1. A carboxyl dihydroxyphallus toxalbumin time-resolved fluorescence immunoassay card is characterized by comprising a bottom plate, a sample pad, a release pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is arranged on the bottom plate, the release pad contains an europium microsphere labeled anti-carboxyl dihydroxyphallus toxalbumin antibody, and the nitrocellulose membrane and the water absorption pad contain a carboxyl dihydroxyphallus toxalbumin detection line T and a quality control line C.

2. The test card of claim 1, wherein: the anti-carboxyl dihydroxyphalloidin toxic peptide antibody is an anti-carboxyl dihydroxyphalloidin toxic peptide monoclonal antibody.

3. The test card of claim 2, wherein:

the release pad containing the europium microsphere labeled anti-carboxyl dihydroxyl phalloidin antibody is formed by spraying an anti-carboxyl dihydroxyl phalloidin antibody solution labeled by europium microspheres on the release pad;

the concentration of the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin antibody solution is 0.1 mg/mL.

4. The test card of claim 3, wherein:

in the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin antibody solution, the mass ratio of the anti-carboxyl dihydroxyphalloidin antibody to the europium microsphere is 1: 25;

and/or the europium microspheres are 200nm carboxylated europium microspheres.

5. The test card of any of claims 1-4, wherein:

the carboxyl dihydroxyphalloidin detection line T is formed by carboxyl dihydroxyphalloidin antigen or solution thereof;

and/or, the quality control line C is formed by IgG antibody or IgG antibody solution;

and/or the cellulose nitrate membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C is a cellulose nitrate membrane coated with a carboxyl dihydroxyphalloidin antigen solution to form a detection line T, and coated with an IgG antibody solution to form a quality control line C, so as to obtain the cellulose nitrate membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C;

preferably, the coating concentration of the carboxyl dihydroxyphalloidin antigen solution is 0.4 mg/mL;

and/or the coating concentration of the IgG antibody solution is 1.0 mg/mL.

6. A method of preparing a test card according to any one of claims 1 to 5, wherein: the method comprises the following steps: firstly, preparing a release pad containing an anti-carboxyl dihydroxyphalloidin antibody marked by europium microspheres and a nitrocellulose membrane containing a carboxyl dihydroxyphalloidin detection line T and a quality control line C; and assembling a sample pad, the release pad containing the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin toxic peptide antibody, the nitrocellulose membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C and a water absorption pad on a bottom plate to obtain the detection card.

7. Use of a test card according to any of claims 1-5 in:

(1) the application in preparing the product for quantitatively or qualitatively detecting or assisting in detecting the carboxyl dihydroxyl phalloidin;

(2) the application in quantitative or qualitative detection or auxiliary detection of carboxyl dihydroxyphalloidin.

8. A method for detecting or assisting in detecting whether a sample to be detected contains carboxyl dihydroxyphalloidin or not is characterized in that: the method comprises the following steps: adding a sample to be tested to a sample pad of the detection card according to any one of claims 1 to 5, reacting, and detecting a reaction product by using a fluorescence immunoassay analyzer;

if the fluorescence signal value of the detection line T of the detection card is less than or equal to the fluorescence signal value of the quality control line C, the sample to be detected contains or is candidate to contain carboxyl dihydroxyphalloidin;

and if the fluorescence signal value of the detection line T of the detection card is greater than the fluorescence signal value of the quality control line C, the sample to be detected does not contain or is candidate to contain carboxyl dihydroxyphalloidin.

9. A method for detecting or assisting in detecting the content of carboxyl dihydroxyl phalloidin in a sample to be detected is characterized in that: the method comprises the following steps:

(1) preparing a standard curve: taking a sample which has the same source as the sample to be detected and has the content of carboxyl dihydroxyl phalloidin less than 0.1 mug/L;

adding carboxyl dihydroxyl phalloidin with different concentrations into the sample to obtain standard substance solutions with different concentrations;

adding the standard solutions with different concentrations to the sample pad of the detection card of any one of claims 1 to 5, reacting, and detecting the reaction product by using a fluorescence immunoassay analyzer to obtain the fluorescence signal values of the detection line T and the quality control line C of the standard solutions with different concentrations;

drawing a standard curve by taking different concentrations of the added carboxyl dihydroxyphalloidin as an X axis and taking the ratio of the fluorescence signal value of the detection line T corresponding to the different concentrations to the fluorescence signal value of the quality control line C as a Y axis;

(2) adding the sample to be detected to the sample pad of the detection card of any one of claims 1 to 5, reacting, and detecting the reaction product by using a fluorescence immunoassay analyzer to obtain a detection line T fluorescence signal value and a quality control line C fluorescence signal value of the sample to be detected;

and (3) substituting the fluorescence signal value of the detection line T and the fluorescence signal value of the quality control line C of the sample to be detected into the standard curve obtained in the step (1) to obtain the content of carboxyl dihydroxyphalloidin in the sample to be detected.

10. The use according to claim 7 or the method according to claim 8 or 9, characterized in that: the sample to be detected is a urine sample, a blood plasma sample or a blood serum sample.

Background

Poisonous mushroom poisoning is one of the leading causes of death in food poisoning events. More than 90% of poisoned and killed mushrooms are caused by mushrooms containing amanitin toxins, phalloidin is one of the amanitin toxins, is a bicyclic heptapeptide, and has been found to have 7 kinds and similar structures. Carboxydihydroxyphalloidin (PCD) is one of the main components, and is present with amatoxin, the lethal dose for mice is 50 μ g, the PCD is also very toxic to human body, and after poisoning, the liver of animals or human is damaged, and salivation, vomiting, hematochezia, cyanosis, spasm and muscle contracture can be caused, so that the death can be caused. It acts in contrast to cytochalasin, binding only to polymeric microfilaments and not to actin monomeric molecules. It, when combined with the polymeric microfilaments, inhibits the disintegration of the microfilaments and thus disrupts the dynamic balance of the polymerization and depolymerization of the microfilaments.

At present, the main detection method of the residual content of carboxyl dihydroxyphalloidin in urine is a chromatographic method, such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC) and the like. The chromatographic method generally needs expensive detection instruments and has the problems of complex pretreatment, complex operation, long detection time and high detection cost, so the chromatographic method is not suitable for detecting large-batch samples in a basic laboratory.

The Time-resolved fluoroimmunoassay (TRFIA) is a novel nonradioactive immunoassay technology established by combining the high specificity of comprehensive immune reaction and the high sensitivity of lanthanide labeling by taking lanthanide (including Eu, Tb, Sm, Dy, and the like) as a marker. The lanthanide has the advantages of large Stokes shift (>200nm) and long fluorescence lifetime (1-2ms), can avoid interference with excitation light and background fluorescence signals to the maximum extent, and greatly improves the detection sensitivity. In view of the above characteristics, TRFIA has been widely used in clinical diagnosis, food safety detection, and other fields. But is not applied to the detection of carboxyl dihydroxyphalloidin at present.

Disclosure of Invention

The invention aims to provide a detection card capable of quickly and sensitively detecting carboxyl dihydroxyphalloidin, a method and application thereof.

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

a carboxyl dihydroxyphalloidin time-resolved fluorescence immunoassay card comprises a bottom plate, a sample pad, a release pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is arranged on the bottom plate, the release pad contains an europium microsphere labeled anti-carboxyl dihydroxyphalloidin antibody, and the nitrocellulose membrane and the water absorption pad contain a carboxyl dihydroxyphalloidin detection line T and a quality control line C (a control line).

In the invention, the anti-carboxyl dihydroxyphalloidin peptide antibody is an anti-carboxyl dihydroxyphalloidin peptide monoclonal antibody.

In the invention, the release pad containing the europium microsphere-labeled anti-carboxyl dihydroxyl phalloidin antibody is formed by spraying an anti-carboxyl dihydroxyl phalloidin antibody solution labeled by europium microspheres on the release pad;

the concentration of the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin antibody solution is 0.1 mg/mL.

In the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin antibody solution, the mass ratio of the anti-carboxyl dihydroxyphalloidin antibody to the europium microsphere is 1: 25;

and/or the europium microspheres are 200nm carboxylated europium microspheres.

In the invention, the carboxyl dihydroxyphalloidin detection line T is formed by carboxyl dihydroxyphalloidin antigen or solution thereof;

and/or, the quality control line C is formed by IgG antibody or IgG antibody solution;

and/or the cellulose nitrate membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C is a cellulose nitrate membrane coated with a carboxyl dihydroxyphalloidin antigen solution to form a detection line T, and coated with an IgG antibody solution to form a quality control line C, so as to obtain the cellulose nitrate membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C;

preferably, the coating concentration of the carboxyl dihydroxyphalloidin antigen solution is 0.4 mg/mL;

and/or the coating concentration of the IgG antibody solution is 1.0 mg/mL.

The invention also provides a method for preparing the detection card, which comprises the following steps: firstly, preparing a release pad containing an anti-carboxyl dihydroxyphalloidin antibody marked by europium microspheres and a nitrocellulose membrane containing a carboxyl dihydroxyphalloidin detection line T and a quality control line C; and assembling a sample pad, the release pad containing the europium microsphere-labeled anti-carboxyl dihydroxyphalloidin toxic peptide antibody, the nitrocellulose membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C and a water absorption pad on a bottom plate to obtain the detection card.

The invention also provides an application of the detection card in the following aspects:

(1) the application in preparing the product for quantitatively or qualitatively detecting or assisting in detecting the carboxyl dihydroxyl phalloidin;

(2) the application in quantitative or qualitative detection or auxiliary detection of carboxyl dihydroxyphalloidin.

The application of the europium microsphere labeled anti-carboxyl dihydroxyphalloidin antibody in the detection card in detecting or assisting in detecting whether a sample to be detected contains carboxyl dihydroxyphalloidin or detecting the content of the carboxyl dihydroxyphalloidin in the sample to be detected is also the protection scope of the invention.

The invention also provides a method for detecting or assisting in detecting whether a sample to be detected contains carboxyl dihydroxyl phalloidin, which comprises the following steps: adding a sample to be detected on the sample pad of the detection card, reacting, and detecting a reaction product by using a fluorescence immunoassay quantitative analyzer;

if the fluorescence signal value of the detection line T of the detection card is less than or equal to the fluorescence signal value of the quality control line C, the sample to be detected contains or is candidate to contain carboxyl dihydroxyphalloidin;

and if the fluorescence signal value of the detection line T of the detection card is greater than the fluorescence signal value of the quality control line C, the sample to be detected does not contain or is candidate to contain carboxyl dihydroxyphalloidin.

A method for detecting or assisting in detecting the content of carboxyl dihydroxyphalloidin in a sample to be detected comprises the following steps:

(1) preparing a standard curve: taking a sample which has the same source as the sample to be detected and has the content of carboxyl dihydroxyl phalloidin less than 0.1 mug/L;

adding carboxyl dihydroxyl phalloidin with different concentrations into the sample to obtain standard substance solutions with different concentrations;

adding the standard substance solutions with different concentrations onto the sample pad of the detection card for reaction, and detecting the reaction product by using a fluorescence immunoassay quantitative analyzer to obtain the fluorescence signal values of the detection line T and the quality control line C of the standard substance solutions with different concentrations;

drawing a standard curve by taking different concentrations of the added carboxyl dihydroxyphalloidin as an X axis and taking the ratio of the fluorescence signal value of the detection line T corresponding to the different concentrations to the fluorescence signal value of the quality control line C as a Y axis;

(2) adding the sample to be detected on the sample pad of the detection card, reacting, and detecting a reaction product by using a fluorescence immunoassay quantitative analyzer to obtain a detection line T fluorescence signal value and a quality control line C fluorescence signal value of the sample to be detected;

and (3) substituting the fluorescence signal value of the detection line T and the fluorescence signal value of the quality control line C of the sample to be detected into the standard curve obtained in the step (1) to obtain the content of carboxyl dihydroxyphalloidin in the sample to be detected.

In the above application or method, the sample to be tested is a urine, plasma or serum sample.

The invention has the beneficial effects that:

the invention utilizes europium microspheres as an antibody marker, establishes a simple, rapid and high-sensitivity fluorescence quantitative immunochromatography detection method for carboxyl dihydroxyphalloidin residual in a sample, evaluates indexes of sensitivity, accuracy and precision of the established method, and provides a rapid detection method suitable for carboxyl dihydroxyphalloidin trace residual.

The invention uses carboxyl microspheres of lanthanide europium (365/610nm, lambda ex/lambda em) as antibody markers to establish an immunochromatography method. Europium element has larger Stokes shift (>200nm), so that an excited signal does not cause interference on a fluorescence signal of emitted light in a detection process; in addition, the fluorescence lifetime of lanthanide is relatively long, which can reach 1-2ms, so that detection can be performed after other fluorescence signals disappear, so as to achieve the purpose of reducing to background signal interference. The advantages of the two aspects greatly improve the sensitivity of the time-resolved fluoroimmunoassay method of the invention.

The detection sensitivity (the limit of quantitation is 0.1 mu g/kg) of the method reaches the sensitivity level of an instrument method; the addition recovery rate (88.8-97.2%) and the variation coefficient (3.5-8.6%) of carboxyl dihydroxyl phalloidin in the method can both meet the detection requirements. Therefore, the time-resolved fluorescence immunochromatographic assay established by the invention has higher application value and application prospect in the detection work of carboxyl dihydroxyl phalloidin.

Drawings

FIG. 1 is a schematic diagram of the time-resolved fluoroimmunoassay rapid detection card according to the present invention.

FIG. 2 is a graph of the results of a gradient assay for PCD addition in a urine sample.

FIG. 3 is a graph showing the results of TRFICA testing standard PCD curve in urine.

FIG. 4 is a graph showing the linear correlation between TRFIA and HPLC detection results.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

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.

Specifically, some materials in the following examples are as follows:

micropipette (Eppendorf, Germany), refrigerated centrifuge (Thermo Fisher Scientific, Germany), ultrasonic cleaner KQ-100E (ultrasonic instruments, Inc., Kunshan), high performance liquid chromatography-tandem mass spectrometer (Thermo Fisher Scientific, USA); the detection card incubator (WH-400) and the time-resolved fluoroimmunoassay quantitative analyzer (FQ-S2, 365/610nm) were developed by Vidervican Biotechnology Ltd.

200nm carboxylated europium microspheres (365/610nm,. lambda.ex/. lambda.em) were purchased from Cretive Diagnostics, USA; nitrocellulose membranes (NC membranes) were purchased from Millipore, usa; the sample pad, the absorbent pad, and the PVC base plate were all purchased from shanghai liang xin science and technology limited.

The carboxyl dihydroxyl phalloidin standard substance is purchased from Sigma company in the United states, and the solution of the standard substance is prepared: accurately weighing 1.0mg of carboxyl dihydroxyl phalloidin peptide standard substance, dissolving with methanol, diluting to 1mL to obtain 1mg/mL standard substance solution, and storing at-20 deg.C.

The carboxyl dihydroxyl phalloidin resisting monoclonal antibody is prepared in the laboratory, and the preparation process comprises the following steps: carboxyl dihydroxyphalloidin is coupled with carrier protein Keyhole Limpet Hemocyanin (KLH) and Bovine Serum Albumin (BSA) by an active ester method to synthesize immunogen PCD-KLH and PCD-BSA, and is coupled with Ovalbumin (OVA) to synthesize coating original PCD-OVA. Diluting the immunogen to 1mg/mL, mixing with Freund's complete adjuvant in equal volume, emulsifying, and injecting multiple points at the back of the neck to immunize Balb/c mice with each immunization dose of 0.2 mL/mouse. Three weeks later, booster immunizations were performed with Freund's incomplete adjuvant. And (4) collecting blood from the orbit of the mouse 1 week after the third boosting immunization is finished, and detecting the antiserum level by an ICELISA method. Selecting a mouse with high titer and good inhibition to prepare spleen cell suspension. Splenocytes were mixed with SP2/0 cells at a ratio of 5:1 and fused with PEG. Specific hybridoma cell clones are screened by a limiting dilution method, and a monoclonal antibody is prepared by an ascites induction method. The monoclonal antibody obtained was stored at-20 ℃ for future use.

The goat anti-mouse secondary antibody is obtained by Beijing Weideweikang biotechnology limited. Urine blank samples were provided from 20 volunteers.

Example 1 preparation of carboxydihydroxyphallus toxicide peptide time-resolved fluoroimmunoassay Rapid detection card and establishment of detection method

Preparation of carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card

1. Antibody labeling of carboxylated europium microspheres

This example was optimized based on 200nm carboxylation time resolution and antibody labeling. The specific labeling method is as follows:

50 mu L of carboxylated europium microspheres (200nm carboxylated europium microspheres (365/610nm, lambda ex/lambda em), the concentration of which is 1 percent), 450 mu L of activation buffer solution (0.05mol/L MES, pH value is 5.0) is added, and the mixture is subjected to ultrasonic treatment for 5 min; secondly, sequentially adding EDC and NHS into the solution after the first step to enable the final concentration to be 0.1mM and 0.2mM respectively, carrying out oscillation reaction at room temperature for 30min, centrifuging at 15000g for 10min, and removing the supernatant; ③ redissolving the sediment obtained by the second step by using 500 mu L of coupling buffer solution (0.04mol/L PB, pH 8.0), carrying out ultrasonic treatment for 5min, adding 10 mu L of 5mg/mL carboxyl dihydroxyphalloidin monoclonal antibody, carrying out oscillation reaction for 2h at room temperature, and centrifuging for 5min at 15000 g; fourthly, 500 mu L of blocking buffer solution (0.01mol/L PB, 2% BSA, pH 8.0) is added into the sediment obtained from the third step, and the mixture is shaken and reacted at 4 ℃ overnight; fifthly, centrifuging the reaction solution obtained from the fourth step for 5min at 15000g, removing the supernatant, adding 500 mu L of Tris-HCl (1% BSA, 0.5% Tween-20, 0.02% Proclin-300, pH 7.0) for redissolving and precipitating, carrying out ultrasonic treatment for 5min, and keeping away from light at 4 ℃ for later use to obtain the carboxylated europium microsphere labeled carboxyl dihydroxyphalloidin monoclonal antibody (the concentration is 0.1 mg/mL).

2. Assembly of carboxyl dihydroxyl phalloidin time-resolved fluorescence immunoassay rapid detection card

Coating carboxyl dihydroxyphalloidin antigen (2.0mg/mL, 1:5 dilution) and goat anti-mouse IgG (20mg/mL, 1:20 dilution) on NC membrane as detection line (T line, 0.7 muL/cm, coating concentration of carboxyl dihydroxyphalloidin antigen is 0.4mg/mL) and quality control line (C line, 0.7 muL/cm, coating concentration of goat anti-mouse IgG is 1.0mg/mL) respectively by using a membrane scribing instrument, and placing in a 37 ℃ oven for overnight drying to obtain the nitrocellulose membrane containing the carboxyl dihydroxyphalloidin detection line T and the quality control line C.

And (2) spraying the carboxyl dihydroxyphalloidin monoclonal antibody marked by the carboxylated europium microspheres prepared in the step (1) onto a release pad (Shanghai gold-labeled Biotech Co., Ltd., SB08, 300 x 200mm) with the spraying amount of 3.0 mu L/cm, and drying in an oven at 37 ℃ for 2h to obtain the release pad containing the europium microsphere-marked anti-carboxyl dihydroxyphalloidin monoclonal antibody.

Then, a sample pad, a release pad containing an europium microsphere-labeled anti-carboxyl dihydroxyphalloidin peptide antibody, a nitrocellulose membrane containing a carboxyl dihydroxyphalloidin detection line T and a quality control line C, and a water absorption pad (Shanghai gold-labeled Biotech Co., Ltd., CH37, 300 x 46mm) were sequentially adhered to a PVC base plate, and cut into test strips of 3.95mm in width by a cutter, to obtain a carboxyl dihydroxyphalloidin peptide time-resolved fluoroimmunoassay rapid detection card, the structure of which is shown in FIG. 1.

Establishment of carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card detection method

1. Establishment of qualitative detection method of carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card

And (3) dropwise adding 100 mu L of sample solution to be detected onto the sample pad of the prepared carboxyl dihydroxyphalloidin time-resolved fluoroimmunoassay rapid detection card (shown in figure 1), accurately reacting in a detection card constant-temperature incubator at 40 ℃ for 5min, taking out, and detecting by using a fluoroimmunoassay quantitative analyzer.

If the fluorescence signal value of the detection line T is less than or equal to the fluorescence signal value of the quality control line C, the sample to be detected contains or is candidate to contain carboxyl dihydroxyphalloidin;

and if the fluorescence signal value of the detection line T is greater than that of the quality control line C, the sample to be detected does not contain or candidate does not contain carboxyl dihydroxyphalloidin.

2. Establishment of carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card quantitative detection method

1) Drawing of standard curve

7 urine negative samples (500 mL each) were taken, and a carboxyl dihydroxyphalloidin standard solution was added thereto to give final concentrations of: 0.0. mu.g/L, 0.1. mu.g/L, 0.3. mu.g/L, 0.9. mu.g/L, 2.7. mu.g/L, 8.1. mu.g/L and 24.3. mu.g/L were thoroughly mixed and subjected to sample extraction according to the method described in the above item 1 to obtain extract solutions of samples of respective concentrations.

The urine samples of the above concentrations were tested according to the above 1. The assay was repeated 5 times for each concentration and averaged. Taking the final concentration of carboxyl dihydroxyphalloidin added in a sample as an X axis, taking the ratio (T/C) of the fluorescence signal value of a detection line T to the fluorescence signal value of a quality control line C as a Y axis, and carrying out nonlinear fitting analysis by origin8.0(origin Lab Corp., Northamapton, MA, USA) to form a four-parameter fitting curve as shown below:

Y＝(A-D)/[1+(X/C)^B]+D；

the fitting of test data shows that the established standard curve of the time-resolved fluorescence immunochromatographic quantitative detection method for carboxyl dihydroxyphalloidin in urine is as follows:

Y＝(0.91–0.014)/[1+(X/0.93)^1.57]+0.014(R²＝0.995)；

power, e.g., (X/0.93) ^1.57 means 1.57 power of (X/0.93).

Wherein X is carboxyl dihydroxyphalloidin concentration value, and Y is T/C value (FIG. 2 and FIG. 3).

2) Detection of

And (3) dropwise adding 100 mu L of urine sample onto the sample pad in the prepared carboxyl dihydroxyphalloidin time-resolved fluoroimmunoassay rapid detection card, accurately reacting in a detection card constant-temperature incubator at 40 ℃ for 5min, taking out, and detecting by using a fluoroimmunoassay quantitative analyzer to obtain a T-line fluorescent signal value and a C-line fluorescent signal value.

And substituting the ratio of the fluorescence signal value of the T line to the fluorescence signal value of the C line into the standard curve to obtain the content of the carboxyl dihydroxyphalloidin in the sample.

Third, the lowest detection limit of carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card

And (3) taking 20 parts of a blank urine sample, and detecting the content of carboxyl dihydroxyphalloidin in the 20 parts of the blank sample according to the method 2 in the second step. The mean and standard deviation of the carboxyl dihydroxyphalloidin content in 20 blank samples were calculated: the mean value plus 3 times the standard deviation is the limit of quantitation.

As shown in Table 1, it can be seen that the quantitative detection limit of carboxyl dihydroxyphalloidin in urine sample obtained by the method is 0.12 μ g/L calculated according to the measured mean and standard deviation of 20 samples.

TABLE 1 measurement of Carboxyphalloidin in urine blank samples (. mu.g/L)

Fourth, accuracy and precision

3 parts of urine blank samples are taken, carboxyl dihydroxyphalloidin solution is respectively added, the final concentration is 0.5 mu g/L, 1.0 mu g/L and 2.0 mu g/L, and 5 replicates are added in each concentration gradient.

And detecting according to the two methods, and finally analyzing the accuracy and precision of the established time-resolved fluorescence immunochromatographic quantitative analysis method according to the added recovery data.

The result is shown in Table 2, the adding recovery rate of carboxyl dihydroxyphalloidin is 88.8% -97.2%, and the coefficient of variation is 3.5% -8.6%, which shows that the method has better accuracy and precision.

TABLE 2 accuracy and precision test results (. mu.g/L)

Concentration of addition	Mean value (n is 5)	Standard deviation of	Recovery (%)	Coefficient of variation (%)
					0.5	0.486	0.017	97.2	3.5
1.0	0.888	0.076	88.8	8.6
					2.0	1.786	0.134	89.3	7.5

The recovery rate was (average value/added concentration) × 100%

Coefficient of variation ═ (standard deviation/average) × 100%;

fifthly, comparing the detection result of the carboxyl dihydroxyl phalloidin time-resolved fluoroimmunoassay rapid detection card with the detection result of HPLC

4 urine blank samples were taken and added to the carboxy dihydroxyphalloidin solution to give final concentrations of 0.5. mu.g/L, 1.0. mu.g/L, 2.0. mu.g/L and 4.0. mu.g/L, 5 replicates per concentration gradient.

The sample is detected by the method (TRFIA) of the invention, and is simultaneously detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and the consistency of the detection results of the two methods is analyzed.

The test results are shown in fig. 4. The two methods areThe linear correlation equation of the detection result is as follows: y is-0.05 +0.98X, correlation coefficient R²0.971. Therefore, the consistency of the detection results of the two methods on the carboxyl dihydroxyphalloidin in urine is higher.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.