CN111273041B - ELISA kit for detecting phalloidin and preparation and application thereof - Google Patents
ELISA kit for detecting phalloidin and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses an ELISA kit for detecting phalloidin, preparation and application of the kit, wherein the kit provided by the invention consists of an ELISA plate (a conjugate of a coated phalloidin drug hapten and carrier protein in a formula I), an antibody working solution (a monoclonal antibody containing the phalloidin drug), an enzyme marker working solution (an anti-antibody containing horseradish peroxidase-labeled anti-phalloidin drug monoclonal antibody), a washing solution, a sample diluent, a sample extracting solution, a phalloidin standard substance solution containing different gradient concentrations, a substrate color development solution and a termination solution. The phalloidin detection kit provided by the invention can be used for detecting the phalloidin in shellfish food, the detection limit of the phalloidin in serum and urine can be defined to be 1.0 mug/L, the inter-batch variation coefficient is less than 10%, the intra-batch variation coefficient is less than 15%, and the kit has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.
Description
Technical Field
The invention belongs to the field of rapid detection of drug residues, and relates to a kit for detecting phalloidin and a preparation method and application thereof.
Background
Mushroom food poisoning has been one of the food safety issues of major concern in many countries around the world, and mushroom poisoning has been the leading cause of death from food poisoning accidents in China for many years. The phalloides belong to a bicyclo polypeptide compound, and exist in some wild bacteria of Amanita (Amanita), coptis (Galerina) and Leptopetalum (Leptopeta), and food poisoning caused by the phalloides is easy to misdiagnose and lacks of targeted treatment, so that early diagnosis is particularly important for treating and curing the poisoning.
The existing detection methods of the phalloidin toxins in biological samples such as blood plasma, urine and the like mainly comprise a capillary electrophoresis method, a radioimmunoassay method, a high performance liquid chromatography and mass spectrometry method and the like, wherein the liquid chromatography-tandem mass spectrometry method can realize high-flux accurate analysis of the toxins and becomes the most main method at present. The existing method has the problems of low detection flux, false positive, low sensitivity, serious matrix effect and the like, and the method with strong specificity and high sensitivity has the defects of complex operation, expensive instrument, inapplicability to screening detection of a large number of samples and incapability of meeting the field detection requirement.
Disclosure of Invention
The invention aims to provide an ELISA kit for detecting phalloidin, and a detection method which has high sensitivity, strong specificity and low detection cost and is suitable for screening a large amount of samples.
In order to achieve the above object, the technical scheme of the present invention is as follows:
an ELISA kit for detecting phalloidin comprises an ELISA plate, a standard substance working solution, an antibody working solution, an enzyme marker working solution, a sample dilution solution, a sample extracting solution, a washing solution, a substrate color development solution and a termination solution.
The ELISA plate is prepared by coating conjugate of phalloidin drug hapten and carrier protein.
The specific preparation method of the phalloidin hapten comprises the following steps: weighing 10mg of phalloidin raw material, dissolving 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid for reaction at room temperature, detecting by TLC, and purifying by a thin-layer preparation plate after the reaction is complete to obtain hapten (formula I).
I is a kind of
The carrier protein is Bovine Serum Albumin (BSA), human Serum Albumin (HSA), murine serum protein (MSA), thyroxine (BCG), rabbit serum protein (RSA), hemocyanin (KLH) or Ovalbumin (OVA).
The conjugate of the phalloidin hapten and carrier protein refers to a product obtained by the phalloidin hapten and carrier protein through an active ester method, and specifically comprises the following steps:
1) Dissolving the phalloidin hapten (formula I) in Dimethylformamide (DMF), and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and magnetically stirring at 20-25 ℃ to react for 2-3 h to obtain a solution I;
wherein the ratio of the phalloidin hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 1.9 mg:0.5 mL:1.0 mg:1.0 mg;
2) Placing the carrier protein in 0.1M sodium bicarbonate buffer solution, stirring at 200 rpm for 10 min, and fully dissolving to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer is 10 mg:2.0 mL;
3) Mixing the solution I and the solution II, specifically, dropwise adding the solution I into the solution II under the condition of stirring at 1000 rpm at the temperature of 0-4 ℃, and stirring at 500 rpm for reaction 24h to obtain a solution III;
4) The solution III was dialyzed with PBS buffer (0.01M PBS, pH 7.2) at 4℃for 3 days with stirring to give the phalloidin coating antigen.
The phalloidin antibody is a specific antibody of a phalloidin drug.
The phalloidin antibody working solution is obtained by diluting the monoclonal antibody of the phalloidin with an antibody dilution solution for 1000 times.
The antibody diluent is PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, and the solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, proclin300 and Triton X-100, and the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively.
The enzyme marker working solution is obtained by diluting an anti-antibody of horseradish peroxidase-marked anti-phalloidin drug monoclonal antibody by 500 times with an anti-antibody diluent.
The anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and the solute is anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentration of the solute in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively. In the kit, the conjugate of the phalloidin drug hapten and carrier protein is coated on an ELISA plate.
In the kit, the specific antibody of the phalloidin drug is the phalloidin monoclonal antibody.
In the kit, the phalloidin monoclonal antibody is composed of a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region can be shown as a sequence 1 in a sequence table. The amino acid sequence of the variable region of the light chain can be shown as a sequence 2 of a sequence table.
In the kit, the solvent of the 6 standard substance working solutions is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is phalloidin; the concentration of the solute in the 6 standard working solutions is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L and 0.1g/L respectively, and the pH value is 7.2.
In the kit, the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.1g/L respectively, and the pH value is 7.2.
In the kit, the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.2g/L respectively, and the pH value is 7.2.
In the kit, the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively.
In the kit, the substrate color development liquid is a mixed aqueous solution of 1.0g/L carbamide peroxide, 5.0g/L sodium acetate, 0.5g/L light stabilizer, 2.5mL/L phosphoric acid and 5.0g/L tetramethyl benzidine, and the solvent is deionized water.
In the kit, the stop solution is a sulfuric acid aqueous solution of 0.05 mol/L.
Another object of the present invention is to provide a method for detecting phalloidin in a sample, comprising the steps of:
1) Pretreating a sample to obtain a solution of the sample;
2) Detecting with any of the above kits;
3) And analyzing the detection result.
In the above method, the detection with the kit comprises the steps of: adding standard working solution or solution of the sample into an ELISA plate coated with the conjugate of the phalloidin drug hapten and carrier protein; adding a specific antibody solution containing the phalloidin drug; after incubation, washing and beating to dry, adding the enzyme-labeled anti-antibody, developing with a substrate, stopping and measuring the absorbance value with an enzyme-labeled instrument.
In the above method, the method for pre-treating the sample specifically comprises the following steps:
accurately weighing 1+/-0.01 g (or mL) sample, adding the sample into 5mL sample extracting solution, carrying out high-speed whirling for 1 min at room temperature (25+/-2 ℃), and centrifuging for 5min at 4000 g to obtain a sample solution. 200 mu L of sample solution is taken, 200 mu L of sample diluent is added, high-speed vortex is carried out for 1 min, and 50 mu L of supernatant is taken for analysis.
The detection principle of the kit provided by the invention is as follows: an indirect competition ELISA method for detecting phalloidin includes such steps as coating ELISA plate with the conjugate of phalloidin medicine hapten and carrier protein, using monoclonal antibody of phalloidin medicine as primary antibody, using the antibody of monoclonal antibody of anti-phalloidin medicine marked with horseradish peroxidase (HRP) as secondary antibody, adding substrate liquid for developing reaction, and using 0.05mol/L H 2 SO 4 The reaction was terminated and absorbance at 450 nm was measured to detect phalloidin.
The analysis process of the detection result provided by the invention comprises the following steps:
the absorbance average value (B) of the standard working solution at each concentration obtained was divided by the absorbance value (B0) of the first standard solution (0 standard) and multiplied by 100%, i.e., the percent absorbance value. The calculation formula is as follows: percent absorbance value (%) = (B/B0) ×100%
And drawing a standard curve graph by taking the half-logarithmic value of the concentration (mu g/L) of the phalloidin standard working solution as an X axis and the percentage absorbance value as a Y axis. The percentage absorbance value of the sample solution is calculated by the same method, and the content of the phalloidin in the sample can be read from the standard curve corresponding to the concentration of each sample.
The analysis of the detection result in the invention can also adopt a regression equation method to calculate the concentration of the sample solution.
The analysis of the detection result in the invention can also utilize the computer professional software, the method is more convenient for the rapid analysis of a large number of samples, and the whole detection process can be completed within 1.5 h only in a short time.
Experiments prove that the kit can detect the phalloidin in serum and urine; the detection limit of the phalloidin in the serum and urine is 1.0 mug/kg, the inter-batch variation coefficient is less than 10%, the intra-batch variation coefficient is less than 15%, and the stability is good.
The phalloidin detection kit provided by the invention can be used for detecting the phalloidin in serum and urine, has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.
Drawings
FIG. 1 is a graph of the standard graph of phalloidin.
FIG. 2 shows a comparison of the present kit with LC-MS/MS detection results.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of ELISA kit for detection of phalloidin
The kit comprises the following components: the kit comprises an ELISA plate (conjugate of coated phalloidin drug and carrier protein), an antibody working solution (monoclonal antibody containing the phalloidin drug), an enzyme marker working solution (monoclonal antibody containing horseradish peroxidase-labeled anti-phalloidin drug), a washing solution, a sample diluent, a sample extracting solution, a standard working solution containing the phalloidin with different gradient concentrations, a substrate color development solution and a termination solution.
The preparation method comprises the following steps:
1. preparation of ELISA plate
1. Preparation of phalloidin coating antigen
(1) Preparation of phalloidin hapten
The 50ml round bottom flask was rinsed clean, blow dried with ethanol, wrapped with tinfoil to prevent visible light, fixed on a stirrer, and added to the stirrer. Weighing 10mg of phalloidin raw material, dissolving 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid for reaction at room temperature, detecting by TLC, and preparing a plate for purification after the reaction is completed, thus obtaining the phalloidin hapten (formula I).
I is a kind of
(2) Preparation of phalloidin coating antigen
Dissolving 1.9. 1.9 mg phalloidin hapten with 0.5 mL DMF, stirring at 200 rpm for 10 min, adding EDC 1.0 mg,NHS 1.0 mg for dissolving, and stirring at room temperature (500 rpm) for activating 2-3 h to obtain solution I; weighing OVA 10mg, dissolving in 2.0 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve, cooling with ice bath to 0-4 ℃, dropwise adding solution I (1 mL/min) under stirring at 1000 rpm, and stirring at 500 rpm for reaction for 24h; the reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d with 1L of 0.01M PBS (1X, pH 7.2) stirred (100 rpm) at 4℃and 3 times daily (each time in the morning, in the evening) and 9 times total, and the dialyzed product was centrifuged at 5000 rpm for 6 min to obtain the phalloidin coating antigen.
2. Preparation of ELISA plate
Diluting the obtained phalloidin coating antigen with coating buffer solution to 10.0 mu g/mL, adding 100 mu L of coating antigen per well, incubating at 37 ℃ for 2 h, pouring off the coating solution, washing with 20 times of diluted washing solution for 2 times for 30 seconds each time, beating to dry, adding 150 mu L of sealing solution per well, incubating at 37 ℃ for 1 h, pouring off the liquid in the wells, drying to obtain the ELISA plate coated with the coating antigen (conjugate of the phalloidin drug hapten and carrier protein), and vacuum sealing and preserving by using an aluminum film.
Coating buffer solution: sodium carbonate buffer solution with pH of 9.6 and 0.03 mol/L;
sealing liquid: a phosphate buffer solution of 0.2 mol/L pH7.7 containing 50 g/L sucrose, 2.5 g/L casein, 0.5% calf serum, 3% sodium azide.
2. Preparation of antibody working solution
1. Preparation of phalloidin monoclonal antibody
(1) Preparation of phalloidin immunogen
Dissolving 1.3. 1.3 mg phalloidin hapten with 0.5 mL DMF, stirring at 200 rpm for 10 min, adding EDC 1.0 mg for dissolving, then adding NHS 1.0 mg, and stirring at room temperature (500 rpm) for activating 2-3 h to obtain solution I; weighing BSA 50 mg, dissolving in 3.5 mL 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to make the BSA dissolved fully, cooling the ice bath to 0-4 ℃, dropwise adding the solution I (1 mL/min) under stirring at 1000 rpm, and stirring at 500 rpm to react for 24h; the reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d with 1L 0.01M PBS (1X, pH 7.2) stirred (100 rpm) at 4℃and 3 times daily (each time in the morning, in the evening) and the total of 9 times, and the dialyzed product was centrifuged at 5000 rpm for 6 min to obtain the phalloidin immunogen.
(2) Immunization of animals
Dissolving the prepared phalloidin immunogen with physiological saline according to the volume of 100 mug/mouse, uniformly mixing the immunogen with Freund's complete adjuvant, subcutaneously injecting the mixture into the back of the neck to immunize 6-8 weeks old Balb/c female mice, uniformly mixing the immunogen with Freund's incomplete adjuvant at the same volume on days 7, 14 and 28 after primary immunization, carrying out additional immunization once each time, and carrying out additional immunization once with 100 mug/mouse of immune complex 3 days before fusion without Freund's adjuvant.
(3) Cell fusion and cloning
Mixing spleen cells of immunized mice with mouse myeloma cells (SP 2/0) in logarithmic phase, slowly adding preheated fusion agent (PEG 4000) for fusion within 45s, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37deg.C and 5% CO 2 Culturing in an incubator, half-changing liquid with HT culture medium after 5 days, and full-changing liquid at 9 days.
After the cells are fused, when the cells grow to 1/4 of the area of the culture hole, the step-by-step screening method is adopted to screen the hybridoma cells. The primary selection uses an indirect ELISA method to coat the antigen (previously using a matrixThe optimal coating concentration and positive serum dilution) of the coated ELISA plate are routinely titrated, the culture supernatant of the tested hole is added, the culture supernatant is incubated, and after washing, goat anti-mouse IgG-HRP and IgM-HRP and OPD are added for color reaction. The positive holes are screened by an indirect competition ELISA method, the cell supernatant is firstly mixed with 100 mug/mL of phalloidin in equal volume, the mixture is subjected to water bath at 37 ℃ for 30 min, and then the mixture is added into the coated ELISA plate. Meanwhile, PBS was used to replace the phalloidin for comparison, and the rest steps were the same as above. OD after blocking with phalloidin 450 If the nm value is reduced to less than 50% of the control hole, the hole is judged to be positive, and the hole which is positive after 2-3 times of detection is immediately subcloned by a limiting dilution method.
(4) Preparation and purification of monoclonal antibodies
Amplifying and culturing hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing; and taking 0.5 mL/mouse of 8-10 week old Balb/c mice to be injected with liquid paraffin in the intraperitoneal injection, and injecting hybridoma cells in the intraperitoneal injection for 7-10 days for 1-2X 10 5 And (3) extracting ascites of the mice after 7-10 days. Collecting cell supernatant or ascites, and measuring its titer by indirect ELISA (P/N for measuring titer)>2.1 Expressed as the maximum dilution of cell supernatants or ascites), the results indicated that the titer of cell supernatants was 1:10000, ascites titer is 1:60000. then purifying the monoclonal antibody by an octanoic acid-saturated ammonium sulfate method, and taking the supernatant to obtain the monoclonal antibody of the purified phalloidin drug.
The determination of the monoclonal antibody titer is carried out by using a chessboard method, and the result shows that: the titer of the monoclonal antibody of the phalloidin drug is 1:150000, half-maximal inhibitory amount (IC 50 ) 0.65. Mu.g/L.
Through detection, the amino acid sequence of the variable region of the heavy chain of the phalloidin monoclonal antibody is shown as the sequence 1 of the sequence table, and the amino acid sequence of the variable region of the light chain of the phalloidin monoclonal antibody is shown as the sequence 2 of the sequence table.
2. Preparation of antibody working solution
And diluting the obtained monoclonal antibody of the phalloidin drug by 1000 times with an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the phalloidin drug.
The antibody diluent is PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, and the solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, proclin300 and Triton X-100, and the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively.
3. Preparation of enzyme-labeled working fluid
1. Preparation of anti-antibodies
The monoclonal antibody of the obtained phalloidin medicine is used as an immunogen, and the goat is used as an immunized animal, so that the goat anti-mouse antibody of the monoclonal antibody of the phalloidin medicine is obtained.
2. Preparation of horseradish peroxidase-labeled anti-antibodies
Coupling the anti-antibody of the monoclonal antibody of the anti-phalloidin drug obtained in the step 1 with horseradish peroxidase (HRP) by adopting an improved sodium periodate method, and the steps are as follows:
dissolving 8 mg horseradish peroxidase in 2 mL distilled water; adding the prepared 100 mmol/L NaIO 4 Solution 0.4 and mL, stirring and reacting for 20 min at room temperature; dialyzing overnight at 4deg.C with 1 mmol/L acetate buffer; removing excess NaIO 4 Simultaneously reducing the self-coupled enzyme; 40. Mu.LPBS buffer (pH 8.6,0.5 mol/L) and 2.0/mL of PBS buffer (pH 8.6,5 mol/L) containing 16. 16 mg of monoclonal antibody against the phalloidin drug were added, and the mixture was stirred at room temperature for reaction for 4 hours; adding 0.1 mol/L NaBH mL which is prepared at present 4 The aqueous solution was reacted at 4℃for 4 hours, and the mixture was purified and stored.
3. Preparation of enzyme-labeled working fluid
Diluting the anti-antibody of the monoclonal antibody of the horseradish peroxidase-marked anti-phalloidin drug obtained in the step 2 by 500 times by using an anti-antibody diluent to obtain an enzyme marker working solution of the anti-antibody of the monoclonal antibody of the horseradish peroxidase-marked anti-phalloidin drug.
The anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and the solute is anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentration of the solute in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively.
4. Preparation of standard working solution
The kit also comprises 6 standard substance working solutions, wherein the solvent of the 6 standard substance working solutions is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is phalloidin; the concentration of the solute in the 6 standard working solutions is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L and 0.1g/L respectively, and the pH value is 7.2.
5. Preparation of other reagents
The kit may further comprise a sample diluent and/or a sample extract and/or a washing solution and/or a substrate color developing solution and/or a stop solution.
The sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.1g/L respectively, and the pH value is 7.2.
The sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.2g/L respectively, and the pH value is 7.2.
The washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively.
The substrate color development liquid is a mixed aqueous solution of 1.0g/L carbamide peroxide, 5.0g/L sodium acetate, 0.5g/L light stabilizer, 2.5mL/L phosphoric acid and 5.0g/L tetramethyl benzidine, and the solvent is deionized water.
The stop solution is 0.05mol/L sulfuric acid aqueous solution.
Example 2, method of Using the kit of example 1
1. Pretreatment of samples
1. Pretreatment of serum and urine samples
Accurately weighing 1+/-0.01 g (or mL) sample, adding the sample into 5mL sample extracting solution, carrying out high-speed whirling for 1 min at room temperature (25+/-2 ℃), and centrifuging for 5min at 4000 g to obtain a sample solution. 200 mu L of sample solution is taken, 200 mu L of sample diluent is added, high-speed vortex is carried out for 1 min, and 50 mu L of supernatant is taken for analysis.
2. Detection Using the example 1 kit
1. The ELISA plate is inserted into the ELISA plate frame, the positions of each standard substance and each sample are recorded, each sample is parallel to 3, and the unused ELISA plate strips are immediately stored in an environment of 2-8 ℃ after being sealed by a self-sealing bag;
2. adding 50 mu L of each standard working solution or sample solution into the corresponding standard or sample hole respectively;
3. add 50. Mu.L of antibody working fluid to each plate well;
4. covering the cover plate film, lightly oscillating the ELISA plate 10 s, fully mixing, and performing light-shielding reaction at room temperature (25+/-2 ℃) for 30 minutes;
5. uncovering the cover plate film, pouring out the liquid in the plate holes, adding 260 mu L of washing working solution (the washing solution is diluted by 20 times by deionized water) into each hole, fully washing for 3-4 times, and soaking for 15-30 s each time; the method comprises the steps of carrying out a first treatment on the surface of the
6. Pouring out the liquid in the plate hole, inverting the ELISA plate on the absorbent paper, and drying;
7. adding 100 mu L of enzyme marker working solution into each plate hole; covering the cover plate film, lightly oscillating the ELISA plate 10 s, fully mixing, and performing light-shielding reaction for 30 min at room temperature (25+/-2 ℃);
8. repeating the steps 5-6;
9. immediately adding 100 mu L of substrate developing solution A, B mixed solution (the substrate developing solution A and the substrate developing solution B are mixed according to the volume of 1:1) into each hole, covering a cover plate film, and carrying out light-proof reaction for 15 min;
10. uncovering the cover plate film, adding 50 mu L of stop solution into each plate hole, gently oscillating the ELISA plate 10 s, and fully and uniformly mixing;
11. the absorbance values of the elisa plates were read with an microplate reader at two wavelengths 405 nm, 630 nm within 5 minutes after termination.
3. Analyzing the detection result
1. Calculation of the percent absorbance values
The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/L), and the absorbance value is multiplied by 100%, so that the percentage of the absorbance corresponding to each standard (or sample to be measured), namely the percentage absorbance value, can be obtained.
Absorbance percentage = B/B 0 ×100%
Wherein: average absorbance value of B-standard (or sample); b (B) 0 Average absorbance value for standard at a concentration of 0 μg/L.
2. Making a standard curve
Drawing a standard curve graph by taking the percentage absorbance value of each standard substance as an ordinate and the concentration (mug/L) of the phalloidin in each standard substance working solution as an abscissa, and carrying out nonlinear fitting analysis by using origin8.0 (origin Lab Corp., northampton, mass., USA) to form a four-parameter fitting curve:
y=(A-D)/[1+(x/C)B]+D
wherein y is the percentage of absorbance; x is the concentration of the substance to be detected; a, B, C and D are four parameters of the standard curve.
Through test data, the standard curve equation of the phalloidin is: y= -0.003+ (2.309+0.003)/(1+ (x/5.550)/(1.03)) linear correlation R 2 0.999.
The standard graph is shown in fig. 2.
3. Calculating the content of phalloidin in the sample
Substituting the percentage absorbance value of the sample to be detected into a standard curve to obtain the corresponding residual concentration of the sample to be detected, and multiplying the residual concentration by the dilution multiple of the corresponding sample to obtain the actual content of the phalloidin in the original sample to be detected.
Example 3 specificity, detection Limit, accuracy, precision detection of the kits of example 1
1. Specificity test of the kit:
the specificity of the phalloidin ELISA kit was determined by cross-reacting with the corresponding substances.
The serial dilutions of phalloidin and its analogues (α -amatoxin, β -amatoxin) were performed respectively, the operations were performed according to example 2, and the serial dilutions of phalloidin and its analogues were used to replace the "phalloidin standard working fluid" therein, to prepare a standard curve, and the 50% Inhibitory Concentrations (IC) were found on the curve 50 ) The specific method comprises the following steps: the concentration (μg/L) of the phalloidin corresponding to the ordinate value of 50%, namely IC, is obtained 50 Values. The cross-reactivity of the kit to the phalloidin and each of the usual rodenticides was calculated by the following formula:
cross reaction ratio (%) = (concentration of phalloidin causing 50% inhibition/concentration of phalloidin analog causing 50% inhibition) ×100%.
The results are shown in Table 1.
Table 1 specificity of the kit
Experiments show that the kit has better specificity on the phalloidin, namely the kit can detect the phalloidin.
2. Detection limit determination of kit
Serum and urine blank samples (negative in LC-MS/MS detection) were taken, tested according to the method of example 2, the measurement values were obtained from the standard curve, the average value was calculated, and the minimum limit of detection (LOD) was obtained by adding 3 times the standard deviation. The results are shown in Table 2.
Table 2 blank sample measurement ([ mu ] g/L)
The result shows that in order to prevent false positive, the detection limit of the kit on the phalloidin in serum and urine can be defined as 1.0 mug/L.
3. Accuracy and precision test of kit
Accuracy refers to the degree of agreement between measured and actual values, accuracy is often expressed in terms of recovery, and precision is often expressed in terms of coefficient of variation. Serum and urine blank samples (LC-MS/MS detection negative) were taken and pre-treated as described in step one of example 2, and then the phalloidin standard was added to the required concentrations of 1.0. Mu.g/kg and 2.0. Mu.g/kg to obtain detection sample solutions.
3 different batches of kits were used for detection, each experiment was repeated 5 times, and the coefficient of variation was calculated separately. The results are shown in Table 3.
The calculation method of the intra-batch variation coefficient comprises the following steps: intra-lot coefficient of variation = coefficient of variation for each parallel sample in the same assay.
The calculation method of the variation coefficient between batches comprises the following steps: inter-lot coefficient of variation = coefficient of variation of the same sample measured in different lots, and the average value is taken.
TABLE 3 accuracy and precision
The results show that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The intra-batch variation coefficient of each additive concentration is lower than 10% and the inter-batch variation coefficient is lower than 15%.
4. The detection of the kit is compared with the detection result of LC-MS/MS
The samples of pork, milk and serum were tested as in example 2, and the results of the tests were confirmed and compared with the results of the LC-MS/MS tests, respectively.
And drawing a scatter diagram by taking the concentration of the phalloidin measured by the kit as an X axis and the concentration of the phalloidin measured by the LC-MS/MS as a Y axis. The measurement results of the two methods are subjected to linear analysis, the results are shown in fig. 2, and the regression equation is as follows: y=0.964x+0.016, it is shown that the method established by the present invention has good agreement with LC-MS/MS detection results.
5. Shelf life test of kit
The storage conditions of the kit of example 1 were 2-8deg.C, and the maximum absorbance (zero standard), 50% inhibition concentration, and the actual measurement of the addition of the phalloidin were all within the normal range after 12 months of measurement. Considering that abnormal preservation conditions appear in the transportation and use processes, the kit is placed for 8 days at 37 ℃ for accelerated aging test, and the result shows that the indexes of the first to fourth steps of the kit completely meet the requirements. Considering the occurrence of the freezing condition of the kit, the kit is placed for 8 days at the temperature of-20 ℃, and all indexes of the steps one to four completely meet the requirements. From the above results, the kit of example 1 can be stored at 2-8℃for at least 12 months.
Sequence listing
<110> Beijing Verdyvelkang Biotechnology Co., ltd
<120> an ELISA kit for detecting phalloidin, its preparation and application
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Gln Gln Ala Gln Ser Gly Ala Glu Leu Val Arg Leu Gln Pro Gly Val
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Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Asp
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Met Ala His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Gly Val
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Trp Thr Ile Ile Ser Tyr Tyr Gly Asp Ala Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Val Asp Lys Ala Thr Met Thr Lys Ser Ser Ser Thr Leu Tyr
65 70 75 80
Met Glu Tyr Ser Tyr Arg Thr Glu Leu Asp Ser Ala Ile Tyr Tyr Cys
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Asp Arg Gly Asp Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr
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Thr Val Thr Val Ser Ser
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<213> Artificial sequence (Artificial Sequence)
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Ile Gln Asp Met Thr Gln Pro Ser Leu Ala Ser Ser Ala Ser Val Gly
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Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Thr Lys Val Tyr
35 40 45
Gln Lys Ile Leu Leu Asn Asp Leu Ala Glu Gly Val Arg Ser Gly Gly
50 55 60
Ser Ser Phe Gly Thr Gln Phe Ser Leu Asn Ser Lys Ile Leu Gln Pro
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Asp Phe Glu Gly Gly Thr Arg Tyr Tyr Cys Ser Gln His His Tyr Pro
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Phe Phe Gly Ser Gly Thr Lys Val Glu Ile Lys Glu
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Claims (9)
1. An enzyme-linked immunosorbent assay kit for detecting phalloidin, comprising: the kit comprises an ELISA plate for detecting phalloidin, a standard substance working solution of the phalloidin series, a working solution of a phalloidin antibody, an enzyme marker working solution, a sample dilution solution, a sample extracting solution, a washing solution, a substrate color development solution and a termination solution, and is characterized in that: the phalloidin detection ELISA plate is prepared by coating conjugate of phalloidin hapten and carrier protein shown in formula I, and the phalloidin antibody is specific antibody of phalloidin.
2. The enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1, wherein the preparation method of the phalloidin hapten comprises the following steps: weighing 10mg of phalloidin raw material, dissolving 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid for reaction at room temperature, and performing thin-layer preparation plate purification after TLC detection reaction is completed to obtain the phalloidin hapten.
3. An enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1, wherein: the specific antibody of the phalloidin is a phalloidin monoclonal antibody, the phalloidin monoclonal antibody consists of a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain is shown as a sequence 1 of a sequence table, and the amino acid sequence of the variable region of the light chain is shown as a sequence 2 of the sequence table.
4. An enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1 or 3, wherein: the phalloidin antibody working solution is obtained by diluting a monoclonal antibody of the phalloidin by 1000 times with an antibody dilution solution;
the antibody diluent is PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, and the solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, proclin300 and Triton X-100, and the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively.
5. An enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1, wherein: the enzyme marker working solution is obtained by diluting an anti-antibody of an anti-phalloidin monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent;
the anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and the solute is anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentration of the solute in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively.
6. An enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1, wherein: the working solution of the standard substance series of the phalloidin is 6 standard substance working solutions of the phalloidin, the solvent of the working solution of the standard substance is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is the phalloidin; the concentration of the solute in the 6 phalloidin standard working solutions is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L and 0.1g/L respectively, and the pH value is 7.2.
7. An enzyme-linked immunosorbent assay kit for detecting phalloidin according to claim 1, wherein: the kit also comprises a sample diluent, a sample extracting solution, a washing solution, a substrate color development solution and a termination solution;
the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.1g/L respectively, and the pH value is 7.2;
the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentration of the solute in the PBS buffer solution is 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.2g/L respectively, and the pH value is 7.2;
the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively;
the substrate color development liquid is a mixed aqueous solution of 1.0g/L carbamide peroxide, 5.0g/L sodium acetate, 0.5g/L light stabilizer, 2.5mL/L phosphoric acid and 5.0g/L tetramethyl benzidine, and the solvent is deionized water;
the stop solution is 0.05mol/L sulfuric acid aqueous solution.
8. A method for detecting residue of phalloidin in a sample, comprising the steps of:
1) Pretreating a sample;
2) Performing a test with the kit of claim 1;
3) And analyzing the detection result.
9. The method for detecting residue of phalloidin in sample according to claim 8, wherein: the detection by the kit comprises the following steps: adding standard working solution or solution of the sample into an ELISA plate coated with the conjugate of the phalloidin hapten and carrier protein; adding specific antibody of phalloidin; washing and beating after incubation, adding the enzyme-labeled anti-antibody, developing color with a substrate of alkaline phosphatase, stopping and measuring a light absorption value with an enzyme-labeled instrument; and then the substrate of horseradish peroxidase is used for color development, termination and measurement of absorbance by an enzyme-labeled instrument.
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| WO1998031380A1 (en) * | 1997-01-16 | 1998-07-23 | Washington State University Research Foundation | Phalloidin derivatives and analogs to treat congestive heart failure |
| CN104628825A (en) * | 2015-02-10 | 2015-05-20 | 广东省微生物研究所 | Preparation method of carboxyl dihydroxy phallotoxin |
| CN109824785A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | A kind of dihydroxy Phallus phallotoxins artificial antigen and the preparation method and application thereof |
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| US20120231996A1 (en) * | 2006-05-04 | 2012-09-13 | Lokey R Scott | Phalloidin derivatives and methods for their synthesis |
| US7964702B2 (en) * | 2006-05-04 | 2011-06-21 | The Regents Of The University Of California | Phalloidin derivatives and methods for their synthesis |
| US8569452B2 (en) * | 2011-02-17 | 2013-10-29 | American Peptide Company, Inc. | Preparation of phalloidin and its derivatives |
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| WO1998031380A1 (en) * | 1997-01-16 | 1998-07-23 | Washington State University Research Foundation | Phalloidin derivatives and analogs to treat congestive heart failure |
| CN104628825A (en) * | 2015-02-10 | 2015-05-20 | 广东省微生物研究所 | Preparation method of carboxyl dihydroxy phallotoxin |
| CN109824785A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | A kind of dihydroxy Phallus phallotoxins artificial antigen and the preparation method and application thereof |
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