CN111273041A

CN111273041A - Enzyme linked immunosorbent assay kit for detecting phalloidin, and preparation and application thereof

Info

Publication number: CN111273041A
Application number: CN202010285671.2A
Authority: CN
Inventors: 马立才; 刘河冰; 温凯; 杨柳; 丁亚芳; 崔乃元; 聂靖东; 邢维维; 刘薇
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2020-06-12
Anticipated expiration: 2040-04-13
Also published as: CN111273041B

Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting phalloidin, and preparation and application of the kit. The phalloidin detection kit provided by the invention can be used for detecting phalloidin in shellfish food, the detection limit of the phalloidin in serum and urine can be defined as 1.0 mug/L, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%, and the kit has the advantages of simple and rapid operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Description

Enzyme linked immunosorbent assay kit for detecting phalloidin, and preparation and application thereof

Technical Field

The invention belongs to the field of rapid detection of drug residues, and relates to a kit for detecting phalloidin, a preparation method and application thereof.

Background

Mushroom food poisoning has been one of the food safety issues of major concern in many countries of the world, and for many years mushroom poisoning has also been the leading cause of death due to food poisoning accidents in our country. The phalloidin belongs to bicyclic polypeptide compounds, exists in some wild bacteria of Amanita (Amanita), paracoccidentalis (Galerina) and stropharia (Lepiota), and is easy to misdiagnose and lack of targeted treatment for food poisoning caused by the phalloidin, so early diagnosis is particularly important for treating the poisoning.

At present, methods for detecting phallotoxin in biological samples such as plasma and urine mainly comprise capillary electrophoresis, radioimmunoassay, high performance liquid chromatography, mass spectrometry and the like, wherein the liquid chromatography-tandem mass spectrometry can realize high-flux accurate analysis of toxin 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 is complex to operate, expensive in instrument, not suitable for screening and detecting mass samples and cannot meet the field detection requirement.

Disclosure of Invention

The invention aims to provide an enzyme linked immunosorbent assay kit for detecting phalloidin, and provides a detection method which is high in sensitivity, strong in specificity, low in detection cost and suitable for screening large-batch samples.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an enzyme linked immunosorbent assay kit for detecting phalloidin comprises an ELISA plate, a standard substance working solution, an antibody working solution, an enzyme marker working solution, a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution.

The ELISA plate is prepared by coating conjugate of phalloidin drug hapten and carrier protein.

The preparation method of the phalloidin hapten comprises the following steps: weighing 10mg of phalloidin raw material, dissolving in 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid, reacting at room temperature, detecting by TLC, and purifying by using a thin-layer preparation plate after complete reaction to obtain the hapten (formula I).

Formula I

The carrier protein is Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), mouse serum protein (MSA), thyroxine (BCG), rabbit serum protein (RSA), hemocyanin (KLH) or Ovalbumin (OVA).

The conjugate of phalloidin hapten and carrier protein is a product obtained by the phalloidin hapten and the carrier protein through an active ester method, and specifically comprises the following steps:

1) dissolving the phalloidin hapten (formula I) in Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and reacting for 2-3 h at 20-25 ℃ by magnetic stirring to obtain a solution I;

wherein the proportion 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) putting the carrier protein into 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 solution 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 0-4 ℃ and stirring at 1000rpm, and stirring at 500 rpm for 24 hours 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 under stirring to obtain the phalloidin-coated antigen.

The phalloidin antibody is a specific antibody of phalloidin medicine.

The phalloidin antibody working solution is prepared by diluting a monoclonal antibody of phalloidin by 1000 times with an antibody diluent to obtain the monoclonal antibody working solution containing phalloidin medicine.

The antibody diluent is a PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, Proclin300 and Triton X-100, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 muL/L and 500 muL/L respectively.

The enzyme marker working solution is obtained by diluting an anti-antibody of an anti-phalloidin drug monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent.

The anti-antibody diluent is a PBS (phosphate buffer solution) containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L respectively. In the kit, the conjugate of the phalloidin drug hapten and carrier protein is coated on an enzyme label 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 variable region of the heavy chain 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 in 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 concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L, and the pH value is 7.2.

In the kit, the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, and the pH value is 7.2.

In the kit, the sample extracting solution is PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, 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 solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

In the kit, the substrate color development liquid is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

In the kit, the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

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 one of the above-mentioned kits;

3) and analyzing the detection result.

In the above method, the detection with the kit comprises the following steps: adding standard working solution or the solution of the sample into an enzyme label plate coated with the conjugate of the phalloidin drug hapten and the carrier protein; adding specific antibody solution containing phalloidin medicine; after incubation, the swatches were washed dry, the enzyme-labeled anti-antibody was added, the substrate was developed, stopped and the absorbance was measured with an enzyme-linked plate reader.

In the above method, the method for pretreating the sample specifically comprises the following steps:

accurately weighing 1+ -0.01 g (or mL) of sample, adding 5 mL of sample extract, whirling at high speed for 1min at room temperature (25 + -2 deg.C), and centrifuging at 4000 g for 5min to obtain sample solution. And adding 200 mu L of sample solution into 200 mu L of sample diluent, whirling at a high speed for 1min, and taking 50 mu L of supernatant to analyze.

The detection principle of the kit of the invention is as follows: an indirect competitive ELISA method for detecting phalloidin includes such steps as coating enzyme label plate with conjugate of haptens of phalloidin medicine and carrier protein, using monoclonal antibody of phalloidin medicine as primary antibody, using the anti-antibody of horseradish peroxidase (HRP) as secondary antibody, adding substrate liquid for developing reaction, and adding 0.05 mol/L H₂SO₄Terminating the reaction, measuringAbsorbance at 450 nm to detect phalloidin.

The analysis process of the detection result provided by the invention comprises the following steps:

the absorbance average (B) of the standard working solution of 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). times.100%

And drawing a standard curve chart by taking a half logarithmic value of the concentration (mu g/L) of the phalloidin standard product working solution as an X axis and a percent absorbance value as a Y axis. The percent absorbance of the sample solution was calculated in the same manner, and the content of phalloidin in the sample was 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 can also utilize 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 in a short time.

Experiments prove that the kit can detect phalloidin in serum and urine; the limit of detection of phalloidin in 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 phalloidin in serum and urine, has the advantages of simple and rapid operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Drawings

FIG. 1 is a standard graph of phalloidin.

FIG. 2 is a comparison of the results of the detection by the present kit with LC-MS/MS.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of enzyme-linked immunosorbent assay kit for detecting phalloidin

The kit comprises the following components: an enzyme label plate (a conjugate of a coated phalloidin medicament and a carrier protein), an antibody working solution (containing a phalloidin medicament monoclonal antibody), an enzyme marker working solution (containing an anti-phalloidin medicament monoclonal antibody marked by horseradish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a standard substance working solution containing phalloidin peptides with different gradient concentrations, a substrate developing solution and a stop solution.

The preparation method comprises the following steps:

firstly, preparation of enzyme label plate

1. Preparation of Coprinus cinereus cyclic peptide coating antigen

(1) Preparation of phalloidin hapten

A50 ml round bottom flask was rinsed clean, blown dry with ethanol, wrapped with tinfoil paper to prevent exposure to light, set on a stirrer, and a stir bar was added. Weighing 10mg of phalloidin raw material, dissolving in 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid, reacting at room temperature, detecting by TLC, and purifying by a thin-layer preparation plate after complete reaction to obtain the phalloidin hapten (formula I).

Formula I

(2) Preparation of Coprinus cinereus cyclic peptide coating antigen

Dissolving 1.9 mg of phalloidin hapten with 0.5 mL of DMF, stirring at 200 rpm for 10 min, adding 1.0 mg of EDC and 1.0 mg of NHS for dissolving, and stirring at room temperature (500 rpm) for activation for 2-3 h to obtain a solution I; weighing OVA 10mg, dissolving in 2.0 mL0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve, cooling at 0-4 deg.C in ice bath, stirring at 1000rpm, dropwise adding solution I (1 mL/min), stirring at 500 rpm, and reacting for 24 h; filling the reaction product into a distilled water washing dialysis bag (10 cm), stirring at 4 ℃ and stirring (100 rpm) for dialysis for 3 d by 1L of 0.01M PBS (1 x, pH7.2), changing the solution 3 times (once in the morning, in the evening) each day, changing the solution 9 times in total, and centrifuging the dialysis product at 5000 rpm for 6 min to obtain the phalloidin coated antigen.

2. Preparation of ELISA plates

Diluting the obtained phalloidin-coated antigen to 10.0 μ g/mL with a coating buffer solution, adding 100 μ L of the antigen into each well, incubating at 37 ℃ for 2 h, pouring out the coating solution, washing with a washing solution diluted by 20 times for 2 times, each time for 30 seconds, patting dry, then adding 150 μ L of a blocking solution into each well, incubating at 37 ℃ for 1 h, pouring out the liquid in the wells, drying to obtain an ELISA plate coated with the antigen (conjugate of phalloidin-drug hapten and carrier protein), and preserving in vacuum sealing with an aluminum film.

Coating buffer solution: 0.03 mol/L sodium carbonate buffer solution with the pH value of 9.6;

sealing liquid: contains 50 g/L sucrose, 2.5 g/L casein, 0.5% calf serum, and 3 ‰ sodium azide in 0.2 mol/LpH7.7 phosphate buffer solution.

Preparation of antibody working solution

1. Preparation of phalloidin monoclonal antibody

(1) Preparation of phalloidin immunogen

Dissolving 1.3 mg of phalloidin hapten with 0.5 mL of DMF, stirring at 200 rpm for 10 min, adding 1.0 mg of EDC, dissolving, adding 1.0 mg of NHS, stirring at room temperature (500 rpm), and activating for 2-3 h to obtain a solution I; weighing 50 mg of BSA, dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve the BSA, cooling in an ice bath at 0-4 ℃, dropwise adding the solution I (1 mL/min) while stirring at 1000rpm, and stirring at 500 rpm for reaction for 24 h; and filling the reaction product into a distilled water washing dialysis bag (10 cm), stirring 1L 0.01M PBS (1 x, pH7.2) at 4 ℃ and dialyzing for 3 d at 100 rpm, changing the solution 3 times (once in the morning, at night) every day, changing the solution 9 times in total, and centrifuging the dialysis product at 5000 rpm for 6 min to obtain the phalloidin immunogen.

(2) Animal immunization

Dissolving the immunogen and Freund's complete adjuvant in normal saline at a ratio of 100 mu g/cell with the prepared phalloidin immunogen, mixing the dissolved immunogen and Freund's complete adjuvant in equal volume, injecting subcutaneously on the neck and back to immunize Balb/c female mice of 6-8 weeks old, mixing the immunogen and Freund's incomplete adjuvant in equal volume at 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once with 100 mu g/cell of immune complex 3 days before fusion without adding Freund's adjuvant.

(3) Cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells of mice (SP 2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator, half-changing the culture medium with HT after 5 days, and completely changing the culture medium after 9 days.

After cell fusion, when the cells grow to 1/4 of the culture hole area, hybridoma cells are screened by a step screening method. The primary selection adopts an indirect ELISA method, an enzyme label plate is coated with coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), culture supernatant of a detected hole is added, incubation and washing are carried out, and then goat anti-mouse IgG-HRP, IgM-HRP and OPD are added for color reaction. The screened positive hole is screened by an indirect competitive ELISA method, the cell supernatant is mixed with the phalloidin of 100 mu g/mL with equal volume, the mixture is taken in water bath at 37 ℃ for 30 min, and then the mixture is added into the coated enzyme label plate. Meanwhile, PBS was used to replace phalloidin as a control, and the rest steps were as above. OD after phalloidin cleavage₄₅₀And (3) judging the wells to be positive when the nm value is reduced to below 50% of the control wells, and subcloning the wells which are positive after 2-3 detections by using a limiting dilution method immediately.

(4) Preparation and purification of monoclonal antibodies

Carrying out expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing and storing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, injecting 1-2 × 10 hybridoma cells into the abdominal cavity after 7-10 days⁵Ascites was extracted 7 to 10 days later. Collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 cell supernatants or abdomenWater maximum dilution) the results indicated a titer of the cell supernatant of 1: 10000, ascites titer 1: 60000. then, the monoclonal antibody of the phalloidin drug is purified by an octanoic acid-saturated ammonium sulfate method, and the supernatant is taken to obtain the purified monoclonal antibody of the phalloidin drug.

The chessboard method is utilized to measure the titer of the monoclonal antibody, and the result shows that: the titer of the phalloidin drug monoclonal antibody is 1: 150000, half maximal Inhibition (IC)₅₀) It was 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 sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain of the phalloidin monoclonal antibody is shown as sequence 2 in the sequence table.

2. Preparation of antibody working solution

Diluting the obtained monoclonal antibody of the phalloidin medicine by 1000 times with an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the phalloidin medicine.

Thirdly, preparation of enzyme marker working solution

1. Preparation of anti-antibodies

The obtained monoclonal antibody of the phalloidin drug is used as immunogen, and the goat is used as immune animal, so as to obtain the goat anti-mouse anti-antibody of the monoclonal antibody of the phalloidin drug.

2. Preparation of horse radish peroxidase-labeled anti-antibody

Coupling the anti-antibody of the monoclonal antibody of the anti-phalloidin drug obtained in the step 1 with Horse Radish Peroxidase (HRP) by adopting a modified sodium periodate method, and the steps are as follows:

dissolving 8 mg of horseradish peroxidase in 2 mL of distilled water; adding 100 mmol/L of the prepared solution NaIO₄Stirring and reacting the solution for 20 min at room temperature, wherein the solution is 0.4 mL; dialyzing with 1 mmol/L acetate buffer solution at 4 deg.C overnight; removing excess NaIO₄Simultaneously reducing the enzyme coupled with the enzyme; adding 40. mu.L of LPBS buffer (pH 8.6, 0.5 mol/L) and 2.0 mL of PBS buffer (pH 8.6, 5 mol/L) containing 16mg of an anti-antibody against the monoclonal antibody of phalloidin drug, and reacting at room temperature with stirring for 4 hours; 0.1 mL of 1 mol/L NaBH prepared now is added₄Reacting the aqueous solution at 4 ℃ for 4 hours, purifying and storing.

3. Preparation of enzyme-labeled working solution

Diluting the anti-antibody of the monoclonal antibody of the anti-phalloidin drug marked by the horseradish peroxidase 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 anti-phalloidin drug marked by the horseradish peroxidase.

The anti-antibody diluent is a PBS (phosphate buffer solution) containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L respectively.

Fourth, preparation of standard substance 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 concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L, and the pH value is 7.2.

Preparation of five, other reagents

The kit may further contain a sample diluent and/or a sample extract and/or a washing solution and/or a substrate developing solution and/or a stop solution.

The sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, 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 solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, 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 solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

The substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

The stop solution is 0.05 mol/L sulfuric acid aqueous solution.

Example 2 and example 1 methods of Using the kits

First, pretreatment of sample

1. Pretreatment of serum and urine samples

Second, detection Using the kit of example 1

1. Inserting an enzyme label plate into an enzyme label plate frame, recording the positions of each standard product and each sample, making 3 samples in parallel, sealing unused enzyme label plates by using self-sealing bags, and immediately storing in an environment at 2-8 ℃;

2. respectively adding 50 mu L of each standard substance working solution or sample solution into the corresponding standard substance or sample hole;

3. adding 50 mu L of antibody working solution into each plate hole;

4. covering a cover plate membrane, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

5. uncovering the plate film, pouring out 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, and fully washing for 3-4 times, wherein each time of soaking is 15-30 s; (ii) a

6. Pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry;

7. adding 100 mu L of enzyme marker working solution into each plate hole; covering a cover plate film, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

8. repeating the step 5-6;

9. immediately adding 100 μ L of substrate color developing solution A, B mixed solution (substrate color developing solution A and substrate color developing solution B are mixed according to volume ratio of 1: 1) into each well, covering with a cover plate membrane, and reacting for 15 min in dark place;

10. uncovering the microplate membrane, adding 50 mu L of stop solution into each plate hole, slightly oscillating the ELISA plate for 10 s, and fully and uniformly mixing;

11. and reading the absorbance values of the ELISA plate at the dual wavelengths of 405 nm and 630 nm by using an ELISA reader within 5min after termination.

Thirdly, analyzing the detection result

1. Calculating percent absorbance value

The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of zero standard (standard with concentration of 0 mug/L) and multiplied by 100 percent, so as to obtain the percentage of absorbance corresponding to each standard (or sample to be measured), namely the percent absorbance value.

Percent absorbance = B/B₀×100%

Wherein: b-mean absorbance value of standard (or sample); b is₀Average absorbance values of standards at a concentration of 0. mu.g/L.

2. Making a standard curve

The percent absorbance values of the standards are used as ordinate, the phalloidin concentration (mug/L) in the working solution of each standard is used as abscissa to draw a standard curve graph, origin8.0 (originLab Corp., Northamapton, MA, USA) is used for nonlinear fitting analysis, and a four-parameter fitting curve is formed:

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 the four parameters of the standard curve.

Through the test data, the standard curve equation of phalloidin is: y = -0.003+ (2.309+0.003)/(1+ (x/5.550) ^1.03), linear correlation R²Is 0.999.

The standard graph is shown in fig. 2.

3. Calculating the content of phalloidin in the sample

Substituting the percent absorbance value of the sample to be detected into the standard curve to obtain the corresponding residual concentration of the sample to be detected, and multiplying the residual concentration by the dilution times of the corresponding sample to obtain the actual content of the phalloidin in the original sample to be detected.

Example 3 and example 1 detection of specificity, detection Limit, accuracy, precision of the kits

Firstly, specific test of the kit:

the specificity of the phalloidin enzyme-linked immunosorbent assay kit is determined by carrying out cross reaction tests on the phalloidin enzyme-linked immunosorbent assay kit and corresponding substances.

Coprinus cinereus Lindl and its analogs (α -amatoxin, β -amatoxin) were diluted serially and processed as in example 2The serial dilutions were used to substitute for "phalloidin standard working solution" to prepare standard curves, and the respective 50% Inhibitory Concentrations (IC) were found on the curves₅₀) The specific method comprises the following steps: the corresponding phalloidin concentration (. mu.g/L), IC, was obtained with a value on the ordinate equal to 50%₅₀The value is obtained. The cross-reactivity of the kit to phalloidin and each of the commonly used muricides was calculated using the following formula:

the cross-reactivity (%). times.100% (phalloidin concentration causing 50% inhibition/phalloidin analogue concentration causing 50% inhibition).

The results are shown in Table 1.

TABLE 1 specificity of the kit

Experiments show that the kit has good specificity to phalloidin, namely the kit can detect phalloidin.

Second, detection limit determination of the kit

A blank sample of serum or urine (negative in LC-MS/MS) was sampled and tested according to the method of example 2, the values were determined from the standard curve, the average value was calculated, and the lowest limit of detection (LOD) was calculated by adding 3 times the standard deviation. The results are shown in Table 2.

TABLE 2 blank sample determination results (μ g/L)

The result shows that in order to prevent the occurrence of false positive, the detection limit of the kit on phalloidin in serum and urine can be defined as 1.0 mug/L.

Thirdly, testing the accuracy and precision of the kit

Accuracy refers to the degree of coincidence between a measured value and a true value, and is often expressed in terms of recovery and precision in terms of coefficient of variation. Serum and a blank urine sample (negative in LC-MS/MS) are respectively pretreated according to the method in the first step of the embodiment 2, and then a phalloidin standard product is added to the required concentration of 1.0 mu g/kg and 2.0 mu g/kg, so as to obtain a detection sample solution.

The detection is carried out by using 3 kits of different batches, each experiment is repeated for 5 times, and the coefficient of variation is calculated respectively. The results are shown in Table 3, respectively.

The method for calculating the intra-batch variation coefficient comprises the following steps: intra-batch coefficient of variation = coefficient of variation for each parallel sample in the same assay.

The method for calculating the inter-batch variation coefficient comprises the following steps: the inter-batch coefficient of variation = coefficient of variation of measurement results of the same sample in different batches, and the average value thereof is taken.

TABLE 3 accuracy and precision

The result shows that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The variation coefficient in batch of each addition concentration is lower than 10%, and the variation coefficient between batches is lower than 15%.

Fourthly, the detection result of the kit is compared with the detection result of LC-MS/MS

The method of example 2 is used for detecting pork, milk and serum samples, and the detection results of LC-MS/MS are respectively used for confirmation and comparison.

A scatter diagram is drawn by taking the concentration of the phalloidin measured by the kit as an X axis and the concentration of the phalloidin drug measured by LC-MS/MS as a Y axis. The measurement results of the two methods were subjected to linear analysis, and the results are shown in fig. 2, and the regression equation is: y =0.964x +0.016, which shows that the method established by the invention has good consistency with the detection result of LC-MS/MS.

Fifth, shelf life test of kit

The storage conditions of the kit of example 1 were 2 to 8 ℃, and the maximum absorbance (zero standard), the 50% inhibitory concentration, and the actual measurement value of phalloidin addition of the kit were within the normal ranges after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed at 37 ℃ for 8 days for accelerated aging test, and the result shows that all indexes of the first step to the fourth step of the kit completely meet the requirements. And (3) considering the occurrence of the freezing condition of the kit, placing the kit at the temperature of-20 ℃ for 8 days, wherein all indexes of the first step to the fourth step completely meet the requirements. As can be seen from the above results, the kit of example 1 can be stored at 2-8 ℃ for at least 12 months.

Sequence listing

<110> Beijing Weideweikang Biotech Ltd

<120> enzyme linked immunosorbent assay kit for detecting phalloidin, and preparation and application thereof

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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 TyrThr 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

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Lys Gly Val Asp Lys Ala Thr Met Thr Lys Ser Ser Ser Thr Leu Tyr

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Thr Val Thr Val Ser Ser

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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

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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

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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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Claims

1. An enzyme linked immunosorbent assay kit for detecting phalloidin, comprising: the kit comprises an enzyme label plate for phalloidin detection, a phalloidin series standard substance working solution, a phalloidin antibody working solution, an enzyme marker working solution, a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution, and is characterized in that: the phalloidin enzyme-linked plate is prepared by coating conjugate of phalloidin drug hapten and carrier protein shown in formula I:

formula I

The phalloidin antibody is a specific antibody of phalloidin medicine.

2. The ELISA 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 in 10ml of methanol, stirring, adding 1mg of sodium hydride and 2.1mg of tetrabromobutyric acid, reacting at room temperature, and detecting by TLC (thin layer chromatography) to obtain the phalloidin hapten after the reaction is completed.

3. The ELISA kit for detecting phalloidin according to claim 1, wherein: the specific antibody of the phalloidin medicine is the phalloidin monoclonal antibody, the phalloidin monoclonal antibody is composed of a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain can be shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain can be shown as the sequence 2 in the sequence table.

4. The ELISA kit for detecting phalloidin according to claim 1 or 3, wherein: the phalloidin antibody working solution is prepared by diluting a monoclonal antibody of phalloidin by 1000 times with an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of a phalloidin drug;

5. The ELISA 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 drug monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent;

6. The ELISA kit for detecting phalloidin according to claim 1, wherein: the phalloidin series standard working solution is 6 phalloidin standard working solutions, the solvent of the solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is phalloidin; the concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L and 0.1 g/L, and the pH value is 7.2.

7. The ELISA kit for detecting phalloidin according to claim 1, wherein: the kit also contains a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution;

the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.1 g/L, 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 solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1 g/L, 4g/L, 0.1 g/L, 0.2g/L, 0.1 g/L and 0.2g/L, 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 solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively;

the substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water;

the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

8. A method for detecting phalloidin residue in a sample, comprising the steps of:

1) pretreating a sample;

2) detecting with the kit of claim 1;

3) and analyzing the detection result.

9. The method of claim 8, wherein the step of detecting phalloidin residue in the sample comprises: the detection by using the kit comprises the following steps: adding standard working solution or the solution of the sample into an enzyme label plate coated with the conjugate of the phalloidin drug hapten and the carrier protein; adding specific antibody of phalloidin medicine; washing and drying after incubation, adding the enzyme-labeled anti-antibody, developing by using a substrate of alkaline phosphatase, terminating, and measuring a light absorption value by using an enzyme-labeling instrument; then, the substrate of horseradish peroxidase is used for developing, stopping and measuring the light absorption value by an enzyme-labeling instrument.

10. The method of claim 8, wherein the step of detecting phalloidin residue in the sample comprises: the kit can detect phalloidin in serum and urine; the limit of detection of phalloidin in serum and urine is 10 mug/kg, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%.