CN108132348B - Sudan red III hapten, coupling antigen, antibody and colloidal gold rapid detection device and application thereof - Google Patents
Sudan red III hapten, coupling antigen, antibody and colloidal gold rapid detection device and application thereof Download PDFInfo
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Abstract
The invention belongs to the foodThe field of product safety detection, in particular to Sudan red III hapten which has a structural formula shown in a formula (I). The invention also relates to a Sudan red III coupling antigen, a Sudan red III antibody, a colloidal gold rapid detection device containing the Sudan red III coupling antigen and the Sudan red III antibody and application thereof. The device has reduced cross reaction with other types of Sudan red, can accurately determine the Sudan red III in food, has low cost, and can quickly and accurately carry out quick online detection and monitoring on suspected objects.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to a Sudan red III hapten, a Sudan red III coupled antigen, a Sudan red III antibody, a colloidal gold rapid detection device containing the Sudan red III coupled antigen and the Sudan red III antibody and application thereof.
Background
Sudan red is an industrial dye with azo structure, and comprises four kinds of Sudan red I, II, III and IV. Sudan red has carcinogenicity, and is a stain prohibited from being used for food in China. The four types of sudan red have great similarity, and the types of sudan red need to be distinguished in detection traceability. At present, when the antibody is used for detecting the sudan red compounds, the cross reaction is serious, and most of the sudan red compounds can not be distinguished.
In addition, the existing methods for detecting sudan red mainly comprise High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), thin layer chromatography, enzyme-linked immunosorbent assay (ELISA) and the like. 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 are complex to operate, high in cost and high in technical requirements on operators, results cannot be displayed immediately, and the method is not suitable for monitoring departments and production enterprises to perform rapid online detection and monitoring on suspected objects.
Accordingly, there is a need in the art to develop a detection system that is specific for sudan red III, has reduced cross-reactivity with other types of sudan red, and accurately characterizes sudan red III in food products. In addition, the system can realize accurate, quick and convenient detection of Sudan red III.
Disclosure of Invention
In order to overcome the defects of the existing detection method, the inventor of the invention repeatedly researches and provides a Sudan red III colloidal gold detection system. The system can be specifically directed to sudan red III, has reduced cross-reactions with other sudan red class compounds, and can accurately detect residual sudan red III in a sample. In addition, compared with the prior art or equipment, the system is more accurate, rapid and convenient.
Accordingly, in a first aspect, the present invention provides a sudan red III hapten having the structure of formula (I):
in a second aspect, the invention provides a sudan red III conjugated antigen comprising a sudan red III hapten of the first aspect of the invention, and a carrier conjugated to the sudan red III hapten.
In a third aspect, the invention provides a sudan red III antibody, which is an antibody specific for a sudan red III conjugated antigen of the second aspect of the invention.
In a fourth aspect, the invention provides a sudan red III 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 a sudan red III coupling antigen; the reaction cup contains a colloidal gold labeled sudan red III antibody specifically directed against the sudan red III conjugate antigen.
In a fifth aspect, the present invention provides a method for detecting sudan red III in a sample, the method comprising detecting using the sudan red III 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 the Sudan red III 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 the Sudan red III in a short time so as to determine whether the Sudan red III is detected. The method can meet the requirement of food safety on the detection of the residual quantity of Sudan red III, and is suitable for government detection mechanisms. In addition, the system has the advantages of low cross reaction with other compounds of Sudan red, and is favorable for accurate qualitative judgment of the Sudan red III dye.
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 Sudan red III colloidal gold rapid detection device of the present invention.
FIG. 2 shows a mass spectrum of an exemplary Sudan red III hapten used in the Sudan red III colloidal gold rapid detection device of the present invention.
Detailed Description
The above-described subject matter of the present invention is 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 described above, the present invention aims to provide a sudan red III detection system which can specifically target sudan red III, has reduced cross-reaction with other types of sudan red, and can accurately characterize sudan red III in food. In addition, compared with the prior art or equipment, the system is more accurate, rapid and convenient.
Accordingly, in one aspect, the invention provides a sudan red III 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, Sudan red III hapten, it is well understood that this is a small molecule that is immunoreactive with, but not immunogenic to, an antibody specific for Sudan red III (i.e., a Sudan red III specific antibody or a Sudan red III antibody, either monoclonal or polyclonal). In other words, in the presence of sudan red III antibodies, such sudan red III haptens may undergo an antigen-antibody binding reaction therewith; however, when the Sudan red III hapten is inoculated into an animal for immunization, the animal cannot be stimulated to produce corresponding antibodies.
Sudan red III itself, as a hapten, does not produce good immunoreactivity, and thus it needs to be modified when 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 sudan red III hapten is obtained by reacting sudan red III with bromoacetic acid.
As described above, sudan red III hapten molecules are only immunoreactive and not immunogenic. Thus, in order to render a sudan red III hapten molecule immunogenic, it is also necessary to couple, bind or crosslink the sudan red III hapten molecule with a suitable carrier molecule, thereby producing a sudan red III conjugated antigen that is both immunoreactive and immunogenic. Methods of coupling, binding or crosslinking between haptens and carrier molecules are known in the art, e.g. from the present application
The method illustrated in the examples section.
Thus, in a second aspect, the invention provides a sudan red III conjugate antigen comprising a sudan red III hapten of the first aspect of the invention, and a carrier conjugated to the sudan red III 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 invention provides a sudan red III antibody, which is an antibody specific for a sudan red III conjugated antigen of the second aspect of the invention.
The sudan red III antibody may be a monoclonal antibody or a polyclonal antibody. In addition, the sudan red III antibody can be prepared by a method conventional in the art. For example, in the case where the sudan red III antibody is a polyclonal antibody, it can be obtained by immunizing a mammal such as a mouse, rat, rabbit, goat, sheep, primate (excluding human), or the like with the sudan red III conjugate antigen, followed by isolating serum. In the case where the sudan III 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 will be understood by those skilled in the art, there is no particular limitation on the source of the sudan red III antibody, which may be derived from any mammal, including, for example, mouse, rat, rabbit, goat, sheep, primate (not including human), and the like, but is not limited thereto. In a specific embodiment, the sudan III antibody is a polyclonal or monoclonal antibody derived from mouse, rat, rabbit, goat, sheep, primate (not including human).
In a specific embodiment, the sudan red III antibody is a murine monoclonal antibody specific for a sudan red III conjugated antigen comprising a sudan red III hapten of formula (I).
In a fourth aspect, the invention relates to a sudan red III 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 sudan red III coupled antigen; the reaction cup contains a colloidal gold labeled sudan red III antibody specifically directed against the sudan red III conjugate 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 nitrocellulose membrane by Sudan red III coupled antigen. The sudan red III conjugate antigen is obtained by conjugating, cross-linking or binding a sudan red III hapten, such as the sudan red III 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 sudan red III conjugated antigen is a sudan red III conjugated antigen of the second aspect of the invention.
The invention relates to a Sudan red III colloidal gold rapid detection device which 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 is obtained by linear spotting of additional antigens or antibodies on the nitrocellulose membrane.
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 sudan red III antibody of the third aspect of the invention. When the colloidal gold-labeled sudan red III 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 described above, the reaction cup contains a colloidal gold labeled sudan red III antibody specifically directed to sudan red III conjugate antigen forming the detection line of the sudan red III colloidal gold rapid detection device.
The sudan red III antibody may be any antibody that can undergo an antigen-antibody binding reaction with the sudan red III conjugate antigen, regardless of whether it is a monoclonal antibody or a polyclonal antibody. However, as will be appreciated by those skilled in the art, the sudan III antibody is preferably a monoclonal antibody in view of the need for greater specificity.
The sudan red III antibody can be prepared by methods conventional in the art. For example, in the case where the sudan red III antibody is a polyclonal antibody, it can be obtained by immunizing a mammal such as a mouse, rat, rabbit, goat, sheep, primate (excluding human), or the like with the sudan red III conjugate antigen, followed by isolating serum. In the case where the sudan III 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 will be understood by those skilled in the art, there is no particular limitation on the source of the sudan red III antibody, which may be derived from any mammal, including, for example, mouse, rat, rabbit, goat, sheep, primate (not including human), and the like, but is not limited thereto. In a specific embodiment, the sudan III antibody is a polyclonal or monoclonal antibody derived from mouse, rat, rabbit, goat, sheep, primate (not including human).
In one embodiment, the sudan red III antibody is a sudan red III antibody of the third aspect of the invention. In a specific embodiment, the sudan red III antibody is a murine monoclonal antibody specific for a sudan red III conjugated antigen comprising a sudan red III hapten of formula (I).
The device of the invention relates to a colloidal gold-labeled sudan red III antibody prepared by coupling the sudan red III antibody of the invention with colloidal gold. The coupling method between the sudan red III antibody and colloidal gold may be performed using related coupling methods known in the art.
In a specific embodiment, the invention provides a sudan red III 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, which are sequentially laid on the bottom plate, the nitrocellulose membrane comprises a detection line and a quality control line in the direction from the sample pad to the absorbent paper, and the detection line is prepared from a sudan red III coupling antigen containing a sudan red III hapten of a formula (I); the reaction cup contains a colloidal gold labeled sudan red III antibody, which is a murine monoclonal antibody specific to the sudan red III conjugate antigen containing the sudan red III hapten of formula (I); and the control line is a rabbit anti-mouse polyclonal antibody directed against the sudan red III antibody.
The Sudan red III 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 Sudan red III coupling 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 sudan red III antibody of the present invention was added to the reaction cup and lyophilized. The ingredients or components used in the preparation method are as described above for the Sudan Red III colloidal gold assay device of the present invention.
In a fifth aspect, the present invention relates to a method for detecting sudan red III in a sample, the method comprising detecting using the sudan red III colloidal gold rapid detection device of the fourth aspect of the present invention.
The sample may be any sample suspected of containing sudan red III, which may be, for example, paprika, chili oil, paprika, zanthoxylum oil, red bean curd, poultry eggs, and products thereof.
Before the Sudan red III detection method is adopted for detection, the sample can be correspondingly pretreated according to the specific source of the sample. For example, in the case of capsicum, the capsicum can be first crushed into powder, and then the sudan red III contained in the capsicum can be extracted, and then the sudan red III detection can be carried out.
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 Sudan red III; (2) if the detection line does not develop color, or the color is lighter than the quality control line, it indicates that the sample contains Sudan red III.
The invention has the beneficial effects that: the invention applies the principle of chromatographic immune colloidal gold, semi-quantitatively detects the residual quantity of the Sudan red III 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 the Sudan red III in a short time so as to determine whether the Sudan red III is detected. The method can meet the requirement of food safety on the detection of the residual quantity of Sudan red III, and is suitable for government detection mechanisms. In addition, the system has the advantages of low cross reaction with other compounds of Sudan red, and is favorable for accurate qualitative judgment of the Sudan red III dye.
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 Sudan Red III hapten
In a 100mL three-necked flask were sequentially added 3.53g of Sudan red III, 1.52g of bromoacetic acid, and 0.1g of sodium carbonate, and reacted in acetonitrile solution at 90 ℃ overnight. And after rotary evaporation is finished, extracting by using ethyl acetate, and purifying by using a column to obtain the Sudan red III hapten.
FIG. 2 shows the mass spectrum of the Sudan red III hapten thus obtained, and it can be understood that the hapten has the following formula:
example 2 Synthesis of Sudan Red III conjugated antigens
An immunizing antigen was prepared using the sudan red III hapten obtained in example 1. 0.1mmol of Sudan red III hapten is dissolved in 2mL of DMF and 27.5mg of Dicyclohexylcarbodiimide (DCC) and 14.4mg of N-hydroxysuccinimide (NHS) are added with stirring. The reaction was magnetically stirred overnight at 4 ℃ and the supernatant was centrifuged to obtain solution A, and then, 140mg of Bovine Serum Albumin (BSA) and hemocyanin (KLH) were weighed and dissolved in 10mL of 0.1mol/L PBS (pH 8.0). Adding 1mL of Dimethylformamide (DMF), stirring to dissolve and prepare solution B, gradually dripping the solution A into the solution B under magnetic stirring, and reacting for 12 hours at 4 ℃. After centrifugation, the supernatant was dialyzed with physiological saline at 4 ℃ for 3 days, and the dialysate was changed 3 times a day, whereby Sudan red III conjugated antigens conjugated with bovine serum albumin and hemocyanin, respectively, were obtained. The resulting Sudan red III conjugated antigen was dispensed into 0.5mL centrifuge tubes at a concentration of 1 mg/mL. Freezing in a refrigerator at-20 deg.C.
EXAMPLE 3 preparation of Sudan Red III monoclonal antibody
The sudan red III monoclonal antibody was prepared using the sudan red III conjugate antigen prepared in example 2 as follows: 4 Kunming mice with 6 weeks old are immunized by using the identified Sudan red III coupling antigen, and blood is collected for measuring the potency after three times of boosting immunization. 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 50mL centrifuge tube according to the proportion of 8:1, adding 30mL serum-free IPMI 1640 culture medium, centrifuging at 1100r/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 1mL of 50% PEG-4000 into the cells, dripping within 1 minute, gently stirring the bottom precipitate, standing for 1 minute, slowly adding 1mL of serum-free culture medium along the tube wall in the first 30 seconds at a constant speed, adding 2mL in the second 30 seconds, rapidly adding 27mL to terminate the fusion process, centrifuging at 1100r/min for 5 minutes, discarding the supernatant, resuspending with HAT selective culture medium, adding into a 96-well cell culture plate paved with feeder cells, and adding 5% volume fraction 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 anti-Sudan red III monoclonal antibody by inducing ascites in vivo is as follows. 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. Purifying ascites by using an n-octanoic acid-ammonium sulfate precipitation method, and measuring the content of the anti-Sudan red III monoclonal antibody by ultraviolet.
Example 4 preparation of Rabbit anti-mouse antibodies
New Zealand white rabbits were immunized with the Sudan red III monoclonal antibody prepared in example 3 at an immunization dose of 50. mu.g-100. mu.g/time and with multiple subcutaneous dorsal spotsInjecting; first-time immunization, emulsifying the Sudan red III monoclonal antibody with equivalent Freund complete adjuvant; enhancing immunity, emulsifying with Sudan red III monoclonal antibody and Freund's incomplete adjuvant, continuously immunizing for 4-5 times, each time at 4-8 weeks interval, 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, 1.6ml 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 the solution was uniformly stirred with a constant speed stirrer while dropwise adding the Sudan red III monoclonal antibody prepared in example 3, polyethylene glycol (PEG) in an amount equivalent to that of the antibody was added after 1 hour, and Bovine Serum Albumin (BSA) in an amount equivalent to that of 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 9000rpm 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 Sudan Red III colloidal gold assay device
Step (I): preparing a nitrocellulose membrane, and linearly spotting the sudan red III conjugate 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) and the reaction cup obtained in the step (III) to form the Sudan red III colloidal gold detection device.
Example 7 detection of Sudan Red III in samples
Taking 5g of a chilli powder sample, extracting Sudan red III with dimethyl sulfoxide (DMSO), carrying out back extraction by using normal hexane, drying, adding a complex solution, sampling, placing in a reaction cup, and incubating for 5 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 humidity 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 Sudan red III; 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 Sudan red III is contained in the sample; if the C line and the T line are not colored, the detection device is failed.
EXAMPLE 8 sensitivity of Sudan Red III colloidal gold assay device
A series of concentration gradients are set, the concentration of Sudan red III 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 Sudan red III is 5 mug/mL, the color depth of the detection line is less than 90% of the quality control line, so the sensitivity is 5 mug/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 specificity test of Sudan Red III colloidal gold assay device
In a negative pepper powder sample of 5g, 15ppb of Sudan Red III, 150ppb of Sudan Red I, 300ppb of Sudan Red II, and 300ppb of Sudan Red IV were added, respectively. The experimental result shows that Sudan red III can be detected, but the samples added with Sudan red I, II and IV cannot be detected.
The above results show that the device of the present invention has better specificity to sudan red III and less cross contamination to different types of sudan red compounds.
Example 10 shelf life test of Sudan Red III colloidal gold test device
Three batches of products produced conventionally are respectively used for carrying out quality guarantee period experiments, the products are placed in an indoor room temperature environment to be kept, 12 devices are taken at intervals of 1 month, negative samples of 2.5 mu g/mL, 5 mu g/mL and 10 mu g/mL are respectively carried out by quality control sample detection, the steps are repeated three times, the color development change of the products is observed, and the quality guarantee period time is inspected. 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 (10)
1. A rapid detection device for Sudan red III 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 Sudan red III coupling antigen; the reaction cup contains a colloidal gold labeled sudan red III antibody prepared from the sudan red III conjugate antigen as an immunogen and specifically directed against the sudan red III conjugate antigen, wherein the sudan red III conjugate antigen contains a sudan red III hapten having a structure represented by the following formula (I), and the proteins bovine serum albumin, human serum albumin, hemocyanin or ovalbumin conjugated with the sudan red III hapten:
2. the sudan red III colloidal gold rapid detection device according to claim 1, wherein the sudan red III antibody is a monoclonal antibody or a polyclonal antibody.
3. The sudan red III colloidal gold rapid detection device according to claim 1 or 2, wherein the sudan red III antibody is derived from a mammal.
4. The sudan red III colloidal gold rapid test device according to claim 3, wherein said mammal is a murine, rabbit, goat, sheep, non-human primate.
5. The sudan red III colloidal gold rapid test device according to claim 4, wherein the mouse is a mouse or a rat.
6. The sudan red III colloidal gold rapid detection device according to claim 1 or 2, wherein the sudan red III antibody is a murine monoclonal antibody.
7. The sudan red III colloidal gold rapid detection device according to claim 1 or 2, wherein the quality control line is prepared from a second antibody specific to the sudan red III antibody.
8. The sudan red III colloidal gold rapid detection device according to claim 1 or 2, wherein the detection line and the quality control line are at least 0.3 cm apart.
9. A method for detecting sudan red III in a sample, the method comprising detecting using the sudan red III colloidal gold rapid detection device of any one of claims 1 to 8.
10. The method of detecting sudan III in a sample according to claim 9 wherein said sample is paprika, chili oil, paprika, zanthoxylum oil, red bean curd, poultry eggs, and products thereof.
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| CN101261271B (en) * | 2008-04-17 | 2012-03-14 | 武汉工业学院 | Sudan red 1 immunity-chromatography test paper detection method |
| CN101839907A (en) * | 2010-04-09 | 2010-09-22 | 中国农业大学 | Enzyme-liked immunosorbent assay method for sudan red I |
| CN103901193A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Tony red immunodetection test paper and preparation method thereof |
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