), Dynabeads TM M-270 Streptavidin (Cat. No.: 65305, 65306), Dynabeads TM M-280 Streptavidin (Cat. No.: 11205D, 11206D, 60210), M-450Epoxy (Cat. No.: 14011), Dynabeads TM MyOne TM Streptavidin T1 (Cat. No.: 65601, 65602, 65604D) were purchased from Thermo Fisher Scientific.

[0049] The ammonium citrate (25102), ammonium formate (70221) and ammonium acetate (73594) described in the present invention were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0050] The EDTA (pH 8.0, 0.5 M) (E1170) described in the present invention was purchased from Beijing Solebow Technology Co., Ltd.

[0051] The methanol (HPLC grade) (>98%) described in the present invention was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0052] The abrin toxin described in the present invention is prepared by this unit.

[0053] Adenine and aciclovir described in the present invention are both purchased from Merck.

[0054] Example 1

[0055] (I) Design and synthesis of different types of oligonucleotide chain substrates

[0056] We designed various types of oligonucleotide chain substrates, including DNA substrates, RNA substrates, straight chains or stem-loop structures of different lengths and different adenine contents. All oligonucleotide substrates were synthesized by GenScript Biotech Co., Ltd.

[0057] Table 1 Single-stranded DNA substrate

[0058]

[0059]

[0060] Table 2 Single-stranded RNA substrates

[0061]

[0062] Table 3 Double-stranded DNA substrate

[0063]

[0064]

[0065] (II) LC-MS / MS conditions

[0066] 1. LC conditions

[0067] The optimized liquid chromatography conditions are as follows: the chromatographic column is Waters -C18 reverse phase column (2.1 mm×150 mm, 3.5 μm); the mobile phase was 10 mM ammonium formate, pH 4, and the organic phase was 100% methanol; the flow rate was 0.2 mL / min; and the injection volume was 2 μL.

[0068] Desolvation Temp.: 250°C; Source Temp.: 150°C.

[0069] 2. MS conditions

[0070] Table 4 MRM detection parameter optimization results

[0071]

[0072] Example 2 Establishment of adenine standard curve

[0073] Adenine standard was diluted with sterile water to 0.5, 1, 10, 25, 37.5, 50, 100, 125, 250 fmoL / μL, and mixed with 50 ng / mL internal standard acyclovir to make the final concentration of acyclovir 10 ng / mL. The chromatogram was obtained by detecting according to the above LC-MS / MS conditions. Then, the concentration of adenine was used as the horizontal coordinate, and (the ratio of the peak area of ​​adenine shed from the positive sample to the corresponding peak area of ​​the internal standard acyclovir): (the ratio of the peak area of ​​adenine shed from the negative sample to the corresponding peak area of ​​the internal standard acyclovir) was used as the vertical coordinate to draw a standard curve, and the linear equation was Y=0.2974*X+2.351, R 2 =0.991. The experimental results are as follows Figure 1 shown.

[0074] Example 3 Determination of reaction conditions

[0075] (I) Reaction substrate

[0076] The designed and synthesized substrates were tested separately, and 25 μg / mL AT was used to act on different types of DNA and RNA substrates (specific sequence information is shown in Tables 1, 2, and 3), and sterile water was used as a negative control, and the reaction was carried out at 55°C. The reaction system size was 50 μL, and the system included 10 μL of ammonium formate buffer, 10 μL of BSA solution with a final concentration of 50 μg / mL, 10 μL of substrate, and 10 μL of AT. After the reaction was completed, 10 μL of ammonia water with a final concentration of 20 mM was added to terminate the reaction. The final concentration of the substrate used was 0.2 μM, and the final concentration of BSA was 50 μg / mL. After the reaction system was configured and 10 μL of AT / sterile water was added, it was placed at 55°C for 1 hour. The ratio of the peak area of ​​depurination after different concentrations of AT acted on the same substrate to the peak area of ​​depurination of the negative control was compared. The substrate with the highest ratio was the best reaction substrate.

[0077] The results are as follows Figure 2 As shown, when DNA20A in the straight single-stranded DNA substrate is used as a reaction substrate, the ratio is relatively the highest, that is, it is the best reaction substrate.

[0078] (II) Reaction system

[0079] The DNA20A obtained by screening was used as the reaction substrate, and the optimal reaction concentration of the reaction substrate and the optimal reaction concentration of BSA were explored, and the reaction effects of different concentrations of ammonium formate buffer (0.5mM), ammonium acetate buffer (5mM), ammonium citrate buffer (15mM), and buffers with metal ions and EDTA added to them were compared. The substrate concentrations were set to 0.02μM, 0.1μM, 0.2μM, 0.25μM, 0.5μM, 1μM, and 2μM, and the BSA concentrations were set to 0μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL, 60μg / mL, 70μg / mL, 80μg / mL, 90μg / mL, and 100μg / mL. After determining the optimal reaction buffer and its reaction concentration, the optimal reaction pH of the buffer was explored, including pH3.25, pH3.85, pH4.05, pH4.45, pH4.65, pH4.85, pH5.05, pH5.25, and pH5.75. Sterile water was used as a negative control in all reactions. The reaction temperature was 55°C, the reaction system size was 50 μg / mL, and the system included 10 μL of buffer, 10 μL of BSA solution, 10 μL of substrate, 10 μL of AT, and 10 μL of ammonia water.

[0080] The reaction system was prepared and 10 μL of AT / sterile water was added and then reacted at 55°C to compare the deadenylation intensity of AT under different types of buffers and their pH values, different concentrations of substrates, and different BSA concentrations.

[0081] The experimental results found that (such as Figure 3 The optimal reaction concentration of DNA20A was 0.2 μM, the optimal reaction concentration of BSA was 10 μg / mL, and the optimal reaction concentration of ammonium citrate + Mg 2+ +EDTA buffer is the best reaction buffer, and the best reaction pH is 4.05.

[0082] (III) Reaction temperature

[0083] The optimal reaction temperature of the reaction system was investigated. The reaction temperatures were set at 4°C, 16°C, 25°C, 37°C, 45°C, 55°C, and 66°C, respectively. The reaction system included ammonium citrate + Mg with a pH of 4.05. 2+ +EDTA buffer, 10μg / mL BSA solution, 0.2μM DNA20A substrate, 25μg / mL AT / sterile water, after the reaction, add 10μL of 20mM ammonia water to terminate the reaction. The reaction system volume is 50μL.

[0084] The experimental results are as follows Figure 4As shown, when the reaction temperature is 45°C, the peak area ratio of positive / negative detached adenine is the highest, that is, the intensity of the reaction is higher, so 45°C is selected as the optimal reaction temperature.

[0085] (IV) Reaction time

[0086] The optimal reaction time of the reaction system was investigated. The reaction times were set to 0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, respectively. The reaction system included ammonium citrate + Mg with a pH of 4.05. 2+ +EDTA buffer, 10μg / mL BSA solution, 0.2μM DNA20A substrate, 25μg / mL AT / sterile water, after the reaction, add 10μL of 20mM ammonia water to terminate the reaction. The reaction system volume is 50μL.

[0087] The results are as follows Figure 5 As shown in the figure, with the extension of reaction time, the amount of substrate depurination gradually increases. When the reaction time reaches 10h, the growth of the amount of depurination tends to be flat. When the reaction time is 2h, the peak area ratio of positive / negative detached adenine is the highest, that is, the intensity of the reaction is higher. Therefore, 2h is selected as the optimal reaction time.

[0088] (V) Reaction Magnetic Beads

[0089] The best coupled magnetic beads for the reaction were explored. Magnetic bead coupling antibody enrichment of toxins can significantly improve the sensitivity of detection, and the selection of magnetic beads is also crucial. Because different magnetic beads have different capture effects on antibodies, this experiment selected magnetic beads with different particle sizes and different surface groups for screening. A magnetic beads are antibody coupling kits, B magnetic beads have carboxylic acid surface groups, with a hydrophilic diameter of 2.8μM; C magnetic beads have toluenesulfonyl surface groups, which are hydrophobic; D magnetic beads have Tosyl groups on the surface; E magnetic beads have toluenesulfonyl surface groups, which are hydrophobic, 2.8μM.

[0090] The five magnetic beads were coated with AT polyclonal antibody for experiments, where 1 mg of A magnetic beads was coated with 25 μg of antibody, 1 mg of B and C magnetic beads were coated with 10 μg of antibody, 1 mg of D magnetic beads was coated with 200 μg of antibody, and 1 mg of E magnetic beads was coated with 40 μg of antibody. The amount of magnetic beads used for each sample was 30 μg, the volume of the sample was 500 μL, the high concentration of AT was set to 250 ng / mL, the low concentration was 50 ng / mL, and the reaction was carried out at 45°C for 2 hours. The reaction system included ammonium citrate + Mg with a pH of 4.05 2++EDTA buffer, BSA solution with a final concentration of 10 μg / mL, DNA20A substrate with a final concentration of 0.2 μM, and internal standard ACV with a concentration of 10 ng / mL. After the reaction, 10 μL of 20 mM ammonia water was added to terminate the reaction. The reaction system volume was 50 μL.

[0091] The results are as follows Figure 6 As shown in the figure, the peak area ratio of positive / negative adenine shedding of B magnetic beads is the highest, that is, the intensity of the reaction is higher. Therefore, B magnetic beads are selected as the best magnetic beads, and B magnetic beads are used for enrichment detection in subsequent experiments.

[0092] Example 4 Sensitivity Detection

[0093] (I) Sample enrichment without using antibody-coated magnetic beads

[0094] Without using antibody-coated magnetic beads for sample enrichment, the optimized reaction system was directly used to explore the detection sensitivity of the detection method. AT was diluted to 3200ng / mL, 1600ng / mL, 800ng / mL, 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.5625ng / mL, 0.78125ng / mL, and reacted at 45°C for 2h. The reaction system included ammonium citrate + Mg with a pH of 4.05 2+ +EDTA buffer, BSA solution with a final concentration of 10 μg / mL, DNA20A substrate with a final concentration of 0.2 μM, and internal standard ACV with a final concentration of 10 ng / mL. After the reaction is completed, 10 μL of 20 mM ammonia water is added to terminate the reaction. The reaction system volume is 50 μL.

[0095] The results are as follows Figure 7 As shown, the detection sensitivity of AT without magnetic beads enrichment was 25 ng / mL.

[0096] (II) Sample enrichment using antibody-coated magnetic beads

[0097] Antibody-coated magnetic beads were used for sample enrichment. The amount of antibody and B magnetic beads coated was 10 μg antibody coated 1 mg magnetic beads. The amount of magnetic beads used for each 500 μL sample was 30 μg. The sample volume was 500 μL. After AT and the corresponding magnetic beads were flipped and incubated for 1 hour at room temperature, the magnetic stand was adsorbed for 2-3 minutes, and the supernatant was removed. Then the reaction system was added. The reaction system included ammonium citrate + Mg with a pH of 4.05. 2++EDTA buffer, BSA solution with a final concentration of 10μg / mL, DNA20A substrate with a final concentration of 0.2μM, and internal standard ACV with a final concentration of 10ng / mL. After the reaction, 10μL of 20mM ammonia water was added to terminate the reaction. The volume of the reaction system is 50μL. The magnetic beads coupled to the antibody and the toxin were gently blown and evenly incubated at 45℃ for 2h. The concentrations of AT were set to 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.563ng / mL, 0.781ng / mL, 0.391ng / mL, 0.195ng / mL, and 0.098ng / mL, respectively. Sterile water was used as a negative control to explore the reaction system and the detection sensitivity of the detection method.

[0098] The experimental results are as follows Figure 8 As shown in the figure, the detection sensitivity of AT after enrichment with magnetic beads is 0.195 ng / mL, the linear range is 0.781-50 ng / mL, and R 2 =0.991.

[0099] 3. Sensitivity testing of complex samples

[0100] Antibody-coated magnetic beads were used for sample enrichment. The coating amount of antibody and B magnetic beads was 10 μg antibody coated 1 mg magnetic beads, and the amount of magnetic beads used for each sample was 30 μg. The volume of the sample was 500 μL. After AT and the corresponding magnetic beads were flipped and incubated for 1 hour at room temperature, the magnetic stand was adsorbed for 2-3 minutes, the supernatant was removed, and 500 μL sterile water was added to wash the magnetic beads 3 times. Then the reaction system was added, which included ammonium citrate + Mg with a pH of 4.05 2+ +EDTA buffer, BSA solution with a final concentration of 10μg / mL, DNA20A substrate with a final concentration of 0.2μM, and internal standard ACV with a final concentration of 10ng / mL. After the reaction, 10μL of 20mM ammonia water was added to terminate the reaction. The volume of the reaction system is 50μL. The magnetic beads coupled to the antibody and the toxin were gently blown and evenly incubated at 45℃ for 2h. Simulated complex samples of AT were made with PBS, milk, and serum, respectively. The concentrations of AT were set to 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.563ng / mL, 0.781ng / mL, 0.391ng / mL, 0.195ng / mL, and 0.098ng / mL, respectively. Sterile water was used as a negative control to explore the reaction system and the detection sensitivity of the detection method.

[0101] The experimental results are as follows Figure 9-11As shown, the detection sensitivity of PBS samples was 0.391 ng / mL, the detection sensitivity of milk samples was 1.563 ng / mL, and the detection sensitivity of serum samples was 1.563 ng / mL.

[0102] In summary, it can be seen that the technical solution of the present invention is used for detection, the reaction components are simple, the reaction time is relatively short, it has more advantages in the analysis of complex matrix samples, the detection effect is better, the sensitivity is higher, and the specificity is stronger.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the activity of Abrinus sibiricum toxin based on LC-MS / MS, characterized in that: The steps include: (1) A series of gradient diluted abrin toxin standards were mixed with reaction system A for reaction, and detected by LC-MS / MS, and then a standard curve was established with the concentration of abrin toxin as the horizontal axis and (the ratio of the peak area of ​​adenine shed from the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​adenine shed from the negative sample to the peak area corresponding to the internal standard acyclovir) as the vertical axis; (2) mixing the sample to be tested with the reaction system A to react, and detecting it by LC-MS / MS, and qualitatively analyzing the sample according to the peak area of ​​shed adenine. If the sample to be tested is a positive sample, substituting (the ratio of the peak area of ​​shed adenine of the positive sample to the peak area corresponding to the internal standard acyclovir): (the ratio of the peak area of ​​shed adenine of the negative sample to the peak area corresponding to the internal standard acyclovir) into the standard curve of step (1) to calculate the concentration of abrin toxin; When the ratio of the shed adenine peak area of ​​the sample to be tested to the shed adenine peak area of ​​the negative control group is greater than 5, it indicates a positive sample; The reaction system A includes ammonium citrate + Mg 2+ +EDTA buffer, BSA solution, oligonucleotide chain substrate and internal standard acyclovir solution.

2. The detection method according to claim 1, characterized in that: The liquid chromatography conditions of the LC-MS / MS are as follows: the chromatographic column is Waters -C18 reverse phase column, inner diameter × length 2.1mm × 150mm, particle size 3.5μm; mobile phase is 10mM ammonium formate, pH=4; organic phase is 100% methanol; flow rate is 0.2mL / min; injection volume is 2μL.

3. The detection method according to claim 1, characterized in that: The MRM mode parameters of the LC-MS / MS are: adenine m / z 136→m / z 92, m / z 136→m / z 119; acyclovir m / z 226.1→m / z 134.9, m / z 226.1→m / z 152.

05.

4. The detection method according to claim 1, characterized in that: The reaction time of step (2) is 1.5-2.2 hours, and the reaction temperature is 42-46°C.

5. The detection method according to claim 1, characterized in that: Step (2) ammonium citrate + Mg 2+ +EDTA buffer pH 4.0-4.1, ammonium citrate + Mg 2+ The final concentration of ammonium citrate in the +EDTA buffer is 0.1-10 mM, ammonium citrate + Mg 2+ +Mg in EDTA buffer 2+ The final concentration is 0.1-5mM, ammonium citrate + Mg 2+ +The final concentration of EDTA in EDTA buffer is 0.1-10mM; The final concentration of the BSA solution is 8-12 μg / mL; The final concentration of the oligonucleotide chain substrate is 0.1-0.3 μM; The final concentration of the internal standard acyclovir solution is 8-12 ng / mL.

6. The detection method according to claim 1, characterized in that: The sequence of the oligonucleotide chain substrate in step (2) is shown in SEQ ID NO.

11.

7. The detection method according to claim 1, characterized in that: The negative control group in step (2) was tested with sterile water.

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