CN114716535A - Synthetic method of patulin artificial antigen and preparation and application of monoclonal antibody thereof - Google Patents
Synthetic method of patulin artificial antigen and preparation and application of monoclonal antibody thereof Download PDFInfo
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Abstract
The invention discloses a synthetic method of an artificial antigen of patulin, and preparation and application of a monoclonal antibody of the artificial antigen, and particularly relates to the technical field of biology. The method comprises the steps of preparing a patulin hapten, dissolving patulin in DMF, adding KOH, dissolving, adding trimercaptopropionic acid and NaI, heating for reaction, drying and purifying to obtain the patulin hapten; preparing a patulin artificial antigen, namely dissolving NHS and EDC in ultrapure water to prepare an ultrapure water solution, slowly dripping the ultrapure water solution into a DMF (dimethyl formamide) solution of a patulin hapten, and activating to obtain a patulin hapten activating solution; coupling the patulin hapten activating solution with the macromolecular protein conjugate, dialyzing and packaging to obtain the patulin artificial antigen. The method for detecting the content of the patulin by the immunoassay has the characteristics of rapidness, sensitivity, accuracy, simple and convenient operation and the like, has low requirement on the purity of a sample, and is particularly suitable for detecting a large quantity of samples.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a synthetic method of an artificial antigen of patulin, and preparation and application of a monoclonal antibody of the artificial antigen.
Background
Patulin, English name (patulin), molecular formula C7H6O4149-29-1, having the following structure:
many penicillins are capable of producing patulin, and the fungi capable of producing patulin include 16 species in total, namely penicillium expansum, penicillium clavuligerum, penicillium soil, penicillium new zealand, penicillium stone, penicillium granular, penicillium mellin, penicillium arc, penicillium chrysogenum, penicillium betulinum, aspergillus clavatus, aspergillus megaterium, aspergillus terreus and epilamellium niveum, and the patulin mainly pollutes fruits and products thereof, especially apples, hawthorns, pears, tomatoes, apple juice, hawthorn slices and the like.
Patulin has strong toxic effect on human and animals, and has respiratory harm effect. Patulin taken into the body causes abnormal membrane substance movement by the permeability change of the cell membrane, thereby indirectly causing physiological respiratory abnormality.
The countries where standards for patulin are established are increasing, but the limiting levels are essentially concentrated at 50. mu.g/kg. The standard of the national food safety standard for mycotoxin (GB 2761-2017) in China stipulates that the limit standard of patulin in apple and hawthorn products is 50 mu g/kg.
Currently, methods for detecting patulin mainly include Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Mass Spectrometry (MS), and High performance Liquid chromatography-Mass spectrometry (HPLC-MS). Although these methods have high accuracy, the instruments and equipment are expensive, the sample pretreatment is complicated and tedious, the detection cost is high, and the operation of professional personnel is required.
With the development of radioimmunoassay, enzyme-labeled immunoassay, carrier pharmacology and targeting, methods and means of protein cross-linking technology are continuously improved and perfected, and are increasingly widely applied in the fields of biology and medicine.
Protein crosslinking refers to the covalent linkage of small molecular substances (such as drugs, haptens, etc.) or large molecular substances (such as enzymes, protein toxins, etc.) to protein molecules to prepare artificial antigens, enzyme-labeled antibodies, carrier-released drugs, antibody-directed drugs, immunotoxins, etc. Since Landsteiner synthesized artificial antigen for the first time 70 years ago, many small molecule substances (haptens) without antigenicity (chemical drugs, neurotransmitters, hormones, etc.) were covalently bonded to carrier macromolecules such as proteins or polysaccharides; make it antigenic, induce animal to produce specific antibody, and can be used for radioimmunoassay, etc. In order to meet the requirements of high sensitivity and strong specificity of radioimmunoassay, a great deal of research is carried out on the method for connecting hapten and protein by predecessors, and crosslinking technologies such as a diazotization method, a glutaraldehyde method, a mixed anhydride method, a diisocyanate method, a halogenated nitrobenzene method and the like are established.
Disclosure of Invention
Therefore, the invention provides a synthetic method of an artificial antigen of patulin and preparation and application of a monoclonal antibody thereof, and aims to solve the problems of expensive equipment, complex sample pretreatment, high detection cost and the like of the conventional patulin detection.
The molecular weight of patulin is 154, and no group capable of being coupled with protein exists, but amino or carboxyl re-coupled protein can be derived to prepare monoclonal antibody.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to a first aspect of the present invention, there is provided a method for synthesizing an artificial antigen of patulin, comprising:
step one, preparation of patulin hapten
Dissolving patulin in DMF, adding KOH, dissolving, adding trimercaptopropionic acid and NaI, heating for reaction, drying, and purifying to obtain patulin hapten;
step two, preparation of patulin artificial antigen
Firstly, dissolving NHS and EDC in ultrapure water to prepare an ultrapure water solution, slowly dripping the ultrapure water solution into a DMF (dimethyl formamide) solution of the patulin hapten, and activating to obtain a patulin hapten activating solution: coupling the patulin hapten activating solution with the macromolecular protein conjugate, dialyzing and packaging to obtain the patulin artificial antigen.
The invention discovers that the existing methods for preparing the artificial antigen of patulin adopt anhydride to react, but the anhydride is difficult to react even does not react at 70 ℃, the trimercaptopropionic acid has stable property, can better react with the patulin at 120 ℃, and byproducts do not exist, and the treatment is convenient.
After the preparation of the patulin artificial antigen activation reaction is finished, an LCMS instrument detects and processes the patulin by drying DMF, adding 5ml of water, adjusting the pH value to 4-5 by 1mol of HCL, extracting by ethyl acetate until no product exists in a water phase, drying, purifying by normal phase Flash, (DCM: MEOH =10: 1), collecting the product, and detecting by LCMS.
Further, in the first step, the conditions for dissolving the patulin in the DMF are the conditions of keeping away from light at 4 ℃;
and/or the dissolving mode is ultrasonic dissolving;
and/or the heating reaction condition is 120 ℃, and the reaction lasts for 8-12 h;
and/or, in the second step, the macromolecular protein conjugate may be any one of macromolecular protein conjugates for coupling patulin, for example, the macromolecular protein conjugate is BSA, KLH or OVA, but is not limited to any one of the three.
Further, in the first step, the structure of the patulin hapten is
Further, in the first step and the second step, the synthetic route of the patulin artificial antigen is
According to a second aspect of the present invention, there is provided an artificial antigen of patulin, wherein the antigen is prepared by the method as described above.
According to a third aspect of the present invention, there is provided a monoclonal antibody prepared using the artificial antigen for patulin as described above.
The preparation method of the monoclonal antibody comprises the steps of immunizing animals by the patulin artificial antigen, fusing and cloning immune animal cells and myeloma cells, and preparing and purifying the monoclonal antibody.
Animal immunization with patulin artificial antigen: dissolving immunogen and Freund's complete adjuvant with physiological saline and mixing in equal volume by using prepared patulin artificial antigen according to 100 mu g/mouse, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at neck and back, mixing immunogen and Freund's incomplete adjuvant in equal volume in 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, performing 100 mu g/mouse of immune complex in 3 days before fusion, and performing additional immunization once without Freund's adjuvant;
fusion and cloning of immune animal cells and myeloma cells: mixing splenocytes of immunized mice with myeloma cells (SP 2/0) of mice in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO2Culturing in an incubator, half replacing the culture medium with HT culture medium after 5 days, and completely replacing the culture medium after 9 days;
after the cells are fused, when the cells grow to 1/4 of the area of the culture hole, screening the hybridoma cells by adopting a step screening method; primarily selecting an indirect ELISA method, coating an ELISA plate by coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), adding culture supernatant of a detected hole, incubating, washing, adding goat anti-mouse IgG-HRP and IgM-HRP, and OPD for color reaction; screening the screened positive holes by using an indirect competitive ELISA method, mixing cell supernatant with 100 mu g/ml patulin in the same volume, carrying out water bath at 37 ℃ for 30min, and adding the mixture into a coated ELISA plate; meanwhile, PBS is used for replacing patulin for comparison, and the rest steps are the same as the above; OD after blocking with patulin450nmIf the value is reduced to below 50% of the control hole, the control hole is judged to be positive, the control hole is detected to be positive for 2-3 times, and subcloning is immediately carried out by a limiting dilution method;
preparation and purification of monoclonal antibody: carrying out amplification culture on the hybridoma after 2-3 times of subclone strain establishment, collecting supernate, measuring the titer by using indirect ELISA, and freezing and storing; injecting 0.5 ml/mouse of liquid paraffin into the abdominal cavity of Balb/c mouse of 8-10 weeks old, injecting hybridoma cells 1-2 × 10 into the abdominal cavity after 7-10 days5Extracting ascites from the mice 7 to 10 days later; collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 the maximum dilution multiple of the cell supernatant or the ascites), the result shows that the titer of the cell supernatant is 1:10000, and the titer of the ascites is 1: 50000; then, the mixture is purified by an octanoic acid-saturated ammonium sulfate method and is stored in an environment with the temperature of 20 ℃ below zero.
The monoclonal antibody provided according to the fourth aspect of the invention may be used in any of the following applications:
a1) the application in detecting patulin;
a2) the application in the preparation of a reagent for detecting patulin;
a3) the application of the test strip in preparing colloidal gold test strips for detecting patulin.
According to a fifth aspect of the present invention, there is provided a kit for detecting patulin, which comprises an artificial antigen using patulin as described above and a monoclonal antibody as described above.
The method of the kit for detecting patulin comprises the following steps:
step one, labeling the antibody with colloidal gold
Preparing a colloidal gold solution, adjusting the pH value of the colloidal gold solution, and dropwise adding the monoclonal antibody solution prepared by the method while stirring; after the addition, dropwise adding BSA aqueous solution, and continuously stirring to obtain a gold-labeled antibody solution; centrifuging the gold-labeled antibody solution, taking the red supernatant, centrifuging at low temperature again, taking the mobile dark red precipitate, adding a phosphate buffer solution containing BSA (bovine serum albumin) to suspend the mobile dark red precipitate to the volume of the original gold-labeled antibody solution, standing, centrifuging at low temperature, and collecting the precipitate; adding BSA and NaN into the precipitate3Suspending the phosphate buffer until the volume of the original gold-labeled antibody solution is 1/40, thus obtaining the colloidal gold-labeled antibody, and storing at low temperature;
step two, spraying gold
Spraying the colloidal gold labeled antibody on a glass fiber membrane to prepare a colloidal gold pad;
step three, spraying the film
Spraying the patulin artificial antigen prepared by the method and spraying the goat anti-mouse antibody on the position of the line C on a T line position on a reaction membrane;
step four, assembling
Assembling a sample absorption pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad according to a conventional method, and cutting to obtain the detection test strip of the patulin;
the test paper strip can also be put into a plastic card to form a test paper card for detection;
step five, assembling the patulin detection kit
And (3) placing the test strip for detecting the patulin into a sealing bag containing a drying agent, sealing, putting 1 part of the specification into the kit together, and plastically packaging to obtain the patulin-containing detection kit.
Further, in the first step, the pH value of the colloidal gold solution is adjusted to 8.2;
and/or the gold-labeled antibody solution is centrifuged at low speed for 20min at the temperature of 20-24 ℃;
and/or the low-temperature centrifugation condition is centrifugation for 40min at 11000r/min at 4 ℃; the red supernatant was again centrifuged at low temperature and divided into three layers: transparent supernatant, flowing dark red sediment at the bottom of the tube and a black compact gold particle layer on the bottom wall of the tube;
and/or, the phosphate buffer solution containing BSA is 0.01mol/L phosphate buffer solution containing 1g/100ml BSA;
and/or, the BSA and NaN contained3The phosphate buffer of (A) is a buffer containing 1g/100ml BSA and 0.02g/100ml NaN30.01mol/L phosphate buffer solution;
and/or the low-temperature storage is 2-8 ℃.
According to the method for detecting the patulin by using the kit for detecting the patulin provided by the sixth aspect of the invention, the sample to be detected is treated, the treated sample solution to be detected is absorbed and dropwise added into a sample absorption pad by 3-5 drops, and the detection result is observed.
The invention has the following advantages:
the invention adopts trimercaptopropionic acid to prepare the artificial antigen of the patulin, the trimercaptopropionic acid has stable property, can better react with the patulin at the high temperature of 120 ℃, and has no by-product and convenient treatment.
The monoclonal antibody is further prepared by utilizing the prepared artificial antigen of the patulin, has the characteristics of strong specificity, high sensitivity and the like, is used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay, and is used for detecting the content of the patulin by an immunoassay method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary and that other implementation drawings may be derived from the provided drawings by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a liquid chromatogram of a patulin hapten LCMS provided in example 1 of the present invention;
FIG. 2 is a diagram showing the OD value detection of one of the artificial antigens of patulin provided in example 1 of the present invention;
FIG. 3 is a schematic diagram of a colloidal gold assay according to an exemplary embodiment of the present invention;
FIG. 4 is a diagram showing the detection of colloidal gold of patulin in the test example of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide
NHS: n-hydroxysuccinimide
DMF: n, N-dimethylformamide
BSA: bovine serum albumin
KLH: keyhole limpet hemocyanin
OVA: egg white albumin of chicken
DCM: methylene dichloride
MEOH: methanol
OPD: o-phenylenediamine
Example 1
The embodiment provides a synthetic method of an artificial antigen of patulin, which comprises the following steps:
step one, preparation of patulin hapten
The path is as follows:
the method comprises the following specific steps:
accurately weighing patulin 10mg (purchased from sigma), dissolving in ultra-dry DMF, stirring at 4 ℃ for 5min in the dark, accurately weighing KOH 8mg, adding into the DMF solution of patulin, stirring for 30min by ultrasound, then weighing trimercaptopropionic acid 16mg and NaI 3mg, adding into the above solution, and reacting at 120 ℃ overnight.
After the reaction is finished, the detection and treatment are carried out by an LCMS instrument, firstly, the patulin DMF is dried, 5ml of water is added, 1mol of HCl is added for adjusting the pH value to 4-5, ethyl acetate is extracted until no product exists in the water phase, the product is dried and purified by normal phase Flash, (DCM: MEOH =10: 1), the product is collected and is detected by an LCMS, the liquid quality result is shown in figure 1, and the liquid quality diagram shows that: the patulin hapten has an absorption peak of 254-214, the purity is 96 percent, and the molecular weight of the hapten is 259 (M + 1).
Step two, preparation of patulin artificial antigen
The path is as follows:
specifically, the method comprises the following steps: weighing 2mg of patulin hapten, dissolving the patulin hapten into 2ml of DMF, and stirring until the patulin hapten is dissolved to obtain a DMF solution of the patulin hapten; accurately weighing NHS 4mg and EDC 6mg, dissolving into 2ml of ultrapure water, slowly adding into DMF solution of patulin hapten after dissolving, and activating overnight to obtain the patulin hapten activating solution.
After activation, removing 1ml of patulin hapten activating solution, adding 6mg of BSA, stirring at room temperature for 4h, dialyzing, subpackaging, and storing at-20 ℃ to obtain patulin-BSA artificial antigen;
removing 2ml of patulin hapten activating solution, adding 3mg of KLH, stirring at room temperature for 4h, dialyzing, subpackaging, and storing at-20 ℃ to obtain patulin-KLH artificial antigen;
removing 1ml of patulin hapten activating solution, adding 3mg of OVA, stirring at room temperature for 4h, dialyzing, subpackaging, and storing at-20 ℃ to obtain the patulin-OVA artificial antigen.
The coupling graph of the patulin-BSA artificial antigen, the patulin-KLH artificial antigen and the patulin-OVA artificial antigen is shown in figure 2, and the coupling is successful as can be seen from the graph.
Example 2
This example provides a method for preparing monoclonal antibodies using the patulin artificial antigen (patulin-BSA artificial antigen) prepared in example 1:
step one, animals are immunized by patulin artificial antigen
Dissolving immunogen and Freund's complete adjuvant with physiological saline solution according to 100 mu g/mouse by using the prepared patulin-BSA artificial antigen, mixing the dissolved immunogen and the Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the neck and the back, mixing the immunogen and the Freund's incomplete adjuvant in equal volume on the 7 th, 14 th and 28 th days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g/mouse of immune complex 3 days before fusion and without adding Freund's adjuvant;
step two, fusing and cloning immune animal cells and myeloma cells
Mixing splenocytes of immunized mice with mouse myeloma cells (SP 2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45s for fusion, uniformly suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO2Culturing in an incubator, half-replacing the culture solution with HT medium after 5 days,performing full liquid change at 9 days;
after the cells are fused, when the cells grow to 1/4 of the area of the culture hole, screening the hybridoma cells by adopting a step screening method; primarily selecting an indirect ELISA method, coating an ELISA plate by coating antigen (the optimal coating concentration and positive serum dilution are titrated conventionally by a square matrix method in advance), adding culture supernatant of a hole to be detected, incubating, washing, adding goat anti-mouse IgG-HRP and IgM-HRP, OPD for color reaction; screening the screened positive holes by using an indirect competitive ELISA method, mixing cell supernatant with 100 mu g/ml patulin in the same volume, carrying out water bath at 37 ℃ for 30min, and adding the mixture into a coated ELISA plate; meanwhile, PBS is used for replacing patulin for comparison, and the rest steps are the same as the above; OD after blocking with patulin450nmIf the value is reduced to below 50% of the control hole, the control hole is judged to be positive, the control hole is detected to be positive for 2-3 times, and subcloning is immediately carried out by a limiting dilution method;
step three, preparation and purification of monoclonal antibody
Performing expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernate, measuring titer by indirect ELISA, and freezing; injecting 0.5 ml/mouse of liquid paraffin into the abdominal cavity of Balb/c mouse of 8-10 weeks old, injecting hybridoma cells 1-2 × 10 into the abdominal cavity after 7-10 days5Extracting ascites from the mice 7 to 10 days later; collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 the maximum dilution multiple of the cell supernatant or the ascites), the result shows that the titer of the cell supernatant is 1:10000, and the titer of the ascites is 1: 50000; then, purifying the monoclonal antibody by an octanoic acid-saturated ammonium sulfate method, purifying and storing the purified monoclonal antibody in an environment at the temperature of 20 ℃ below zero to obtain the monoclonal antibody.
Example 3
This example provides a method for preparing a colloidal gold assay kit using the artificial antigen for patulin prepared in example 1 and the monoclonal antibody prepared in example 2:
step one, labeling the antibody with colloidal gold
(1) Preparation of colloidal gold solution
Heating 100ml of 0.01% chloroauric acid aqueous solution to boiling with a constant temperature electromagnetic stirrer, adding 2.5ml of 1% trisodium citrate aqueous solution under the condition of continuous stirring, continuously stirring and heating for 20min to obtain a transparent red solution, cooling at room temperature, recovering the volume to the original volume (100 ml) with deionized water, and storing at 2-8 ℃.
(2) Preparation of gold-labeled antibody solution
With 0.1mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.2 by using the aqueous solution, then adding 10ml of the colloidal gold solution into a 50ml beaker, stirring the colloidal gold solution by using an electromagnetic stirrer at 250r/min, dropwise adding the monoclonal antibody solution, dropwise adding 3ml of 5g/100 ml of BSA aqueous solution, and continuously stirring the mixture for 10 min.
(3) Centrifuging the gold-labeled antibody solution at low speed (1500 r/min) at 20-24 deg.C for 20min, discarding precipitate formed by coagulated gold particles, and collecting red supernatant solution.
(4) And (3) centrifuging the red supernatant solution at 4 ℃ and 11000r/min for 40min, dividing the solution into three layers (transparent supernatant, a mobile dark red precipitate at the bottom of a tube and a black compact gold particle layer on the bottom wall of the tube), transferring the mobile dark red precipitate into another centrifuge tube, suspending the mobile dark red precipitate to the volume (100 ml) of the original gold-labeled antibody solution by using 0.01mol/L phosphate buffer solution containing 1g/100ml BSA, centrifuging at 4 ℃ and 11000r/min for 40min after overnight, and collecting the precipitate.
(5) Using a mixture of 1g/100ml BSA and 0.02g/100ml NaN3Suspending the precipitate obtained in the step (4) to 1/40 (100 ml) of the original gold-labeled antibody solution by using 0.01mol/L phosphate buffer solution, and storing at 2-8 ℃ to obtain the colloidal gold-labeled antibody.
Step two, spraying gold
And (4) spraying the colloidal gold labeled antibody obtained in the step one onto a glass fiber membrane to prepare a colloidal gold pad.
Step three, spraying the film
The artificial antigen of patulin-BSA prepared in example 1 is sprayed on the position of the T line on the reaction membrane, and the goat anti-mouse antibody is sprayed on the position of the C line.
Step four, assembling
Assembling a sample absorption pad (cellulose filter membrane), a colloidal gold pad, a nitrocellulose membrane and a water absorption pad according to a conventional method, and then cutting into strips to obtain the detection product; the test strip can also be put into a plastic card to form a test card for detection.
Step five, assembling the patulin detection kit
And (3) placing the test strip for detecting the patulin into a sealing bag containing a drying agent, sealing, putting 1 part of the specification into the kit together, and plastically packaging to obtain the colloidal gold detection kit.
Test examples
In this test example, the colloidal gold assay kit prepared in example 3 was used to detect the content of patulin in apple and hawthorn products.
An HPLC detection method is used as a comparative example, the specific detection method is a method for detecting patulin in hawthorn and apple products with the patent number of CN 201711209603.2;
20 apple and hawthorn samples were tested and numbered, and the results are shown in table 1.
The specific method for detecting the colloidal gold detection kit comprises the following steps:
1. the sample pretreatment method comprises the following steps: the collected liquid samples of the apple and hawthorn products can be directly detected, and the samples to be detected are uniformly mixed and then detected; the collected solid samples of apple and hawthorn products need to be homogenized or ground to obtain homogenized or ground tissues, the homogenized or ground tissues are centrifuged, and supernate is taken for detection.
2. Taking out the test paper card, unsealing, horizontally placing on a table, sucking the sample solution to be tested, and dropwise adding 3-5 drops of the sample solution into the sample hole; judging result in 5-10min, and judging result after 15min is invalid.
And (4) judging the standard of the result:
negative: c line is developed, the T line can be seen by naked eyes, and the color of the T line is darker than that of the C line, so that the result is judged to be negative;
positive: c line is colored, when T line is not colored or the color of T line is equal to or lighter than C line, the T line is judged to be positive, and the lighter the T line is, the stronger the positive is;
and (4) invalidation: the C line did not develop color, and the test strip was judged to be ineffective regardless of whether the T line developed color or not. The schematic diagram is shown in fig. 3.
TABLE 1
The test result is shown in fig. 4 as the test paper for detecting patulin colloidal gold. As can be seen from the figure, the test strip for detecting the patulin colloidal gold meets the limited requirement of the patulin specified by the national standard, and can be applied to the rapid detection of the patulin in clinical samples. The colloidal gold test strip is high in sensitivity, and the detection concentration of the colloidal gold test strip can be adjusted according to different concentration limits.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (9)
1. A synthetic method of an artificial antigen of patulin is characterized by comprising the following steps:
step one, preparation of patulin hapten
Dissolving patulin in DMF, adding KOH, dissolving, adding trimercaptopropionic acid and NaI, heating for reaction, drying, and purifying to obtain patulin hapten;
step two, preparation of patulin artificial antigen
Firstly, dissolving NHS and EDC in ultrapure water to prepare an ultrapure water solution, slowly dripping the ultrapure water solution into a DMF (dimethyl formamide) solution of the patulin hapten, and activating to obtain a patulin hapten activating solution; coupling the patulin hapten activating solution with the macromolecular protein conjugate, dialyzing and packaging to obtain the patulin artificial antigen.
2. The method for synthesizing the artificial antigen of patulin of claim 1, wherein in the first step, the conditions for dissolving patulin in DMF are conditions of 4 ℃ and protection from light;
and/or the dissolving mode is ultrasonic dissolving;
and/or the heating reaction condition is 120 ℃, and the reaction lasts for 8-12 h;
and/or, in the second step, the macromolecular protein conjugate is BSA, KLH or OVA.
3. The method for synthesizing the artificial antigen of patulin as claimed in claim 1, wherein in the first step, the structure of the hapten of patulin is
4. The method for synthesizing the artificial antigen of patulin of claim 1, wherein the synthetic route of the artificial antigen of patulin is as follows
5. An artificial antigen of patulin, characterized in that it is prepared by the method of any one of claims 1 to 4.
6. A monoclonal antibody prepared by using the artificial antigen of patulin of claim 5.
7. The monoclonal antibody of claim 6 for any one of the following uses:
a1) the application in detecting patulin;
a2) the application in the preparation of a reagent for detecting patulin;
a3) the application in the preparation of the test strip for detecting the colloidal gold of the patulin.
8. A kit for detecting patulin, comprising the patulin artificial antigen of claim 5 and the monoclonal antibody of claim 6.
9. The method for detecting patulin by using the kit for detecting patulin of claim 8, wherein a sample to be detected is treated, the treated sample solution to be detected is absorbed and dropwise added onto a sample absorption pad by 3 to 5 drops, and the detection result is observed.
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