CN108120832B - Rapid detection device for thiabendazole hapten, coupled antigen, antibody and colloidal gold and application thereof - Google Patents
Rapid detection device for thiabendazole hapten, coupled antigen, antibody and colloidal gold and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of food safety detection, and particularly relates to a thiabendazole hapten which has a structural formula shown in a formula (I). The invention also relates to a probenazole coupled antigen, a probenazole antibody, a colloidal gold rapid detection device containing the probenazole coupled antigen and the probenazole antibody and application of the probenazole coupled antigen and the probenazole antibody. The device has low cost, and can quickly and accurately carry out quick online detection and monitoring on the suspected object.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to a thiabendazole hapten, a thiabendazole coupled antigen, a thiabendazole antibody, a colloidal gold rapid detection device containing the thiabendazole coupled antigen and the thiabendazole antibody, and application of the colloidal gold rapid detection device.
Background
The thiabendazole is an imidazole bactericide, and is a high-efficiency, broad-spectrum and universal bactericide. The thiabendazole is used for preventing and treating various diseases of crops and economic crops caused by fungi, and is used for preserving and refreshing fruits and vegetables. With the unreasonable use and even abuse of the antibacterial synergist drugs, the residue of the drugs inevitably occurs. If the food containing the residual probenazole is eaten for a long time, the harm to the health of human beings is possible. Therefore, in order to strengthen the monitoring of the medicines in the food, a method for detecting the thiabendazole residues needs to be established.
At present, the detection method of the thiabendazole residues at home and abroad mainly comprises a High Performance Liquid Chromatography (HPLC), a liquid-mass spectrometry (LC-MS) method and an enzyme-linked immunosorbent assay (ELISA) method. In food safety detection, ELISA is used for preliminary screening, and then positive samples are confirmed by HPLC or LC-MS. The instruments and equipment used in the method have complex operation, high cost and high technical requirements on operators, can not immediately display results, and are not suitable for fast online detection and monitoring of suspected objects by food, commodity inspection, epidemic prevention and stockman producers.
In conclusion, there is a need in the art for an accurate, rapid and convenient method for detecting thiabendazole residues.
Disclosure of Invention
In order to overcome the defects of the existing detection method, the inventor provides a probenazole colloidal gold detection device through repeated research, and the probenazole colloidal gold detection device can be used for quickly and accurately detecting residual probenazole in food.
Accordingly, in a first aspect, the present invention provides a thiabendazole hapten having the structure of formula (I):
in a second aspect, the present invention provides a thiabendazole-conjugated antigen comprising a thiabendazole hapten of the first aspect of the present invention, and a carrier conjugated to the thiabendazole hapten.
In a third aspect, the present invention provides a thiabendazole antibody that is an antibody specific for a thiabendazole-conjugated antigen of the second aspect of the invention.
In a fourth aspect, the invention provides a probenazole colloidal gold rapid detection device, which comprises a test strip and a reaction cup, wherein the test strip comprises a sample pad and a nitrocellulose membrane, the nitrocellulose membrane comprises a detection line and a quality control line, and the detection line is prepared from probenazole coupled antigen; the reaction cup contains a colloidal gold labeled thiabendazole antibody that is specific for the thiabendazole-conjugated antigen.
In a fifth aspect, the present invention provides a method for detecting probenazole in a sample, which comprises detecting probenazole by using the probenazole colloidal gold rapid detection device of the fourth aspect of the present invention.
The invention has the beneficial effects that: the invention applies the principle of chromatographic immune colloidal gold, semi-quantitatively detects the residual quantity of probenazole in a sample by the color comparison between a detection line and a quality control line in a test strip, and quickly and accurately detects whether the sample contains probenazole in a short time so as to determine whether the probenazole exceeds the standard. The invention can meet the requirement of food safety on the detection of the residual quantity of thiabendazole, and is suitable for the planting industry, the breeding industry and government detection mechanisms.
Compared with the prior art, the invention has the advantages of obvious effect, convenient use, economy, rapidness, easy manufacture and low cost.
Drawings
Fig. 1 shows a specific embodiment of the probenazole colloidal gold rapid detection device of the invention.
FIG. 2 shows the mass spectrum of an exemplary thiabendazole hapten used in the thiabendazole colloidal gold rapid detection device of the present invention.
Detailed Description
The probenazole colloidal gold rapid detection device, the preparation method and the application thereof are described in more detail below. It should be noted that the summary above and the detailed description below are merely intended to specifically illustrate the present invention and are not intended to limit the invention in any way. The scope of the invention is to be determined by the appended claims without departing from the spirit and scope of the invention.
As mentioned above, the present invention aims to provide a probenazole detecting device which is more accurate, faster and more convenient than the prior art or devices.
Accordingly, in one aspect, the present invention provides a thiabendazole hapten having the structure of formula (I):
as understood by those skilled in the art, by hapten is meant a class of small molecule substances: it alone does not induce an immune response, i.e. is not immunogenic; however, when it is crosslinked or conjugated with a carrier such as a macromolecular protein or non-antigenic polylysine, immunogenicity can be obtained, thereby inducing an immune response. Such small molecule substances can bind to responsive effector products and are antigenic, i.e., immunoreactive, but not immunogenic.
In particular, the thiabendazole hapten is easily understood to be a small molecule substance which has immunoreactivity to an antibody (namely, a thiabendazole-specific antibody or a thiabendazole antibody, a monoclonal antibody or a polyclonal antibody) which is specifically directed to thiabendazole, but has no immunogenicity. In other words, such thiabendazole haptens can undergo an antigen-antibody binding reaction with them in the presence of thiabendazole antibodies; however, when the thiabendazole hapten is inoculated into an animal for immunization, the animal cannot be stimulated to produce corresponding antibodies.
Since thiabendazole itself does not produce good immunoreactivity as a hapten, it needs to be modified when it is used as a hapten. It is noted, however, that the modification should be as few as possible and as many sites as possible remain available for binding to the antibody.
In the present invention, the thiabendazole hapten is obtained by reacting a thiabendazole analogue (2- (5-thiazolyl) -1H-benzimidazole) with hydrobromic acid.
As mentioned above, the thiabendazole hapten molecule is only immunoreactive and not immunogenic. Thus, in order to confer immunogenicity on a thiabendazole hapten, it is also necessary to couple, bind or crosslink the thiabendazole hapten with a suitable carrier molecule, thereby producing a thiabendazole-coupled antigen that is both immunoreactive and immunogenic. Methods of coupling, binding or cross-linking the hapten to the carrier molecule are known in the art, for example, as exemplified in the examples section of this application.
Thus, in a second aspect, the present invention provides a thiabendazole-conjugated antigen comprising a thiabendazole hapten of the first aspect of the present invention, and a carrier conjugated to the thiabendazole hapten.
The term "carrier" as referred to herein is any substance capable of coupling, conjugating or cross-linking to a hapten and thereby producing both immunogenic and immunoreactive properties, including for example macromolecular proteins or non-antigenic polylysines and the like. By way of example, carriers that may be used include, but are not limited to, macromolecular proteins such as Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), hemocyanin (KLH), egg albumen (OVA).
In a third aspect, the present invention provides a thiabendazole antibody that is an antibody specific for a thiabendazole-conjugated antigen of the second aspect of the invention.
The thiabendazole antibody may be a monoclonal antibody or a polyclonal antibody. In addition, the thiabendazole antibody can be prepared by a method that is conventional in the art. For example, in the case where the thiabendazole antibody is a polyclonal antibody, it can be obtained by immunizing a mammal such as a mouse, rat, rabbit, goat, sheep, primate (excluding human), etc., with the thiabendazole-conjugated antigen, followed by isolating serum. In the case where the thiabendazole antibody is a monoclonal antibody, the monoclonal antibody may be obtained by producing and culturing hybridoma cells and collecting the culture medium, or the hybridoma cells thus produced may be inoculated into the body of a mammal such as a mouse, a rat, a rabbit, a goat, a sheep, a primate (excluding human beings) or the like by intraperitoneal injection, and ascites may be collected when the abdomen of the inoculated animal is significantly enlarged, thereby obtaining the monoclonal antibody.
In addition, as can be understood by those skilled in the art, there is no particular limitation on the source of the thiabendazole antibody, which can be derived from any mammal, including, for example, murines such as mice and rats, rabbits, goats, sheep, primates (excluding humans), and the like, but is not limited thereto. In a particular embodiment, the thiabendazole antibody is a polyclonal or monoclonal antibody derived from a murine species such as mouse and rat, rabbit, goat, sheep, primate (excluding human).
In a particular embodiment, the thiabendazole antibody is a murine monoclonal antibody specific for a thiabendazole-conjugated antigen comprising a thiabendazole hapten of formula (I).
In a fourth aspect, the invention relates to a probenazole colloidal gold rapid detection device, which comprises a test strip and a reaction cup, wherein the test strip comprises a sample pad and a nitrocellulose membrane, the nitrocellulose membrane comprises a detection line and a quality control line, and the detection line is prepared from probenazole coupled antigen; the reaction cup contains a colloidal gold labeled thiabendazole antibody that is specific for the thiabendazole-conjugated antigen.
As mentioned above, the test strip comprises a sample pad and a nitrocellulose membrane, but the test strip may also comprise other components. For example, the test strip may further comprise a base plate and absorbent paper. In this case, the sample pad, the nitrocellulose membrane, and the absorbent paper may be sequentially laid on the base plate by lap bonding.
As described above, the nitrocellulose membrane comprises a detection line and a quality control line. Typically, the detection line is disposed on a side near the sample pad.
The detection line is prepared by carrying out linear spotting on the cellulose nitrate membrane by using probenazole coupled antigen. The thiabendazole-coupled antigen is obtained by coupling, cross-linking or binding a thiabendazole hapten, such as the thiabendazole hapten of the first aspect of the invention, to a carrier molecule. Such carrier molecules include, for example, macromolecular proteins or non-antigenic polylysines and the like. By way of example, carriers that may be used include, but are not limited to, macromolecular proteins such as Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), hemocyanin (KLH), egg albumen (OVA).
In a preferred embodiment, the thiabendazole-conjugated antigen is the thiabendazole-conjugated antigen of the second aspect of the present invention.
The probenazole colloidal gold rapid detection device comprises a quality control line. The purpose of the control line is to indicate that the device is in use and that the detection regime is in place, to ensure the reliability and correctness of the results obtained.
The control line can be obtained by linearly spotting an antigen or an antibody on the nitrocellulose membrane. In one embodiment, the control line is spotted from a second antibody directed against the thiabendazole antibody of the third aspect of the invention. When the colloidal gold-labeled thiabendazole antibody moves to the quality control line by siphoning, it undergoes a binding reaction with the second antibody forming the quality control line, thereby developing a color.
If the quality control line is colored, the detection system is indicated to be established, and the detection result is available. On the contrary, if the quality control line does not develop color, the detection system is indicated to be not established, and the detection result is unavailable.
The detection line and the quality control line are parallel to each other and are at a proper distance, so that the optimal detection is realized. In one embodiment, the detection line and the quality control line are at least 0.3 cm apart.
As mentioned above, the reaction cup contains a probenazole antibody labeled with colloidal gold, which specifically targets a probenazole-coupled antigen forming a detection line of the probenazole colloidal gold rapid-detection apparatus.
The thiabendazole antibody may be any antibody, regardless of whether it is a monoclonal antibody or a polyclonal antibody, as long as it can undergo an antigen-antibody binding reaction with the thiabendazole-coupled antigen. However, as will be appreciated by those skilled in the art, the thiabendazole antibody is preferably a monoclonal antibody from the viewpoint that higher specificity is required.
For the thiabendazole antibody, it can be prepared by a method conventional in the art. For example, in the case where the thiabendazole antibody is a polyclonal antibody, it can be obtained by immunizing a mammal such as a mouse, rat, rabbit, goat, sheep, primate (excluding human), etc., with the thiabendazole-conjugated antigen, followed by isolating serum. In the case where the thiabendazole antibody is a monoclonal antibody, the monoclonal antibody may be obtained by producing and culturing hybridoma cells and collecting the culture medium, or the hybridoma cells thus produced may be inoculated into the body of a mammal such as a mouse, a rat, a rabbit, a goat, a sheep, a primate (excluding human beings) or the like by intraperitoneal injection, and ascites may be collected when the abdomen of the inoculated animal is significantly enlarged, thereby obtaining the monoclonal antibody.
In addition, as can be understood by those skilled in the art, there is no particular limitation on the source of the thiabendazole antibody, which can be derived from any mammal including, for example, mouse, rat, rabbit, goat, sheep, primate (excluding human), and the like, but is not limited thereto. In a specific embodiment, the thiabendazole antibody is a polyclonal or monoclonal antibody derived from a mouse, rat, rabbit, goat, sheep, primate (excluding human).
In one embodiment, the thiabendazole antibody is a thiabendazole antibody of the third aspect of the invention. In a particular embodiment, the thiabendazole antibody is a murine monoclonal antibody specific for a thiabendazole-conjugated antigen comprising a thiabendazole hapten of formula (I).
In the device of the invention, colloidal gold-labeled thiabendazole antibodies are involved, which are prepared by coupling the thiabendazole antibodies of the invention with colloidal gold. The coupling method between the thiabendazole antibody and the colloidal gold can be performed using related coupling methods known in the art.
In a specific embodiment, the invention provides a probenazole colloidal gold rapid detection device, which comprises a test strip and a reaction cup, wherein the test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the nitrocellulose membrane and the absorbent paper are sequentially laid on the bottom plate; the reaction cup contains a colloidal gold-labeled thiabendazole antibody, which is a murine monoclonal antibody specific for the thiabendazole-coupled antigen comprising the thiabendazole hapten of formula (I); and the quality control line is a rabbit anti-mouse polyclonal antibody against the thiabendazole antibody.
The probenazole colloidal gold detection device can be prepared by the following preparation method: preparing a nitrocellulose membrane, performing linear sample application preparation detection line on the nitrocellulose membrane by adopting the thiabendazole coupled antigen, and preparing a quality control line by linear sample application; sequentially overlapping and sticking a sample pad, a nitrocellulose membrane and absorbent paper on a bottom plate along the same direction, thereby assembling a test strip; the colloidal gold-labeled thiabendazole antibody of the present invention was added to the reaction cup and lyophilized. The components or assemblies used in the preparation method are as described above for the probenazole colloidal gold detection device of the present invention.
In a fifth aspect, the present invention relates to a method for detecting probenazole in a sample, which comprises detecting probenazole by using the probenazole colloidal gold rapid detection device of the fourth aspect of the present invention.
The sample may be any sample suspected of containing thiabendazole, which may be a food product of animal origin or a food product of plant origin. For example, the food of animal origin may be mutton, beef, chicken, pork, duck, fish, seafood, etc., but is not limited thereto. Plant-derived foods include fruits and vegetables, such as apples, bananas, pears, grapes, citrus, lychee, sugar cane, pineapples and the like, vegetables such as radishes, carrots, turnips, burdock, root celery, root mustard, chinese cabbage, cauliflower, tuber mustard, potherb mustard, cabbage, lettuce, celery, spinach, garland chrysanthemum, coriander, amaranth, water spinach, basella vulgaris, tomatoes, peppers, eggplants, green peppers, bell peppers, pumpkins, cucumbers, melons, wax gourds, luffa, balsam pears and the like.
Before the probenazole detection method is adopted for detection, the probenazole can be correspondingly pretreated according to the specific source of the sample. For example, for a food of animal origin, the tissue may be minced, the probenazole contained therein extracted, and interfering substances such as fats contained therein removed, followed by probenazole detection. For plant-derived foods, no fat removal operation is required.
After the corresponding pretreatment of the sample, it is added to the reaction cuvette and incubated for a few minutes, e.g. 3-5 minutes, followed by insertion of the test strip and incubation for a further few minutes, e.g. 3-5 minutes. After the incubation is finished, if the quality control line is observed to show a purple red strip, the detection system is indicated to be established and available, and at this time, the following judgment is carried out: (1) if the detection line shows a purple red strip as the quality control line and the color depth of the detection line is equal to or deeper than that of the quality control line, the sample does not contain thiabendazole; (2) if the detection line is not colored or is lighter in color than the quality control line, the sample contains probenazole.
The invention has the beneficial effects that: the invention applies the principle of chromatographic immune colloidal gold, semi-quantitatively detects the residual quantity of probenazole in a sample by the color comparison between a detection line and a quality control line in a test strip, and quickly and accurately detects whether the sample contains probenazole in a short time so as to determine whether the probenazole exceeds the standard. The invention can meet the requirement of food safety on the detection of the residual quantity of thiabendazole, and is suitable for slaughtering enterprises and government detection organizations.
Compared with the prior art, the invention has the advantages of obvious effect, convenient use, economy, rapidness, easy manufacture and low cost.
The present invention will be described in more detail and with reference to the following examples and drawings.
Examples
Example 1 preparation of Thiediazole hapten
The preparation steps of the thiabendazole hapten are as follows: 2.73 g of thiabendazole analogue (2- (5-thiazolyl) -1H-benzimidazole), 1.67 g of propylbromous acid, 0.1 g of sodium carbonate were added in succession to a 100 mL three-necked flask, and allowed to react overnight to completion in 50 mL of acetonitrile solution at 70 ℃. After the rotary evaporation is finished, the thiabendazole hapten can be obtained by extracting and passing through a column by ethyl acetate.
The mass spectrum of the thus obtained thiabendazole hapten is given in fig. 2 and it can be seen that the hapten has the following formula:
example 2 Synthesis of Thiediazole conjugate antigen
The thiabendazole hapten prepared in example 1 was used to prepare a conjugated antigen as follows: 0.1 mmol of thiabendazole hapten is dissolved in 2mL of DMF and 27.5 mg of Dicyclohexylcarbodiimide (DCC) and 14.4 mg of N-hydroxysuccinimide (NHS) are added with stirring. The reaction was magnetically stirred at 4 ℃ overnight, and the supernatant was retained after centrifugation and labeled as solution A. Bovine Serum Albumin (BSA) and hemocyanin (KLH) were weighed out at 140 mg, and dissolved in 10mL of PBS (pH8.0) at a concentration of 0.1 mol/L, followed by addition of 1mL of Dimethylformamide (DMF), followed by stirring and dissolution to prepare solution B. Under magnetic stirring, solution A was gradually dropped into solution B, and reacted at 4 ℃ for 12 hours. After centrifugation, the supernatant was collected and dialyzed against physiological saline at 4 ℃ for 3 days, and the dialysate was changed 3 times a day, whereby probenazole-conjugated antigens conjugated with bovine serum albumin and hemocyanin, respectively, were obtained. The obtained probenazole coupled antigen was dispensed into a 0.5 mL centrifuge tube at a concentration of 1 mg/mL, and frozen in a-20 ℃ refrigerator.
Example 3 preparation of Thiediazole monoclonal antibody
The probenazole conjugated antigen prepared in example 2 is used to prepare a probenazole monoclonal antibody, which is specifically as follows: 4 Kunming mice with 6 weeks of age are immunized by using the identified thiabendazole coupling antigen, and after three times of boosting immunization, blood is collected for measuring the titer. When the serum titer does not rise any more, immunizing a mouse with two times of the dose of antigen without adjuvant, removing the neck to kill the mouse after three days, taking the spleen to prepare splenocytes under the aseptic condition, mixing the splenocytes with mouse myeloma cells growing vigorously in a 50 mL centrifuge tube according to the proportion of 8:1, adding 30 mL serum-free IPMI 1640 culture medium, centrifuging at 1100 r/min for 5 minutes, discarding supernatant, slightly shaking and loosening cell clusters, and placing the cell clusters in a water bath at 37 ℃. Slowly adding 50% PEG-4000 into cells, dripping within 1 minute, gently stirring bottom precipitate, standing for 1 minute, slowly adding 1mL serum-free culture medium along tube wall at uniform speed for the first 30 seconds, adding 2mL for the second 30 seconds, rapidly adding 27mL to terminate fusion process, centrifuging at 1100 r/min for 5 minutes, discarding supernatant, re-suspending with HAT selective culture medium, adding into 96-well cell culture plate paved with feeder cells, and adding 5% volume fraction of CO at 37 deg.C2Culturing under the condition. And 7 days later, changing the culture medium into an HT culture solution, when the number of the hybrid cells in the hole reaches more than 300, screening by using an indirect ELISA method, selecting the hole with strong positive, good inhibition effect and vigorous cell growth for limited dilution and cloning, carrying out cloning culture and detection for more than 3 times, wherein the cell in the hole which is positive is the hybrid tumor cell secreting the monoclonal antibody, and carrying out expanded culture on the hybrid tumor cell for preparing the monoclonal antibody.
The method for producing the probenazole-resisting monoclonal antibody by adopting the in-vivo induced ascites method comprises the following specific steps. Selecting 4 Kunming mice, injecting liquid paraffin oil into abdominal cavity 0.5 mL/mouse, injecting hybridoma cells 3-5 × 10 into abdominal cavity 7 days later6Ascites was collected after 10 days when the abdomen of the mice had significantly enlarged. The ascites is purified by using an n-octanoic acid-ammonium sulfate precipitation method, and the content of the probenazole resisting monoclonal antibody is determined by ultraviolet.
Example 4 preparation of Rabbit anti-mouse antibodies
New Zealand white rabbits were immunized with the thiabendazole monoclonal antibody prepared in example 3 at an immunization dose of 50. mu.g-100. mu.g/time, subcutaneously on the backInjecting at multiple points; emulsifying the first-stage immunization by using a probenazole monoclonal antibody and an equivalent amount of Freund's complete adjuvant; reinforcing immunization, emulsifying with thiabendazole monoclonal antibody and equivalent Freund incomplete adjuvant, continuously immunizing for 4-5 times, each time at intervals of 4-8 weeks, and measuring titer to 10 by enzyme-linked immunosorbent assay (ELISA) method 10-15 days after last immunization5In the above steps, blood is collected, hyperimmune serum is separated and collected, and IgG antibody is extracted by salting out with saturated ammonium sulfate, and the IgG antibody is frozen at-20 ℃ for later use.
Example 5 preparation of colloidal gold-labeled monoclonal antibody
5.1 preparation of colloidal gold
1ml of 1% chloroauric acid solution was taken, and 99ml of ultrapure water was added to the resulting solution to give a chloroauric acid solution having a final concentration of 0.01%. After the solution is heated to boil, 1.6 ml of 1% trisodium citrate is rapidly added into the boiled chloroauric acid solution at one time, and the heating is continued until the solution is changed from light yellow to blue black and finally to bright red. And after the color is stable, continuing to heat for 5min, cooling at room temperature, and supplementing water to the original volume.
5.2 coupling of colloidal gold and monoclonal antibodies
The pH of the colloidal gold solution obtained in 5.1 was adjusted to 8.0, and uniformly stirred with a constant speed stirrer while dropwise adding the probenazole monoclonal antibody prepared in example 3, polyethylene glycol (PEG) in an amount equivalent to the antibody was added after 1 hour, and Bovine Serum Albumin (BSA) in an amount equivalent to the antibody was added after sufficiently reacting for 30 minutes. After the addition was complete, stirring was continued for 30 minutes. The mixture was centrifuged at 9000 rpm for 30 minutes to obtain a homogeneous gold-labeled antibody precipitate, and then p-nitrophenol butyrate (PNPB) was added thereto for resuspension.
Example 6 preparation of probenazole colloidal gold detection device
Step (I): preparing a nitrocellulose membrane, and linearly spotting the thiabendazole-coupled antigen prepared in example 2 on the nitrocellulose membrane, thereby forming a detection line; and the rabbit anti-mouse IgG antibody prepared in example 4 was linearly spotted, thereby forming a quality control line.
Step (II): and sequentially overlapping and adhering the sample pad, the nitrocellulose membrane and the absorbent paper on one bottom plate along the same direction, thereby preparing the test strip.
Step (three): the colloidal gold-labeled monoclonal antibody prepared in example 5 was added to a reaction cuvette and lyophilized, thereby forming a desired reaction cuvette.
And (3) combining the test strip obtained in the step (II) with the reaction cup obtained in the step (III) to form the probenazole colloidal gold detection device.
Example 7 detection of Thiediazole in samples
Fresh vegetable or fruit samples were minced and extracted with acetonitrile and acetone, after which the extract samples were placed in the reaction cups prepared in example 6 and incubated for 3 minutes at room temperature. The test strips prepared in example 6 were then inserted into reaction cups and incubated at room temperature for 5 minutes. Taking out the test strip, lightly scraping the dehumidifying spongy cushion, and judging the result: if the detection line (T line) and the quality control line (C line) simultaneously display a purple red strip, and the color depth of the detection line is equal to or deeper than that of the quality control line, the result is negative, which indicates that the sample does not contain thiabendazole; if the color of the T line is lighter than that of the C line, or the C line is colored and the T line is not colored, the result is positive, and the sample contains probenazole; if the C line and the T line are not colored, the detection device is failed.
Example 8 sensitivity of probenazole colloidal gold detection device
A series of concentration gradients are set, the concentration of the probenazole is 1.25, 2.5, 5, 10 and 20 mu g/ml respectively, a colloidal gold test strip is used for testing, and an instrument is used for comparing the color depth of a detection line and a quality control line after testing. When the concentration of the probenazole is 5 mu g/ml, the color depth of the detection line is less than 90 percent of the quality control line, so the sensitivity is 5 mu g/ml.
And selecting 5 mu g/ml parallel experiments for 5 times, and counting the ratio of the color depth of the detection limit to the color depth of the quality control line, wherein the CV value is less than 15 percent.
Example 9 shelf life experiment of probenazole colloidal gold detection device
And (3) respectively carrying out quality guarantee period experiments on three batches of conventionally produced products, placing the products in an indoor room temperature environment for keeping, taking 12 devices at intervals of 1 month, detecting by using a quality control sample, respectively carrying out negative tests, 2.5 ug/mL, 5 ug/mL and 10 ug/mL samples, repeating the steps for three times, observing the color development change of the products, and inspecting the time of the quality guarantee period. The negative color development is reduced from 13 months, and the experimental result shows that the product quality has no obvious change within 1 year, so that the shelf life is determined to be 1 year.
The invention has been described in detail by way of general illustration and specific embodiments, but 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.
Claims (15)
1. A thiabendazole hapten having a structure represented by formula (I):
2. a thiabendazole-conjugated antigen comprising the thiabendazole hapten of claim 1, and a carrier conjugated to the thiabendazole hapten.
3. The thiabendazole-conjugated antigen of claim 2, wherein the carrier is a non-antigenic polylysine or a protein.
4. The thiabendazole-conjugated antigen of claim 3, wherein the protein is bovine serum albumin, human serum albumin, hemocyanin, or egg protein.
5. A thiabendazole antibody that is an antibody specific for the thiabendazole-coupled antigen of any one of claims 2 to 4.
6. The thiabendazole antibody of claim 5, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
7. The thiabendazole antibody of claim 5, wherein the antibody is derived from a mammal.
8. The thiabendazole antibody of claim 7, wherein the mammal is a murine, rabbit, goat, sheep, non-human primate.
9. The thiabendazole antibody of claim 8, wherein the murine is selected from the group consisting of a mouse and a rat.
10. The thiabendazole antibody of claim 5, wherein the antibody is a murine monoclonal antibody.
11. The rapid prober for probenazole colloidal gold comprises a test strip and a reaction cup, wherein the test strip comprises a sample pad and a nitrocellulose membrane, the nitrocellulose membrane comprises a detection line and a quality control line, and the detection line is prepared from probenazole coupled antigen; the reaction cup comprises a colloidal gold labeled thiabendazole antibody specifically directed to the thiabendazole-coupled antigen, wherein the thiabendazole-coupled antigen is the thiabendazole-coupled antigen of any one of claims 2 to 4, and the thiabendazole antibody is the thiabendazole antibody of any one of claims 5 to 10.
12. The probenazole colloidal gold rapid detection device according to claim 11, wherein the quality control line is prepared from a second antibody specific to the probenazole antibody.
13. The probenazole colloidal gold rapid detection apparatus according to claim 11 or 12, wherein the detection line and the quality control line are separated by at least 0.3 cm.
14. A method for detecting thiabendazole in a sample, comprising detecting using the thiabendazole colloidal gold rapid-detection device of any one of claims 11 to 13.
15. The method for detecting thiabendazole in a sample according to claim 14, wherein said sample is a food of animal origin or a food of plant origin.
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| CN110294762B (en) * | 2019-06-14 | 2020-12-15 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Artificial antigen for detecting acidosis of rice fermentum and application thereof |
| CN112921103A (en) * | 2021-03-25 | 2021-06-08 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Primer group for quantitatively analyzing bovine-derived components in meat products and application of primer group |
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| Title |
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| Determination of Thiabendazole in Fruit Juices by a New Monoclonal Enzyme Immunoassay;ANTONIO ABAD, et al.;《JOURNAL OF AOAC INTERNATIONAL》;20011231;第84卷(第1期);第156-161页 * |
| Strip-based immunoassay for rapid detection of thiabendazole;Martina Blaˇzková，et al.;《Biosensors and Bioelectronics》;20100224;第25卷;第2122-2128页 * |
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