CN116514907B - Immunochromatography test strip for rapidly detecting mycotoxin tenatoxin - Google Patents
Immunochromatography test strip for rapidly detecting mycotoxin tenatoxin Download PDFInfo
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- CN116514907B CN116514907B CN202310390538.7A CN202310390538A CN116514907B CN 116514907 B CN116514907 B CN 116514907B CN 202310390538 A CN202310390538 A CN 202310390538A CN 116514907 B CN116514907 B CN 116514907B
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- tenatoxin
- hapten
- test strip
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- pad
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Classifications
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- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an immunochromatographic test strip for rapidly detecting mycotoxin tenatoxin, which is prepared by hapten with a structure shown as a formula (I), is developed by adopting a colloidal gold immunochromatographic technology, can rapidly and qualitatively or semi-quantitatively detect the tenatoxin residue in a sample, is simple to operate, is economical and practical, is suitable for field detection, and can be widely applied to detection and rapid screening of tenatoxin pollution in grains.
Description
Technical Field
The invention relates to the technical field of food safety monitoring and immunoassay detection, in particular to an immunochromatography test strip for rapidly detecting mycotoxin tenxin.
Background
Tentoxin (Ten) is a mycotoxin produced by Alternaria alternata (Alternaria) and has a cyclic tetrapeptide structure. Alternaria alternata is a fungus widely distributed in soil and various crops, can widely pollute grains and feeds through the ways of field infection, storage contact and the like, can generate more than 70 toxins, and has chronic or acute toxic effects such as mutagenicity, carcinogenicity, teratogenicity and the like on human beings and animals. TEN can reduce intracellular Adenosine Triphosphate (ATP) enzyme activity, inhibit photosynthetic phosphorylation, and cause plant chlorosis. In vitro experiments have shown that TEN forms hydroxylated and demethylated metabolites by cytochrome P450-3A action. However, toxicity tests of TEN on mammals have not been reported.
Ten is mainly present in crops such as grains, tomatoes, oranges, cherries and fruits, and has very common pollution to wheat, high detection rate and large detection amount, and is reported all over the world. The European Food Security Agency (EFSA) of 2016 has issued scientific opinion regarding the health risks to humans of the toxins of the genus Alternaria in foods, TEN being a representative toxin produced by Alternaria and having a maximum daily intake threshold initially set at 1500ng/kg bw/d. Food pollutant monitoring in the country of 2016-2018 shows that the pollution of Ten in wheat and products thereof is serious, the detection rate exceeds 90%, and the maximum detection value exceeds 200 mug/kg. Currently, the detection of TEN mainly adopts the traditional large-scale instrument detection method, including high performance liquid chromatography (HPLC-UV), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the like. The methods are operated under laboratory conditions, the pretreatment of the samples is tedious and time-consuming, expensive instruments and equipment are needed to be equipped, the detection cost is high, the time consumption is long, the operation is complex, the method has great limitation in the practical application process, and the method is difficult to meet the requirements of rapid detection of a large number of samples and on-site samples. The detection result of the immunochromatography test strip based on the specific binding reaction of the colloidal gold labeled antibody and the antigen is visible, large-scale instrument and equipment are not needed, the detection cost is low, the analysis time is short, and therefore qualitative, online and rapid detection of various mycotoxins can be realized, but no related report of detecting tenatoxins by using the immunochromatography test strip is available at present.
Disclosure of Invention
The invention aims to provide a tenatoxin hapten, and a preparation method and application thereof.
The invention also aims to provide an immunochromatographic test strip for rapidly detecting tenatoxin and application thereof.
In order to achieve the purpose of the invention, the toxin hapten has the structure shown as the formula (I):
the invention also provides a total antigen of the tenatoxin, which is obtained by coupling the hapten of the tenatoxin with carrier protein, namely, the carboxyl of the hapten and the free amino of the carrier protein are subjected to condensation reaction.
Wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin; bovine Serum Albumin (BSA) and Ovalbumin (OVA) are preferred.
Specific antibodies prepared from the tenatoxin holoantigen comprise polyclonal antibodies and monoclonal antibodies. Wherein, the tenatoxin monoclonal antibody is obtained by taking a tenatoxin hapten-carrier protein conjugate (such as a tenatoxin hapten-BSA conjugate) as an immunogen and combining an immune test animal with a hybridoma cell preparation technology; preferably, the monoclonal antibody has a titer of 1:810000.
The tenatoxin hapten of the invention can be prepared by the following method: taking 0.39g of anhydrous aluminum trichloride, adding 100mL of 1, 2-dichloroethane and 0.5g of succinic anhydride, and stirring for 30min; then 10mL of 1, 2-dichloroethane solution of 0.414g of tenatoxin is added, stirred for 1h at room temperature, 200mL of pure water is slowly added under stirring, and the mixture is stirred and then left to stand; separating the water phase, washing the organic phase again, drying with anhydrous sodium sulfate, evaporating to dryness, loading on a silica gel small column, eluting with a dichloromethane-methanol mixed solution with the volume ratio of 10:1, and separating to obtain the hapten product. The synthesis route of the tenatoxin hapten is shown in figure 1.
The invention also provides a test reagent or a kit for the tenatoxin prepared from the tenatoxin hapten, the tenatoxin holoantigen or the specific antibody.
The invention also provides application of the tenatoxin hapten, the tenatoxin holoantigen, the specific antibody or the detection reagent or the kit in immunoassay detection of the tenatoxin.
The invention also provides application of the tenatoxin hapten, the tenatoxin holoantigen, the specific antibody or the detection reagent in preparing a colloidal gold detection test strip of tenatoxin.
The invention also provides an immunochromatography test strip (figure 2) for rapidly detecting the tenatoxin, which comprises a sample pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate. Preferably, 1/3 to 1/2 of the conjugate release pad is covered under the sample pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
Wherein, the conjugate release pad is coated with a tenatoxin specific antibody-colloidal gold marker, and the tenatoxin specific antibody can be a polyclonal antibody or a monoclonal antibody; the detection line is coated with the antitoxin holoantigen (such as an antitoxin hapten-OVA conjugate), and the quality control line is coated with a goat anti-mouse secondary antibody.
The preparation method of the tenatoxin specific antibody-colloidal gold marker comprises the following steps: mixing the specific antibody with colloidal gold solution, adding 10% BSA until the final concentration of BSA in the solution is 1%, standing, centrifuging to obtain precipitate, washing with redissolving buffer solution, and re-suspending the precipitate;
preferably, the re-dissolving buffer solution is 0.1-0.5% of BSA-containing phosphate buffer solution with the mass fraction of 2-4% of sucrose and the pH value of 7.2.
The invention also provides a preparation method of the immunochromatography test strip, which comprises the following steps:
1) Preparing a conjugate release pad sprayed with a tenatoxin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a tenatoxin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse secondary antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample pad, the water absorption pad and the bottom plate which are prepared in the steps (1) and (2) into the test strip.
Specifically, the preparation method of the immunochromatographic test strip comprises the following steps:
1) The method comprises the steps of performing acylation reaction on a benzene ring of the tenatoxin under the catalysis of aluminum trichloride by utilizing Friedel-crafts reaction, and introducing a succinic anhydride spacer arm to prepare the tenatoxin hapten;
2) Coupling the tenascin hapten with carrier protein to prepare a tenascin hapten-carrier protein conjugate;
3) Immunizing a mouse by using a tenatoxin hapten-carrier protein conjugate, and fusing and screening spleen cells of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting a tenatoxin monoclonal antibody;
4) Coating a tenatoxin hapten-carrier protein conjugate and a goat anti-mouse secondary antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;
5) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;
6) Adding the prepared tenatoxin monoclonal antibody into the prepared colloidal gold to obtain a tenatoxin monoclonal antibody-colloidal gold marker;
7) Spraying a toxin-free monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 2 hours, taking out, and storing in a dry environment for standby;
8) Soaking the sample pad in 0.02mol/L phosphate buffer solution containing 1% BSA and having a pH of 7.2 for 2 hours, and drying at 37 ℃ for 2 hours for later use;
9) A sample pad, a conjugate release pad, a reaction membrane, and a water absorbing pad are sequentially stuck on the bottom plate, and 1/3 area of the conjugate release pad from the initial end is covered by the sample pad. Finally cutting into small strips with the width of 3.95mm, placing the small strips in a specially made plastic card-making shell, sealing the small strips by an aluminum foil bag, and preserving the small strips for 12 months at the temperature of 4-30 ℃. The 1/3 of the conjugate release pad is covered by the sample pad, so that the observation time of the detection result can be prolonged, the sample pad can fully absorb the detection liquid and fully react with the gold-labeled antibody, and the error is reduced.
The invention further provides a method for detecting the toxin in the grains by using the immunochromatographic test strip, a sample to be detected is crushed, 1.0+/-0.05 g is weighed into a 15mL polystyrene centrifuge tube, 10mL 80% methanol is added, vortex oscillation is carried out for 3min,3000r/min room temperature centrifugation is carried out for 5min, and the supernatant is the sample liquid to be detected; 100 mu L of sample liquid to be detected is sucked by a micropipette and vertically dripped on a sample pad of a test strip, the liquid flow starts to time, the reaction is carried out for 10min, and the color development judgment result is carried out according to a detection line and a quality control line.
The test strip for rapidly detecting the tenatoxin adopts a highly specific antibody antigen reaction and immunochromatographic analysis technology, a tenatoxin specific antibody-colloidal gold marker is fixed on a conjugate release pad, and the tenatoxin in a sample is combined with the tenatoxin specific antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the tenatoxin-antibody-colloidal gold marker. The test method comprises the steps that the antitoxin in a sample competes with an antitoxin hapten-carrier protein conjugate on a reaction membrane detection line for binding with an antitoxin specific antibody-colloidal gold marker, and whether the sample liquid to be tested contains the antitoxin or not is judged according to the red stripe depth of the detection line.
During detection, a sample is dripped into a sample pad after being treated, when the concentration of the tenatoxin in the sample is lower than the detection limit or is zero, the specific antibody-colloidal gold marker is combined with the tenatoxin hapten-carrier protein conjugate fixed on the reaction membrane in the chromatographic process, a red strip appears on a detection line (T) and a quality control line (C), and the color development of the T line is deeper than or consistent with that of the C line; if the concentration of tenatoxin in the sample is at or above the limit of detection, the specific antibody-colloidal gold-tag will bind all of the tenatoxin, and thus will not bind to the tenatoxin hapten-carrier protein conjugate at the T-line due to the competition reaction without a red band or a lighter color than the C-line. As shown in fig. 3.
Negative: and when the quality control line (C) shows red stripes, the detection line (T) also shows red stripes, and the color of the (T) line is close to or deeper than that of the (C), the judgment is negative.
Positive: and judging positive when the quality control line (C) shows red stripes and the detection line (T) does not develop color or the color of the detection line (T) is lighter than that of the detection line (C).
Invalidation: when the quality control line (C) does not display red stripes, whether the detection line (T) displays red stripes or not, the test strip is judged to be invalid.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
(1) The invention starts from designing the hapten structure of the tenatoxin, prepares immunogen and coating antigen, screens the high-specificity and high-affinity tenatoxin monoclonal antibody, has good specificity, and the developed colloidal gold immunochromatography test strip can be directly used for detecting the tenatoxin and has great practical application value.
(2) The colloidal gold immunochromatographic test strip for detecting the tenatoxin in the grains can realize detection in one step, has accurate result, does not need washing and standard reference, can rapidly and qualitatively or semi-quantitatively detect single or batch samples in real time, and has high detection sensitivity and detection limit of 10 mug/kg. The test strip has no cross reaction to other Alternaria alternata toxins.
(5) The colloidal gold immunochromatographic test strip for detecting the tenatoxin in the grains has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, no limit of detection equipment, suitability for various units, simple storage and long shelf life, can play an important role in rapid detection and screening of the tenatoxin in a large number of samples on site, and can better assist food enterprises and government function supervision departments in China to develop related detection works.
Drawings
Figure 1 shows the synthesis of tenatoxin hapten.
Fig. 2 is a schematic diagram of a cross-sectional structure of a test strip, in which: 1. a sample pad; 2. a conjugate release pad; 3. a reaction membrane; 4. a water absorbing pad; 5. a detection line; 6. a quality control line; 7. a bottom plate.
Fig. 3 is a test strip detection result judgment chart.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.
Example 1 preparation of immunochromatographic test strip for rapidly detecting Tengtoxin
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a tenatoxin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a tenatoxin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse secondary antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample pad, the water absorption pad and the bottom plate which are prepared in the steps (1) and (2) into the test strip.
The following is a stepwise detailed description:
1. synthesis of Tengtoxin hapten (synthetic route see figure 1)
Taking 0.39g of anhydrous aluminum trichloride, adding 100mL of 1, 2-dichloroethane and 0.5g of succinic anhydride, and stirring for 30min; then 10mL of 1, 2-dichloroethane solution of 0.414g of tenatoxin is added, stirred for 1h at room temperature, 200mL of pure water is slowly added under stirring, and the mixture is stirred and then left to stand; separating the water phase, washing the organic phase again with water, drying with anhydrous sodium sulfate, evaporating to dryness, loading on a small silica gel column, eluting and separating with a dichloromethane-methanol mixed solution with the volume ratio of 10:1 to obtain hapten product of 0.24g and yield of 47%.
2. Preparation of immunogens
Taking 19mg of tenatoxin hapten, adding 1mL of N, N-Dimethylformamide (DMF), dissolving and clarifying, adding 9.26mg of N-hydroxysuccinimide (NHS) and 15.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving and mixing uniformly, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 6mL of 0.1mol/L PB buffer solution with pH of 8.0 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the tenatoxin hapten-BSA conjugate, namely the immunogen.
3. Preparation of coating Material
Taking 11mg of tenatoxin hapten, adding 1mL of DMF for dissolving and clarifying, adding 5.2mg of NHS and 8.2mg of EDC, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of Ovalbumin (OVA), adding 6mL of 0.1mol/L CB buffer solution with pH of 9.1 for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the tenatoxin hapten-OVA conjugate, namely the coating antigen.
4. Preparation of Tengtoxin monoclonal antibody
(1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.
(2) Cell fusion and cloning
Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation
The hybridoma cells were prepared into 1X 10 by using a frozen stock solution 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, so that the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
(5) Determination of antibody titers
Coating 100 mu L of coating antigen diluted to a certain concentration in each hole of the ELISA plate, and coating at 4 ℃ overnight; washing the cleaning solution for 1 time, adding 150 mu L of sealing solution into each hole, incubating at 37 ℃ for 2 hours, and spin-drying; 100 mu L of antibody diluted by antibody diluent is added to each well, and the wells are incubated for 0.5h at 37 ℃; after washing 3 times by the cleaning solution, respectively adding enzyme-labeled secondary antibodies which are diluted 2500 times by enzyme-labeled secondary antibody diluent into each hole, and incubating for 0.5h at 37 ℃; after washing 3 times, adding a color development solution (50 mu L of substrate solution A and 50 mu L of substrate solution B), and reacting for 15min at room temperature in a dark place; adding 50 mu L of stop solution to stop the reaction; the absorbance was measured with a microplate reader at an absorption wavelength of 450/630nm. The titer of the prepared tenatoxin monoclonal antibody can reach 1:810000.
5. Preparation of Tengtoxin monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 1.5mL 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering to original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight.
(2) Preparation of Tengtoxin monoclonal antibody-colloidal gold marker
Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution, adding the antitragus monoclonal antibody into the colloidal gold solution according to the standard of adding 30 mug antibody into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30 minutes; after 10min of standing, 10% BSA was added to give a final concentration of 1% in the colloidal gold solution, and the mixture was left for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.
Reconstitution buffer: 0.02mol/L phosphate buffer solution containing 0.2% of BSA, 3% of sucrose and pH 7.2.
6. Preparation of conjugate release pads
The conjugate release pad was soaked in 0.02mol/L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, uniformly soaked for 2h, and dried at 37℃for further use. And uniformly spraying the prepared tenatoxin monoclonal antibody-colloidal gold marker on a conjugate release pad by using a Bio dot film-drawing instrument, spraying 0.01mL of the tenatoxin monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing the conjugate release pad in an environment with the temperature of 37 ℃ for 2 hours (humidity is less than 20%), taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.
7. Preparation of sample pad
The sample pad was soaked in 0.2mol/L phosphate buffer containing 1% BSA at pH 7.2 for 2h and dried at 37℃for 2 h.
8. Preparation of reaction film
Coating the tenatoxin hapten-OVA conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse secondary antibody on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the tenatoxin hapten-OVA conjugate to 1mg/mL by using 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coating the tenatoxin hapten-OVA conjugate on a detection line (T line) on a nitrocellulose membrane by using a Bio dot-based membrane-drawing instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse secondary antibody was diluted to 200. Mu.g/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 1.0. Mu.L/cm using a Bio dot-based membrane marker. And (5) drying the coated reaction film for 16 hours at 37 ℃ for standby.
9. Assembly of test strips
According to the cross-section structure of the test strip shown in figure 2, a sample pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially stuck on a PVC bottom plate (7); the 1/3 area of the initial end of the conjugate release pad is covered by the sample pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting test paper into strips with the width of 3.95mm by a machine, placing the strips in a specially-made plastic card shell, sealing the strips by an aluminum foil bag, and storing the strips at the temperature of 4 ℃ for later use.
Example 2 detection of Tengtoxin in grains
1. Sample pretreatment
Crushing a sample to be detected, weighing (1.0+/-0.05) g to 15mL of polystyrene centrifuge tube, adding 10mL of 80% methanol, carrying out vortex oscillation for 3min, and centrifuging at room temperature for 5min at 3000r/min, wherein the supernatant is the sample liquid to be detected.
2. Detection by test strips
100 mu L of sample liquid to be detected is sucked by a micropipette and vertically dripped on a sample pad of a test strip, the liquid flow starts to time, the reaction is carried out for 10min, and the result is judged.
3. Analyzing the detection result
Negative (-). The color development of the T line is deeper than or consistent with that of the C line, which indicates that the concentration of the tenascin in the sample is lower than the detection limit, as shown in figures 3a and 3b.
Positive (+): the T line is lighter than the C line or the T line is not, which indicates that the concentration of tenatoxin in the sample is equal to or higher than the detection limit, as shown in FIGS. 3C and 3d.
Invalidation: the absence of line C indicates an incorrect procedure or that the test strip has failed due to deterioration, as shown in fig. 3e, 3f.
Example 3 sensitivity, accuracy, and specificity analysis of immunochromatographic test strips
1. Limit of detection test
Blank wheat and corn samples are taken, and tenatoxin is added to the samples to obtain final concentrations of 2.5 mug/kg, 5 mug/kg, 10 mug/kg and 20 mug/kg respectively, test strips are taken for detection, and each sample is repeatedly measured three times.
When the test strip is used for detecting wheat and corn samples, when the test strip has no tenatoxin and the addition concentration is 2.5 mug/kg or 5 mug/kg, the test strip shows that the color development of the T line is deeper than or consistent with that of the C line, and the test strip is negative; when the adding concentration of the tenatoxin is 10 mug/kg and 20 mug/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed, and the test strip is positive, so that the detection limit of the test strip on the tenatoxin in the grains is 10 mug/kg.
2. False positive rate and false negative rate test
Taking blank wheat and corn samples and 20 positive wheat and corn samples with final concentration of 10 mug/kg by adding tenatoxin, respectively detecting with test strips produced in 3 batches, and calculating the negative-positive rate.
The results show that: when 3 batch production test strips are used for detecting positive wheat and corn samples, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when blank wheat and corn samples are detected, the results are all negative, and the negative coincidence rate is 100% and the false positive rate is 0. The test strip for detecting the tenatoxin can be used for rapidly and accurately detecting the tenatoxin residue in grains.
3. Specificity test
When the test strip is used for detecting 1000 mug/kg of alternariol, alternariol monomethyl ether, alternariene, tenatoxin, tenatonic acid and other alternaria toxins, the test strip shows that the color development of the T line is deeper than that of the C line, and the test strip is negative, so that the test strip has no cross reaction to other alternaritoxins and has good specificity.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (9)
1. The tenatoxin hapten is characterized in that the tenatoxin hapten has a structure as shown in a formula (I):
formula (I).
2. A total antigen of tenatoxin, characterized in that it is obtained by coupling a hapten of tenatoxin according to claim 1 with a carrier protein; wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
3. A process for preparing a tenatoxin hapten according to claim 1, wherein 0.39g of anhydrous aluminum trichloride is added with 100ml of 1, 2-dichloroethane and 0.5g of succinic anhydride and stirred for 30min; then 10mL of 1, 2-dichloroethane solution of 0.414g of tenatoxin is added, stirred for 1h at room temperature, 200mL of pure water is slowly added under stirring, and the mixture is stirred and then left to stand; separating the water phase, washing the organic phase again, drying with anhydrous sodium sulfate, evaporating to dryness, loading on a silica gel small column, eluting with a dichloromethane-methanol mixed solution with the volume ratio of 10:1, and separating to obtain the hapten product.
4. A test agent or kit for tenatoxin prepared from the tenatoxin hapten of claim 1 or the tenatoxin holoantigen of claim 2.
5. Use of a tetanus toxin hapten as defined in claim 1, a tetanus toxin holoantigen as defined in claim 2, or a detection reagent or kit as defined in claim 4 in the immunoassay detection of tetanus toxin.
6. Use of a tetanus hapten as defined in claim 1, a tetanus holoantigen as defined in claim 2, or a detection reagent as defined in claim 4 for the preparation of a colloidal gold test strip for detecting tetanus.
7. The immunochromatographic test strip is characterized by comprising a sample pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate; wherein, the conjugate release pad is coated with a tenatoxin specific antibody-colloidal gold marker; the detection line is coated with the total antigen of the tenxin according to claim 2, and the quality control line is coated with the goat anti-mouse secondary antibody.
8. A test strip as in claim 7 wherein said method of preparing a tenatoxin-specific antibody-colloidal gold marker comprises the steps of: mixing the specific antibody with colloidal gold solution, adding 10% bovine serum albumin until the final concentration of the bovine serum albumin in the solution is 1%, standing, centrifuging to obtain precipitate, washing with redissolving buffer solution, and re-suspending to obtain precipitate; the re-dissolving buffer solution is 0.02mol/L phosphate buffer solution containing 0.1-0.5% of bovine serum albumin, 2-4% of sucrose and 7.2 of pH.
9. A method for detecting tenatoxin by using the test strip as claimed in claim 7 or 8, which is characterized in that a sample to be detected is crushed, 1.0 g +/-0.05 g to 15mL polystyrene centrifuge tube is weighed, 10mL 80% methanol is added, vortex oscillation is carried out for 3min,3000r/min room temperature centrifugation is carried out for 5min, and supernatant fluid is sample liquid to be detected; 100 mu L of sample liquid to be detected is sucked by a micropipette and vertically dripped on a sample pad of a test strip, the liquid flow starts to time, the reaction is carried out for 10min, and the color development judgment result is carried out according to a detection line and a quality control line.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310390538.7A CN116514907B (en) | 2023-04-13 | 2023-04-13 | Immunochromatography test strip for rapidly detecting mycotoxin tenatoxin |
Applications Claiming Priority (1)
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| CN113588956A (en) * | 2021-07-08 | 2021-11-02 | 上海市农产品质量安全中心 | Alternaria alternata toxin colloidal gold immunochromatographic assay rapid detection method |
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| CN113588956A (en) * | 2021-07-08 | 2021-11-02 | 上海市农产品质量安全中心 | Alternaria alternata toxin colloidal gold immunochromatographic assay rapid detection method |
Non-Patent Citations (4)
| Title |
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| A novel hapten and monoclonal antibody-based indirect competitive ELISA for simultaneous analysis of alternariol and alternariol monoethyl ehter in wheat;Wang JY等;Food control;第94卷;65-70页 * |
| 粮油产品T-2毒素单克隆抗体研制及应用;张亮等;《中国油料作物学报》;第37卷(第5期);724-729页 * |
| 链格孢霉毒素样品处理及分析方法研究进展;王昌钊等;《化学分析计量》;第31卷(第10期);93-100页 * |
| 食品中真菌毒素抗体制备及其快速检测方法研究;孔德昭;《中国博士学位论文全文数据库工程科技I辑》;B024-14页 * |
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