A hapten and artificial antigen for detecting toxoflavin and their application

Abstract

The present invention discloses a hapten for detecting toxoflavin, and an artificial antigen and antibody for detecting toxoflavin are prepared based on the hapten. The hapten completely retains the structural characteristics of toxoflavin, and the artificial antigen prepared by the hapten has an intact structure and completely retains the original characteristics of toxoflavin, which is conducive to presenting the structural characteristics of toxoflavin and improving the immunogenicity of the artificial antigen. The artificial antigen can quantitatively detect toxoflavin in a sample to be tested. When used in enzyme-linked immunosorbent assay (ELISA) of toxoflavin, IC 50 The value is 0.41μg / L; at the same time, based on the artificial antigen, an immunochromatographic test strip for the quantitative detection of toxoplasmone is provided. The detection sensitivity of toxoplasmone standard reaches 0.1μg / L, and the detection limit of toxoplasmone in the test sample reaches 25ng / kg, which can achieve high-sensitivity detection of toxoplasmone.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a hapten and an artificial antigen for detecting toxoplasmone and applications thereof. Background Art

[0002] Fermented rice and flour poisoning is a type of bacterial food poisoning with a very high mortality rate. Pseudomonas cocovenenans subspecies fermented rice and flour is the cause of rice and flour poisoning and spoiled white fungus poisoning.

[0003] The pathogenic substances of Pseudomonas cocovenenans subspecies are two toxins produced during its growth and reproduction: bongkrekic acid (BA) and toxoflavin (TF). These two toxins are small molecular substances of fatty acids, which are highly toxic, heat-resistant and toxic. They cannot be destroyed by general cooking methods. Therefore, food contamination can easily cause poisoning, and there is currently no specific antidote for these two toxins.

[0004] Currently, accurate detection of fumonisin in food can be achieved by instrumental detection or immunoassay. Detection of toxoplasmonic acid is mainly achieved through spectrophotometry, high-performance liquid chromatography, or a combination of the two methods, which not only requires expensive detection instruments, but also takes a long time and requires professional personnel to operate, making on-site detection impossible. Immunoassay methods for toxoplasmonic acid lack reliable toxoplasmonic acid haptens, and usually have too high a detection limit to accurately detect toxoplasmonic acid in food.

[0005] Therefore, developing a highly specific cypermethrin hapten to achieve high-sensitivity detection of cypermethrin has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a hapten, an artificial antigen and applications thereof for detecting toxoplasm.

[0007] The first object of the present invention is to provide a hapten for detecting toxoplasmin.

[0008] The second object of the present invention is to provide a method for preparing the above hapten.

[0009] The third object of the present invention is to provide the use of the above hapten in preparing an artificial antigen for detecting toxoplasmin.

[0010] The fourth object of the present invention is to provide an artificial antigen for detecting toxoplasm.

[0011] The fifth object of the present invention is to provide the use of the above artificial antigen in preparing antibodies for detecting toxoplasmin.

[0012] The sixth object of the present invention is to provide an artificial antigen combination for detecting toxoplasmin.

[0013] The seventh object of the present invention is to provide an immunochromatographic test strip for quantitative detection of toxoplasmone.

[0014] The eighth object of the present invention is to provide the use of the above-mentioned artificial antigen combination and / or immunochromatographic test strip in the preparation of a kit for the quantitative detection of toxoplasmin.

[0015] The ninth object of the present invention is to provide a kit for quantitatively detecting toxoplasmin.

[0016] In order to achieve the above object, the present invention is implemented through the following scheme:

[0017] A hapten for detecting toxoplasmin, the structural formula of which is shown in Formula I;

[0018]

[0019] Wherein R in the structural formula shown in Formula I is a halogen ion.

[0020] Preferably, R in the structural formula shown in Formula I is a bromide ion.

[0021] The present invention also claims a method for preparing the above-mentioned hapten, comprising subjecting toxic flavin and compound a of formula II to a quaternization reaction to obtain a hapten of formula I;

[0022]

[0023] Wherein R in the structural formula shown in Formula II is a halogen atom.

[0024] Preferably, R in the structural formula shown in Formula II is a bromine atom.

[0025] Preferably, the molar ratio of toxoplasmin to compound a is 1:(1.5-3).

[0026] More preferably, the molar ratio of toxoplasmin to compound a is 1:2.

[0027] The present invention also claims to protect the use of the above hapten in preparing an artificial antigen for detecting toxoplasmin.

[0028] The present invention also claims protection for an artificial antigen for detecting toxoplasmin, which is obtained by coupling the above hapten with a carrier protein, and has a structural formula as shown in Formula III;

[0029]

[0030] In the structural formula shown in Formula III, R is a halogen ion, and Protein is a carrier protein.

[0031] Preferably, in the structural formula shown in Formula III, R is a bromide ion.

[0032] Preferably, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin or lactoferrin.

[0033] A method for preparing an artificial antigen for detecting toxoplasmin comprises the following steps:

[0034] S1. The hapten for detecting toxoplasmin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are fully reacted to obtain solution A;

[0035] S2. dissolving the carrier protein in phosphate buffer to obtain solution B;

[0036] S3. Solution A obtained in step S1 and solution B obtained in step S2 are mixed, reacted fully, and dialyzed to obtain an artificial antigen for detecting toxoplasmin.

[0037] Preferably, the mass ratio of the hapten for detecting toxoplasmin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide for detecting toxoplasmin in step S1 is 1:(1-2):(1-2).

[0038] More preferably, the mass ratio of the hapten for detecting toxoplasmin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is 1:1:1.

[0039] Preferably, the carrier protein in step S2 is bovine serum albumin, ovalbumin, hemocyanin or lactoferrin.

[0040] Preferably, the sufficient reaction in step S3 is carried out at room temperature for 16 to 24 hours.

[0041] Preferably, in step S3, the dialysis method is to dialyze using phosphate buffer after solution A and solution B have fully reacted.

[0042] The present invention also claims protection for the use of the artificial antigen in preparing antibodies for detecting toxoplasmin.

[0043] An artificial antibody for detecting toxoflavin is prepared by immunizing an animal with the artificial antigen.

[0044] The present invention also claims protection for the use of the above-mentioned antibody in detecting toxoplasmin, wherein the detection is for the purpose of non-disease treatment and diagnosis.

[0045] The present invention also claims protection for an artificial antigen combination for detecting toxoflavin, comprising an immunogen and a coating agent, wherein the immunogen is obtained by coupling the above hapten to lactoferrin, and the coating agent is obtained by coupling the above hapten to bovine serum albumin.

[0046] The present invention also seeks protection for an immunochromatographic test strip for quantitatively detecting toxoplasmin, comprising a PVC base plate and a sample pad, a cellulose membrane and a water-absorbing pad arranged in sequence on the PVC base plate, a detection line and a quality control line being provided on the cellulose membrane, the detection line being coated with the above-mentioned coating agent, and the quality control line being coated with IgG antibodies.

[0047] Preferably, the concentration of the coating agent on the detection line is 0.05-0.4 mg / ml.

[0048] More preferably, the concentration of the coating agent on the detection line is 0.2 mg / ml.

[0049] Preferably, the concentration of the IgG antibody on the quality control line is 0.05-0.2 mg / ml.

[0050] More preferably, the concentration of the IgG antibody on the quality control line is 0.1 mg / ml.

[0051] The method of using the immunochromatographic test strip is as follows:

[0052] S1. Mix at least five gradient concentrations of a cypermethrin standard solution and a fluorescently labeled artificial antibody, then add the mixture dropwise to the sample pad of an immunochromatographic test strip. After sufficient reaction, measure the fluorescence value of the immunochromatographic test strip to obtain the test line fluorescence value (T value) and the control line fluorescence value (C value) of the cypermethrin standard.

[0053] S2. Construct a fluorescence quantitative standard curve using the concentration of the flavin standard solution in step S1 as the horizontal axis and the T value / C value obtained in step S1 as the vertical axis;

[0054] S3. Replace the tocopherol standard solution with the sample to be tested, repeat step S1, obtain the detection line fluorescence value and the quality control line fluorescence value of the sample to be tested, and calculate the tocopherol concentration of the sample to be tested by combining the fluorescence quantitative standard curve obtained in step S2.

[0055] The present invention also seeks to protect the use of the artificial antigen combination and / or the immunochromatographic test strip in detecting toxoplasm, wherein the detection is for the purpose of non-disease treatment and diagnosis.

[0056] The present invention also seeks to protect the use of the artificial antigen combination and / or the immunochromatographic test strip in the preparation of a kit for the quantitative detection of toxoplasmin.

[0057] A kit for quantitatively detecting toxoplasmin contains the artificial antigen combination and / or the immunochromatographic test strip.

[0058] Preferably, the kit further contains a stop solution, a washing solution, a blocking solution and an enzyme-labeled secondary antibody.

[0059] More preferably, the stop solution is sulfuric acid.

[0060] More preferably, the washing solution is PBST solution.

[0061] More preferably, the blocking solution is a phosphate buffer containing 0.5-1.5% BSA by mass.

[0062] More preferably, the enzyme-labeled secondary antibody is goat anti-mouse IgG labeled with horseradish peroxidase.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] The present invention provides a hapten for detecting toxoflavin, and an artificial antigen and antibody for detecting toxoflavin are prepared based on the hapten. The hapten completely retains the structural characteristics of toxoflavin, and the artificial antigen prepared by the hapten has an intact structure and completely retains the original characteristics of toxoflavin, which is conducive to presenting the structural characteristics of toxoflavin and improving the immunogenicity of the artificial antigen. The artificial antigen can quantitatively detect toxoflavin in a sample to be tested. When used in enzyme-linked immunosorbent assay (ELISA) of toxoflavin, IC 50 The value is 0.41μg / L; at the same time, based on the artificial antigen, an immunochromatographic test strip for the quantitative detection of toxoplasmone is provided. The detection sensitivity of toxoplasmone standard reaches 0.1μg / L, and the detection limit of toxoplasmone in the test sample reaches 25ng / kg, which can achieve high-sensitivity detection of toxoplasmone. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a synthetic route for the hapten used to detect toxoplasmin in Example 1;

[0066] Figure 2 This is the mass spectrometry spectrum of the hapten used to detect toxoplasmin in Example 1;

[0067] Figure 3 This is a synthetic route for the artificial antigen used to detect toxoplasmin in Example 2;

[0068] Figure 4 This is the standard curve of cypermethrin in Example 4;

[0069] Figure 5 This is the fluorescence quantitative standard curve of cypermethrin in Example 5. DETAILED DESCRIPTION

[0070] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0071] Example 1 Synthesis and Identification of a Hapten for Detecting Toxoplasmin

[0072] 1. Experimental Methods

[0073] 1. Synthesis of hapten for detection of toxoflavin

[0074] The synthetic route of the hapten used to detect toxic flavin is as follows Figure 1 The specific steps are as follows:

[0075] Place 100 g of cypermethrin (CAS: 84-82-2, 0.52 mmol) in a 50 mL round-bottom flask, add 10 mL of ultrapure water, and after the cypermethrin is fully dissolved, add 172 mg of 4-bromobutyric acid (CAS: 2623-87-2, 1.04 mmol). After thorough mixing, place at 60 ° C for 5 to 8 hours.

[0076] After the reaction was completed, the mixture was evaporated to dryness under reduced pressure, washed twice with ethyl acetate, and dried to obtain the hapten for detecting toxoplasmin.

[0077] 2. Identification of haptens for the detection of toxoflavin

[0078] The prepared hapten for detecting toxoplasmin was subjected to electrospray ionization mass spectrometry (ESI-MS), and the mass spectrum was recorded.

[0079] 2. Experimental Results

[0080] The structural formula of the hapten used to detect toxoplasmin is shown in Formula IV;

[0081]

[0082] The mass spectrometry spectrum of the hapten used to detect toxoplasmone is as follows: Figure 2 As shown, the results show that: a peak data appears at 279.1 in the mass spectrum, indicating that the relative molecular mass of the hapten used to detect toxoplasmin is 279.1, which is consistent with the relative molecular mass of the hapten used to detect toxoplasmin as shown in the structural formula IV, indicating that the hapten used to detect toxoplasmin was successfully prepared.

[0083] Example 2 Synthesis of artificial antigens for detecting toxoplasmin

[0084] The synthetic route of artificial antigens for detecting toxoplasm Figure 3 As shown, specifically:

[0085] 1. Synthesis of artificial antigen 1 (coating antigen)

[0086] The synthesis steps of artificial antigen 1 (coating antigen) include:

[0087] S1. 5 mg of the hapten synthesized in Example 1 (Formula IV) was dissolved in 0.1 mL of dimethylformamide (DMF). After sufficient dissolution, 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added and stirred at room temperature for 4 h to obtain a hapten activated ester solution.

[0088] S2. Dissolve 25 mg of bovine serum albumin (BSA) in 2.5 mL of 0.01 mol / L PBS solution to obtain a BSA solution;

[0089] S3. The hapten activated ester solution obtained in step S1 is added dropwise to the BSA solution obtained in step S2, and stirred at room temperature for 16 to 24 hours. The solution is then transferred to a dialysis bag and dialyzed against 0.01 mol / L PBS for 3 days, with the dialysate being changed three times a day. After the dialysis is completed, the solution in the dialysis bag is collected to obtain an artificial antigen 1 having the structural formula shown in Formula V.

[0090]

[0091] 2. Synthesis of Artificial Antigen 2 (Immunogen)

[0092] The synthesis steps of artificial antigen 2 (immunogen) include:

[0093] S1. 5 mg of the hapten synthesized in Example 1 (Formula IV) was dissolved in 0.1 mL of dimethylformamide (DMF). After sufficient dissolution, 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added and stirred at room temperature for 4 h to obtain a hapten activated ester solution.

[0094] S2. 20 mg of lactoferrin (LF) was dissolved in 2 mL of PBS solution having a concentration of 0.01 mol / L to obtain a LF solution;

[0095] S3. The hapten activated ester solution obtained in step S1 is added dropwise to the LF solution obtained in step S2, and stirred at room temperature for 16 to 24 hours. The mixture is then transferred to a dialysis bag and dialyzed against 0.01 mol / L PBS for 3 days, with the dialysate being replaced 3 times a day. After the dialysis is completed, the solution in the dialysis bag is collected to obtain an artificial antigen 2 having the structural formula shown in Formula VI.

[0096]

[0097] Example 3 Preparation of a Monoclonal Antibody for Detecting Toxoplasmin

[0098] The artificial antigen 2 prepared in Example 2, represented by the structural formula VI, and Freund's adjuvant were mixed and emulsified in equal volumes, and BALB / C mice were immunized at a dose of 50 μg to 100 μg per mouse. Booster immunizations were then performed every two weeks for a total of three immunizations. After the antibody titer no longer increased, subcutaneous booster immunizations were performed with 100 μg of the whole antigen.

[0099] Five days after the booster immunization, the mouse spleen cells were fused with human osteosarcoma SP20 cells. The fused cells were collected and screened using HAT medium in a 48-well plate. After five days, the HAT medium was replaced with complete medium for culture.

[0100] Then, the supernatant of each well was tested by ELISA. The cells in the wells with strongly positive ELISA test results were cloned and cultured by limiting dilution. After three clone culture tests, the cells in the positive wells were recorded as hybridoma cells secreting monoclonal antibodies.

[0101] The hybridoma cells are amplified and cultured and inoculated into the peritoneal cavity of mice. The mouse ascites is collected and purified by octanoic acid-ammonium sulfate precipitation to obtain the toxoplasmin monoclonal antibody.

[0102] Example 4 ELISA performance evaluation

[0103] 1. Experimental Methods

[0104] 1. Antigen coating

[0105] The artificial antigen 1 (coating source) prepared in Example 2 was diluted to a concentration of 0.05 μg / mL using carbonate buffer at pH 9.6 as the coating diluent. 100 μL / well was then added to a 48-well plate, coated overnight at 4°C, dried, and washed three times with PBST.

[0106] 2. Closed

[0107] Phosphate buffer containing 1% (w / w) BSA was added at 280 μL / well, blocked at 37°C for 1 hour, dried, washed three times with PBST, dried, and vacuum-packed for storage to obtain an ELISA plate coated with artificial antigen 1.

[0108] 3. Primary Antibody Dilution

[0109] The toxoflavin monoclonal antibody prepared in Example 3 was diluted to 0.1 μg / mL using phosphate buffer (pH=7.4) containing 0.05% (w / w) sodium azide to obtain a primary antibody solution.

[0110] 4. Preparation of standard solution

[0111] The toxoplasmin standard (CAS: 84-82-2) was dissolved in 0.01 M PBS to obtain toxoplasmin standard solutions with concentrations of 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L and 4.05 μg / L, respectively; the toxoplasmin standard solution with a concentration of 0 μg / L was 0.01 M PBS.

[0112] 5. Add cypermethrin standard solution of various concentrations to the ELISA plate coated with artificial antigen 1 at 80 μL / well (3 parallel wells for each concentration), then add primary antibody solution at 20 μL / well, incubate at 37°C for 0.5 h, spin dry, and wash three times with PBST as washing solution at 280 μL / well, then pat dry.

[0113] 6. Add horseradish peroxidase (HRP enzyme)-labeled goat anti-mouse IgG enzyme-labeled secondary antibody at 100 μL / well, react at 37°C for 0.5 h, use PBST as washing solution, add 280 μL / well and wash three times, then pat dry.

[0114] 7. Add TMB colorimetric solution at 100 μL / well, react at 37°C for 15 min, then add 1 M sulfuric acid at 50 μL / well to stop color development and obtain the developed ELISA plate.

[0115] 8. Place the developed ELISA plate in an ELISA reader and read the OD value of each well at 450 nm to obtain the OD value of the cypermethrin standard solution at each concentration (take the average value of 3 parallel readings).

[0116] 9. Using the OD value of each concentration of cypermethrin standard as the ordinate and the concentration of cypermethrin standard as the abscissa, use ELISACalc software to perform logistic curve fitting to obtain the standard curve equation of cypermethrin standard and draw a standard curve graph.

[0117] 2. Experimental Results

[0118] The OD values ​​of the cypermethrin standard solutions at various concentrations are shown in Table 1.

[0119] Table 1 OD values ​​of cypermethrin standard solutions at various concentrations

[0120] Concentration (μg / L) 0 0.05 0.15 0.45 1.35 4.05 OD value (toxoflavone) 1.27 0.91 0.69 0.50 0.34 0.22

[0121] The standard curve of toxic flavonoids is as follows Figure 4 The standard curve equation of cypermethrin standard is shown in Formula I,

[0122] Formula 1: y = (AD) / {1 + (x / C)^B} + D, r 2 =0.99963; wherein A=1.27091, B=0.65705, C=0.16441, D=0.09752, x is the concentration of toxoplasmin, and y is the OD value.

[0123] The results showed that when the ELISA method was used to detect the concentration of toxic flavonoids, IC 50 When the value is 0.41μg / L and the concentration of toxic flavonoids is in the range of 0.05μg / L to 0.45μg / L, the OD 450 The values ​​show a linear relationship.

[0124] Example 5 Preparation of Immunochromatographic Test Strips for Quantitative Detection of Toxoplasmin

[0125] 1. Experimental Methods

[0126] 1. Construction of immunochromatographic test strips

[0127] A nitrocellulose membrane (NC membrane) was used as the reaction membrane. A test line (T line) and a quality control line (C line) were drawn on the reaction membrane. The interval between the test line and the quality control line was 5 mm.

[0128] The artificial antigen 1 (coating source) prepared in Example 2 was diluted with coating buffer to obtain an artificial antigen 1 with a concentration of 0.2 mg / mL, and the rabbit IgG antibody was diluted with coating buffer to obtain a rabbit IgG antibody with a concentration of 0.1 mg / mL; wherein the coating buffer is 0.01 M PBS buffer containing 0.5% (w / w) PEG20000, 1% (w / w) sucrose, 0.05% (w / w) BSA and 0.05% (w / w) sodium azide.

[0129] 0.2 mg / mL of artificial antigen 1 was sprayed on the test line at a spraying volume of 1 μL / cm, and 0.1 mg / mL of rabbit IgG antibody was sprayed on the quality control line. The mixture was placed in a 45°C oven for 24 to 48 hours to obtain a reaction membrane coated with the antigen.

[0130] The blank sample pad was soaked in the sample pad treatment solution for 5 minutes and then dried at 37°C for 16 hours to obtain a sample pad; the sample pad treatment solution was 0.01 M PBS buffer containing 0.3% (w / w) Tween 20, 1% (w / w) sucrose, 0.5% (w / w) BSA and 0.05% (w / w) sodium azide.

[0131] The reaction membrane coated with the antigen is pasted on the middle part of the PVC base plate, and the sample pad and the absorbent pad are pasted on both ends of the PVC base plate. The reaction membrane coated with the antigen, the absorbent pad and the sample pad are overlapped to obtain an immunochromatographic test strip for quantitative detection of toxoplasmin; wherein the sample pad is located at one end close to the test line, and the absorbent pad is located at one end close to the quality control line.

[0132] 2. How to use immunochromatographic test strips

[0133] (1) Preparation of time-resolved fluorescent microsphere-labeled cytoxan monoclonal antibody:

[0134] To 500 μL of time-resolved fluorescent microsphere solution (containing 5 mg of time-resolved fluorescent microspheres), 5 mg of NHS and 4 mg of DEC were added with stirring, the solution temperature was controlled at 4-10° C., and activated for 40 min. Then, the pH was adjusted to 8-9 with 0.1 M potassium carbonate solution and the solution temperature was controlled at 4-10° C., followed by the addition of 0.3 mg of the toxoplasmin monoclonal antibody prepared in Example 3. After thorough stirring, the mixture was stirred at room temperature for 4-6 h, followed by centrifugation at 12000 rpm for 10 min. After discarding the supernatant, 0.5 mL of fluorescent buffer was added for ultrasonic resuspending, the supernatant was discarded, 0.5 mL of fluorescent buffer was added for ultrasonic resuspending, the supernatant was discarded, 0.5 mL of fluorescent buffer was added for ultrasonic resuspending, the supernatant was discarded, 0.5 mL of fluorescent buffer was added for ultrasonic resuspending, the supernatant was discarded, 0.5 mL of fluorescent buffer was added for ultrasonic resuspending, the supernatant was discarded, and 0.5 mL of fluorescent buffer was added for ultrasonic resuspending to obtain fluorescent microsphere-labeled toxoplasmin monoclonal antibody.

[0135] The fluorescence buffer is 0.01 M PBS buffer containing 0.5% (w / w) PEG20000, 2% (w / w) sucrose, 0.1% (w / w) Tween 20, 0.5% (w / w) BSA and 0.05% (w / w) sodium azide.

[0136] (2) Preparation of time-resolved fluorescent microsphere-labeled goat anti-rabbit IgG:

[0137] In the process of preparing the toxoflavin monoclonal antibody labeled with time-resolved fluorescent microspheres, 0.3 mg of the toxoflavin monoclonal antibody prepared in Example 3 was replaced with an equal amount of goat anti-rabbit IgG to prepare goat anti-rabbit IgG labeled with fluorescent microspheres.

[0138] (3) Preparation of cytoxan time-resolved fluorescence micro-detection solution:

[0139] Remove the fluorescent microsphere-labeled cytoxan monoclonal antibody and fluorescent microsphere-labeled goat anti-rabbit IgG and allow them to cool to room temperature. Then dilute them with fluorescence buffer to form a cytoxan time-resolved fluorescence detection solution. The fluorescent microsphere-labeled cytoxan monoclonal antibody should be diluted 200-fold (control the fluorescence signal value of the T line to be 10,000-12,000); the fluorescent microsphere-labeled goat anti-rabbit IgG should be diluted 800-fold (control the fluorescence signal value of the C line to be 3,000-4,000).

[0140] (4) Establishment of standard curve

[0141] Using 0.1 M PBS buffer as a solvent, toxoplasmin standard solutions with concentrations of 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L and 8.1 μg / L were prepared using toxoplasmin standard.

[0142] Mix 100 μL of a 0.1 μg / L toxoplasmin standard solution and 20 μL of a toxoplasmin time-resolved fluorescence detection solution, let it stand for 3 minutes to obtain a mixed solution, take 100 μL of the mixed solution and drop it onto the sample pad of the immunochromatographic test strip for quantitative detection of toxoplasmin prepared in step 1, let it stand for 10 minutes, and use a time-resolved fluorescence quantitative detector to detect the fluorescence value (T value) of the test line and the fluorescence value (C value) of the quality control line on the immunochromatographic test strip, and calculate the T value / C value; perform three parallel measurements, and take the average value as the (T / C) value of the toxoplasmin standard solution with a concentration of 0.1 μg / L.

[0143] According to the above method, the (T / C) values ​​of the toxoplasmin standard solution with a concentration of 0.3 μg / L, the (T / C) value of the toxoplasmin standard solution with a concentration of 0.9 μg / L, the (T / C) value of the toxoplasmin standard solution with a concentration of 2.7 μg / L, and the (T / C) value of the toxoplasmin standard solution with a concentration of 8.1 μg / L were obtained respectively; and the (T / C) value of the toxoplasmin standard solution when the concentration of the toxoplasmin standard solution was 0 μg / L (i.e., 100 μL of the toxoplasmin standard solution with a concentration of 0.1 μg / L was replaced with 100 μL of 0.1 M PBS buffer) was detected.

[0144] The concentration of the toxoplasmin standard solution was used as the abscissa and the (T / C) value was used as the ordinate. Logistic curve fitting was performed using ELISACalc software to obtain the fluorescence quantitative standard curve of toxoplasmin, and a fluorescence quantitative standard curve graph of toxoplasmin was drawn.

[0145] (5) Determination of cypermethrin concentration in the test sample

[0146] The cypermethrin standard described in (4) is replaced with the sample to be tested, and the fluorescence value (T 测 ) and the fluorescence value of the quality control line (C测 ), combined with the fluorescence quantitative standard curve of cypermethrin obtained in (4), the cypermethrin concentration of the sample to be tested was calculated.

[0147] 2. Experimental Results

[0148] The (T / C) values ​​of various concentrations of cypermethrin standard solutions on the immunochromatographic test strips are shown in Table 2.

[0149] Table 2 (T / C) values ​​of various concentrations of cypermethrin standard solutions on immunochromatographic test strips

[0150]

[0151] The (T / C) value is the ratio of the fluorescence value of the test line (T value) to the fluorescence value of the quality control line (C value) on the immunochromatographic test strip; B / B0 is the ratio of the (T / C) value to the (T / C) value when the concentration of the toxoplasmin standard solution is 0 μg / L.

[0152] The standard curve of fluorescence quantitative analysis of cypermethrin is shown in Figure 2. Figure 5 The fluorescence quantitative standard curve of cypermethrin is shown in Formula II.

[0153] Formula II: y1=(A1-D1) / {1+(x1 / C1)^B1}+D1, r 2 =0.999; wherein A1=3.60848, B1=0.89160, C1=0.24692, D1=0.28795, x1 is the concentration of toxoplasmin (μg / L), and y1 is the (T / C) value when performing fluorescence quantitative detection using the immunochromatographic test strip constructed in step 1.

[0154] The results showed that when the concentration of the toxoplasmin standard solution was 0.1 μg / L, its B / B0 value was 71.2%, indicating that when the immunochromatographic test strip was used to detect a sample with a toxoplasmin concentration of 0.1 μg / L, the (T / C) value was significantly different from the (T / C) value of a sample without toxoplasmin (i.e., the toxoplasmin concentration was 0); this was because the immunochromatographic test strip had a detection sensitivity of 0.1 μg / L for toxoplasmin when using fluorescent quantitative detection of toxoplasmin concentration.

[0155] Example 6 Performance Test of Immunochromatographic Test Strips for Quantitative Detection of Toxoplasmin

[0156] 1. Experimental Methods

[0157] 1. Stability test

[0158] An immunochromatographic test strip for the quantitative detection of toxoflavin was constructed according to the method shown in Example 5, and an accelerated aging experiment was conducted. Specifically, the immunochromatographic test strip for the quantitative detection of toxoflavin was sealed and placed continuously at room temperature and 45°C for 60 days, and three immunochromatographic test strips were placed repeatedly at each temperature.

[0159] The fluorescence values ​​of the test line (T value) and the quality control line (C value) on the immunochromatographic test strips placed at different temperatures were measured on the 0th, 5th, 10th, 30th and 60th days after placement, and the average (T / C) value was calculated.

[0160] 2. Precision test

[0161] The intra-plate error and inter-plate error of the (T / C) value of the immunochromatographic test strip constructed by the method shown in Example 5 represent the precision of the immunochromatographic test strip, and the intra-plate error and inter-plate error are respectively reflected by the intra-plate coefficient of variation and the inter-plate coefficient of variation.

[0162] The intra-assay coefficient of variation (CV) is determined by analyzing the dispersion of the T / C values ​​of 10 immunochromatographic test strips from the same plate. The inter-assay coefficient of variation (CV) is determined by analyzing the dispersion of the T / C values ​​of 10 immunochromatographic test strips from different plates. The intra-assay and inter-assay coefficients of variation are calculated using the formula CV% = (standard deviation SD value / mean value) × 100%.

[0163] 3. Spiked sample test

[0164] Rice noodles, rice noodles, black fungus and white fungus were used as test samples respectively, and each test sample included a dry sample (dry sample) and a moist sample (wet sample).

[0165] Take the spiked test on dry rice noodles as an example, the details are as follows:

[0166] Toxoflavin standard was added to dry rice noodle samples to prepare spiked rice noodle samples with toxoflavin concentrations of 25 ng / kg, 50 ng / kg, and 100 ng / kg. Dry rice noodle samples without toxoflavin standard (0 ng / kg) were used as blank controls.

[0167] Place 3 g of dry rice noodle sample spiked with a concentration of 25 ng / kg in a 15 mL centrifuge tube, then add 9 mL of ethyl acetate and 0.2 mL of acetic acid. Shake well for 1 min, centrifuge at room temperature and 4000 r / min for 5 min, take 6 mL of supernatant and blow dry with nitrogen at 65°C. Then add 0.4 mL of PBST buffer (0.01 M) to the dried residue and mix thoroughly to obtain the sample solution.

[0168] The immunochromatographic test strips shown in Example 5 were used in combination with the method for use to quantitatively detect the concentration of toxoplasmin in the sample test solution. The test was repeated 5 times and the quantitative test results were recorded each time.

[0169] According to the above method, the concentration of toxin in the spiked rice noodle samples with toxin concentrations of 50 ng / kg and 100 ng / kg and the blank control was quantitatively detected.

[0170] Rice noodle wet samples, rice noodle dry samples, rice noodle wet samples, wood ear dry samples, wood ear wet samples, white ear dry samples and white ear wet samples were spiked according to the above method and quantitatively detected for toxoplasmone in combination with the immunochromatographic test strips shown in Example 5, and the test results were recorded.

[0171] 2. Experimental Results

[0172] 1. Stability test results

[0173] The average (T / C) values ​​of the immunochromatographic test strips placed at various temperatures are shown in Table 3.

[0174] Table 3 Average (T / C) values ​​of immunochromatographic test strips placed at various temperatures

[0175]

[0176] The results showed that the immunochromatographic test strip for quantitative detection of toxoplasmin constructed in Example 5 was sealed and stored for 60 days at room temperature and 45°C, and the (T / C) value of the immunochromatographic test strip did not change significantly, indicating that the immunochromatographic test strip can be stably stored at 45°C for at least 60 days; therefore, the immunochromatographic test strip for quantitative detection of toxoplasmin constructed in Example 5 can be stably stored at room temperature for more than one year.

[0177] 2. Precision test results

[0178] The results of intra-plate and inter-plate coefficients of variation are shown in Table 4.

[0179] Table 4 Results of intra-plate and inter-plate coefficients of variation

[0180] In-board (T / C) value Inter-board (T / C) value 1 3.67 3.66 2 3.51 3.57 3 3.77 3.82 4 3.68 3.91 5 3.62 3.67 6 3.66 3.56 7 3.84 3.94 8 3.74 3.82 9 3.91 3.75 10 3.62 3.69 CV value 3.14％ 3.53％

[0181] The results showed that the (T / C) values ​​of the immunochromatographic test strip constructed by the method shown in Example 5 varied little within the same plate and between different plates, with an intra-plate coefficient of variation of 3.14% and an inter-plate coefficient of variation of 3.53%. This indicates that the immunochromatographic test strip has small intra-plate and inter-plate coefficients of variation and high precision, which can meet the quantitative requirements of the immunochromatographic test strip.

[0182] 3. Test results of spiked samples

[0183] The quantitative detection results of dry rice noodle samples and wet rice noodle samples are shown in Table 5, the quantitative detection results of dry rice noodle samples and wet rice noodle samples are shown in Table 6, the quantitative detection results of dry wood ear samples and wet wood ear samples are shown in Table 7, and the quantitative detection results of dry white fungus samples and wet white fungus samples are shown in Table 8.

[0184] Table 5 Quantitative detection results of dry rice flour sample and wet rice flour sample

[0185]

[0186]

[0187] Table 6 Quantitative test results of dry and wet rice noodle samples

[0188]

[0189] Table 7 Quantitative detection results of dry and wet samples of fungus

[0190]

[0191] Table 8 Quantitative detection results of Tremella dry sample and Tremella wet sample

[0192]

[0193] Note: "<15" means that the concentration of toxoplasmin in the sample to be tested is <15 ng / kg. It cannot be accurately quantified in the immunochromatographic test strip constructed in Example 5, so "<15" is judged as negative.

[0194] The results showed that the immunochromatographic test strip for quantitative detection of toxoflavin constructed in Example 5 had a recovery rate ranging from 96% to 132% when performing quantitative detection on wet rice noodle samples, wet rice noodle samples, dry wood ear samples, wet wood ear samples, dry white fungus samples, and wet white fungus samples spiked with 25 ng / kg, 50 ng / kg, and 100 ng / kg of toxoflavin standards. The recovery rate range fluctuated slightly, and the test strip could be used for accurate and rapid quantitative detection.

[0195] After spiking dry rice noodle samples and dry rice noodle samples with 25 ng / kg and 50 ng / kg of toxoflavin standards, quantitative detection was performed using the immunochromatographic test strip for quantitative detection of toxoflavin constructed in Example 5. The recovery rate ranged from 68% to 130%, with large fluctuations in the recovery rate range. After spiking with 100 ng / kg, the recovery rate ranged from 88% to 124%, with small fluctuations.

[0196] However, the quantitative detection results of all spiked samples showed that the concentration of toxoplasmin was greater than 15 ng / kg, and no false negatives due to missed detection occurred. Therefore, the detection limit of the immunochromatographic test strip for quantitative detection of toxoplasmin constructed in Example 5 was 25 ng / kg.

[0197] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A hapten for detecting toxoplasmin, characterized in that: Its structural formula is shown in Formula I; Wherein R in the structural formula shown in Formula I is a halogen ion.

2. The method for preparing the hapten according to claim 1, characterized in that: The hapten with the structural formula of Formula I is quaternized by reacting toxin with Compound a of Formula II to obtain a hapten of Formula I; Wherein R in the structural formula shown in Formula II is a halogen atom.

3. Use of the hapten according to claim 1 in the preparation of an artificial antigen for detecting toxoplasmin.

4. An artificial antigen for detecting toxoplasmin, characterized in that: Obtained from the hapten-coupled carrier protein according to claim 1, the structural formula is shown in Formula III; In the structural formula shown in Formula III, R is a halogen ion, and Protein is a carrier protein.

5. The artificial antigen according to claim 4, characterized in that The carrier protein is bovine serum albumin, ovalbumin, hemocyanin or lactoferrin.

6. Use of the artificial antigen according to any one of claims 4 to 5 in the preparation of antibodies for detecting toxoflavan.

7. An artificial antigen combination for detecting toxoplasmin, characterized in that: The invention comprises an immunogen and a coating agent, wherein the immunogen is obtained by coupling the hapten according to claim 1 with lactoferrin, and the coating agent is obtained by coupling the hapten according to claim 1 with bovine serum albumin.

8. An immunochromatographic test strip for quantitative detection of toxoplasmin, comprising a PVC base plate and a sample pad, a cellulose membrane, and a water-absorbing pad sequentially arranged on the PVC base plate, wherein a test line and a quality control line are provided on the cellulose membrane, characterized in that: The detection line is coated with the coating agent according to claim 7, and the quality control line is coated with IgG antibodies.

9. Use of the artificial antigen combination according to claim 7 and / or the immunochromatographic test strip according to claim 8 in the preparation of a kit for the quantitative detection of toxoplasmin.

10. A kit for quantitative detection of toxoplasmin, characterized in that: Containing the artificial antigen combination according to claim 7 and / or the immunochromatographic test strip according to claim 8.

Images