Patent Application
20250012789
The disclosure belongs to the technical field of food safety testing, and relates to a magnetic chemiluminescence immunoassay kit based on a bifunctional fusion protein for mycotoxins, and a use thereof.
Mycotoxins, secondary metabolites produced by toxigenic fungi under certain environmental conditions, have widely polluted plant-based products such as agricultural products, food, traditional Chinese medicine material and feed. Deoxynivalenol (DON) (which is also known as vomitoxin), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEN), T-2 toxin, HT-2 toxin or aflatoxin M1 (AFM1), patulin, etc. are common mycotoxins in the agricultural products, the food and the feed. The mycotoxins can cause acute or chronic intoxication in humans and animals, which can damage livers, kidneys, nerve tissues, hematopoietic tissues and skin tissues of bodies, thereby producing serious effects on human and animal health. Owning to the harm of the mycotoxins, countries around the world have imposed strict limits on their content. It is of great significance to conduct research on mycotoxin residue pollution and develop highly-sensitive, low-cost and reliable automatic test technologies in ensuring the quality and safety of the agricultural products, the food, the traditional Chinese medicine and the feed, breaking down foreign technological barriers, protecting China's economic interests in international trade, and increasing export earnings.
At present, thin-layer chromatography, high-performance liquid chromatography, enzyme-linked immunosorbent assay, capillary electrophoresis, and liquid chromatography mass spectrometry serve to test the mycotoxins. The thin-layer chromatography serves to test the mycotoxins at the earliest, and has the advantages of suitability for operation of personnel without being specially trained, low cost and no need for expensive instruments. However, the thin-layer chromatography has tedious sample treatment, a complex experimental process, a long required test cycle, and easy interference from impurities. The use of visual semi-quantitative measurement in the test process has the disadvantages of significant subjective influence and low sensitivity, which can no longer satisfy modern test requirements. The enzyme-linked immunosorbent assay has the advantages of excellent specificity test, high sensitivity and low test cost, and is suitable for screening and surveying a large number of samples in a grassroots institution, which can greatly save time and cost. However, the enzyme-linked immunosorbent assay mainly causes false positives easily. Consequently, it mainly serves to screen and test the samples at a grassroots level. Instrument analysis methods such as the high-performance liquid chromatography, the capillary electrophoresis and the liquid chromatography mass spectrometry have the advantages of high accuracy, high sensitivity and micro determination, making them common in test of toxins in the food. However, with their high requirements for sample purity and the need for some pretreatment processes, the instrument analysis methods have high test cost and a long cycle, resulting in an incapability of satisfying the requirements of quickly screening a large quantities of samples. Magnetic particles have superparamagnetic microparticle properties and magnetic particle field responsiveness in a magnetic particle field. The magnetic particles are used as a solid-phase carrier for immune detection, which will greatly increase a surface area of reaction, and make it easier to separate a solid phase from a liquid phase, thereby improving sensitivity of test. Small magnetic particles are used as the solid phase, which can increase a surface area of a coating, thereby increasing adsorption capacity of antigens or antibodies, which not only increases a reaction speed, but also makes cleaning and separation easier. At present, a method for preparation an immune enzyme-labeled reagent couples antibodies with enzymes according to a chemical labeling method, such as use of a bifunctional reagent, such as glutaraldehyde, periodate, {N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate} (SMCC) reagent and a 2-iminothiolane (2-IT) reagent (iminothiophene hydrochloride). However, a chemical coupling method has complex operation and low coupling efficiency. With an unfixed coupling site and a harsh condition, it is easy to reduce activity of the antibodies or the enzymes, and conjugates of the antibodies and the enzymes are uneven. Consequently, it is necessary to separate and remove unbound enzymes and antibodies. Moreover, free antibodies will compete with enzyme-labeled antibodies for corresponding antigens, which reduces an amount of the enzyme-labeled antibodies bound to the solid phase, thereby reducing sensitivity of test. As a result, it is crucial to remove the unbound antibodies.
With the significant harm of the mycotoxins, it is of great significance to find a simple, quick, accurate and automatic test method in study of mycotoxin test and pollution control.
A first objective of the disclosure is to provide a magnetic chemiluminescence immunoassay kit for mycotoxins, which integrates chemiluminescence, magnetic particle separation and gene engineering technologies, and has excellent accuracy, sensitivity and repeatability.
A second objective of the disclosure is to provide a use of the kit in test of mycotoxins.
In order to achieve the above objectives, the disclosure uses the following technical solutions:
In a first aspect, the disclosure provides a magnetic chemiluminescence immunoassay kit for mycotoxins. The kit includes streptavidin magnetic particles, a biotin-labeled mycotoxin antigen, a mycotoxin standard solution, a mycotoxin nanobody-alkaline phosphatase (ScFV-AP) bifunctional fusion protein, a sample diluent, a washing solution and a substrate solution.
The disclosure covalently binds —COOH active groups provided by surface organic matters with streptavidin —NH2 by taking micron-sized magnetic particles as a carrier, and carries out “bridging” through biotin-streptavidin to form a magnetic particle-streptavidin-biotin-mycotoxin-ScFV-AP bifunctional fusion protein complex. The technology has the novelty: (1) A contact area between an antigen and an antibody and a light-emitting area of a substrate are increased by using the magnetic particles as the solid-phase carrier, thereby improving sensitivity of reaction; and the magnetic particles have an effect of stirring and separating the magnetic particle-streptavidin-biotin-mycotoxin-ScFV-AP bifunctional fusion protein complex from a mycotoxin-ScFV-AP bifunctional fusion protein complex through a rotating magnetic particle field. (2) The ScFV-AP bifunctional fusion protein improves specificity and stability of reaction. (3) Edible oil can be directly added for test without operations of extraction, centrifugation, etc., thereby greatly improving testing efficiency.
Further, the ScFV-AP bifunctional fusion protein is obtained by fusion expression of a mycotoxin nanobody (ScFV) and an alkaline phosphatase (AP) by using the AP as a catalyst for bioluminescence.
Preferably, the mycotoxin nanobody (ScFV) is selected from heavy chain antibodies of camelids or sharks.
Further, the mycotoxin nanobody-alkaline phosphatase bifunctional fusion protein of the disclosure may have any one or more of the following amino acid sequences:
According to a particular embodiment of the disclosure, corresponding biotin-labeled mycotoxin antigens, mycotoxin standard solutions, and mycotoxin nanobody-alkaline phosphatase bifunctional fusion proteins are selected according to types of toxins to be tested. For example, when deoxynivalenol is tested, the kit of the present application should include a biotin-labeled deoxynivalenol antigen, a deoxynivalenol standard solution, and a deoxynivalenol nanobody-alkaline phosphatase bifunctional fusion protein.
Further, the ScFV-AP bifunctional fusion protein may be synthesized according to the amino acid sequence provided above, or may be prepared through the following method:
The disclosure can overcome various defects of a chemical labeling method and obtain an enzyme-labeled antibody having high specific activity by means of fusion expression of the mycotoxin nanobody and the alkaline phosphatase. The fusion protein prepared can be applied to the field of immunodiagnosis, such as chemiluminescence immunoassay, enzyme linked immunosorbent assay or enzyme fluorescence immunoassay, as a test reagent. Compared with a traditional chemical coupling method of an alkaline phosphatase, the ScFV-AP bifunctional fusion protein has the following advantages: (1) The traditional chemical coupling method has a long process flow, a complex coupling process and a severe condition, resulting in an unstable process, low yield of a coupling product and poor stability between batches. The fusion expression of the mycotoxin nanobody and the alkaline phosphatase is simple and has a stable process, thereby avoiding tedious and inefficient chemical crosslinking of the enzyme and the protein. (2) In order to obtain the excellent coupling product, the traditional chemical coupling method usually needs to purify a target coupling product to remove unlinked enzyme and antibody molecules, has incomplete purification, and thus may produce a false positive result during test. The fusion expression of the mycotoxin nanobody and the alkaline phosphatase overcomes the disadvantage. (3) An enzyme and antibody complex after coupling is heterogeneous in the traditional chemical coupling method. A ratio of the mycotoxin nanobody to a monomer alkaline phosphatase molecule in a fusion expression product of the mycotoxin nanobody and the alkaline phosphatase is 1:1 or 1:2, and the mycotoxin nanobody can be separated from the monomer alkaline phosphatase molecule during nickel column purification, thereby ensuring a homogeneous enzyme and antibody complex. (4) In the traditional chemical coupling method, a binding position of a coupling chemical active reagent is unfixed, and may be bound to an antibody variable region, thereby affecting binding of the antibody to the target antigen; alternatively, a coupling chemical active reagent is bound to a vicinity of an alkaline phosphatase active site, thereby affecting bonding of the alkaline phosphatase to a substrate; and both the cases can cause reduction in the activity of a coupling complex, and thus a false negative result is easily obtained during test. The fusion expression mode of the mycotoxin nanobody and the alkaline phosphatase does not affect activity of the mycotoxin nanobody and the alkaline phosphatase. (5) The ScFV-AP fusion protein according to the disclosure has excellent specificity and signal amplification, and a hypersensitive bioluminescence immunoassay kit is established through the ScFV-AP fusion protein, is configured to test contents of mycotoxins in substrates such as grain and oil, food, feed and Chinese herbal medicine, and has excellent application value and prospect. (6) Compared with a single-chain variable fragment, the nanobody has great advantages in aspects of antibody stability, batch-to-batch repeatability, titer, etc., and all other antibody molecules apart from those indicated in the disclosure can produce an alkaline phosphatase coupled molecule in this way.
Further, peripheries of magnetic particles are coated with polystyrene or dextran by taking a ferroferric oxide or ferric oxide superparamagnetic material as a core, the magnetic particles are activated according to a physical or chemical method to generate —NH2, tosyl, —COOH or —CH(O) groups on surfaces of the magnetic particles, and the magnetic particles have a particle size ranging from 1 m to 2 m.
Further, the streptavidin magnetic particles are obtained by coupling streptavidin with magnetic particles.
Further, the biotin-labeled mycotoxin antigen is obtained by coupling mycotoxins with bovine serum albumin, and then coupling an obtained bovine serum albumin-mycotoxin complex with biotin.
Further, the mycotoxin standard solution is prepared by dissolving a mycotoxin standard into a methanol-water mixed solution. According to a particular embodiment of the disclosure, the mycotoxin standard solution in the kit can be a mother solution having a certain concentration, is diluted into standard working solutions having different concentrations according to requirements during use, and is configured to draw a standard curve; or a series of concentrations of mycotoxin standard solutions may be directly subpackaged and placed in the kit, and are configured to draw standard curves.
Preferably, a volume ratio of methanol to water in the methanol-water mixed solution is 50:50.
Further, the substrate solution is (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridine methylene) disodium phosphate salt solution having a concentration ranging from 0.5 mmol/L to 2 mmol/L.
Preferably, the sample diluent consists of 0.01 M phosphate buffer, 0.1% Tween-20 and 0.5% bovine serum albumin.
Preferably, the washing solution consists of 0.01 M tris-HCl buffer and 0.1% Tween-20.
Further, the kit further includes a reaction tube; and preferably, the reaction tube is made of transparent polystyrene, polyethylene, polypropylene or glass.
In a second aspect, the disclosure further sets forth a use of the kit in test of mycotoxins, particularly, a use of the kit in test of mycotoxins in edible oil, food, grain, feed or Chinese herbal medicine.
Further, the edible oil includes one of peanut oil, corn oil, soybean oil, rapeseed oil, sunflower oil, rapeseed oil, sesame oil and olive oil.
Further, the mycotoxins include one or more of aflatoxin, zearalenone, deoxynivalenol, fumonisin, ochratoxin A and T-2 toxin.
According to the disclosure, the mycotoxins in the food, the agricultural products and the feed are determined according to an indirect competition principle, and a biotin-labeled mycotoxin antigen working solution, the ScFV-AP bifunctional fusion protein and a sample to be tested are added into a streptavidin magnetic particle suspension. Mycotoxin in the sample and the biotin-labeled mycotoxin antigen compete for a limited number of ScFV-AP bifunctional fusion proteins, and forms a magnetic particle-streptavidin-biotin-mycotoxin-ScFV-AP bifunctional fusion protein complex and a mycotoxin-ScFV-AP bifunctional fusion protein complex respectively by means of affinity reaction of streptavidin and biotin and antigen-antibody reaction. Magnetic particles directly precipitate in an external magnetic particle field, the complex is adsorbed at a bottom of a reagent tube through the magnetic particle field, free components are washed away, the substrate solution is added, the AP catalyzes hydrolysis of phosphate radical of the substrate, a decomposition reaction immediately occurs to release 475 nm of photons, and a relative light unit (RLU) of each sample hole is determined within 5 min. The RLU of the sample is negatively correlated with a concentration of the mycotoxin in the sample. The concentration of the mycotoxin in the sample is quantified according to a four-parameter mathematical model established from a concentrations of a mycotoxin standard and the corresponding RLU to test a content of the mycotoxin in the sample.
Further, a fully-automatic chemiluminescence immunoassay analyzer may be used for the above test, thereby fully-automatically testing the mycotoxins. The kits do not interfere with each other, and multiple samples can be tested simultaneously in real time, thereby highly satisfying the development requirements of quick field test of the mycotoxins. Furthermore, the kit of the disclosure used for testing the mycotoxins further has the following advantages: (1) A test speed is quick, stable and free of radioactive pollution; magnetic particles are suspended in a liquid under the condition that no magnetic particle field exists, such that the antigen-antibody reaction is similar to homogeneous reaction; and the magnetic particles can be conveniently separated and washed quickly under the action of the external magnetic particle field, and a test result may be determined within 24 min. (2) Sensitivity is high. (3) Specificity is high. (4) Precision is excellent. (5) A streptavidin and biotin cascade amplification system, the streptavidin magnetic particles, and the biotin-labeled mycotoxin antigen are directly linked to the mycotoxin antigen compared with the magnetic particles, such that reaction efficiency is greatly improved, and an operation is simple; and moreover, expiration date of the kit is increased, and it is confirmed through an accelerated stability test at 37° C. and a real stability test at 2° C.-8° C. that the product can be stored at 37° C. for 7 days or above, and can be stored for 1 year at 2° C.-8° C. (6) Cost is low, and compared with a similar product on the market, the kit has excellent performance, low cost and practical application value.
The particular embodiments of the disclosure will be further described in detail below in combination with the accompanying drawings.
In order to illustrate the disclosure more clearly, the disclosure is further described below in combination with the examples and the accompanying drawings. In the accompanying drawings, the similar components are designated by the same reference numerals. Those skilled in the art should understand that the contents specifically described below are illustrative rather than restrictive, and should not limit the scope of protection of the disclosure.
The experimental materials used in the following examples, unless otherwise specified, are purchased by conventional biochemical reagent suppliers.
The AFB1-ScFV-AP bifunctional fusion protein was obtained by fusion expression of an AFB1 nanobody (AFB1-ScFV) and an alkaline phosphatase (AP) by taking the alkaline phosphatase as a catalyst for bioluminescence.
The AFB1-ScFV-AP bifunctional fusion protein was prepared through the following method:
(1) An AFB1 nanobody gene was designed and synthesized: according to an amino acid sequence SEQ ID NO:7 of the AFB1 nanobody, a structure of a gene was optimized to efficiently express the gene in Escherichia coli (E. coli) as follows: a preferred codon of the E. coli was used; a possible secondary structure was eliminated o, the GC/AT ratio was balanced. And a nucleotide fragment (nucleotide sequence shown as SEQ ID NO:8) of the AFB1 nanobody gene was designed and synthesized.
(2) An AFB1-ScFV-AP expression vector was constructed: the AFB1 nanobody gene synthesized in step (1) was cloned into a prokaryotic expression vector pET25b(+) to obtain pET25b(+)—ScFV; then an alkaline phosphatase expression vector pET25b(+)-AP (provided by Suzhou Hongxun Biotechnology Co., Ltd.) was digested with restriction enzymes XhoI and NotI to obtain an alkaline phosphatase gene fragment; and the alkaline phosphatase gene fragment was recovered by agrose gel and linked to the pET25b(+)—ScFV expression vector digested with the restriction enzymes XhoI and NotI through T4 DNA ligase in a molar ratio of 1:3. A linked product was transformed into Escherichia coli (E. coli) DH5a competent cells according to a heat shock method (42° C., 45 s), and transformed bacteria were inoculated into a lysogeny broth (LB) plate containing 100 μg/mL ampicillin (Amp), and cultured overnight at 37° C. Clones were primarily screened through colony polymerase chain reaction (PCR) and double enzyme digestion verification, screened positive clones were sequenced, and a bifunctional fusion expression vector AFB1-ScFV-AP was finally obtained.
(3) The AFB1-ScFV-AP bifunctional fusion protein was expressed: the bifunctional fusion expression vector was transformed into competent cells of an expression strain E. coli BL21 (Rosetta, DE3) through heat shock, evenly spread on an LB plate containing 100 μg/mL Amp and 34 μg/mL chloramphenicol (CAP), and cultured upside down at 37° C. for 12 h. A single colony was selected and inoculated into a 5 mL LB/Amp/CAP liquid medium, and shaken and cultured at 37° C. and 220 rpm for 12 h; the culture was transferred to a 200 mL LB/Amp/CAP liquid medium according to 1% inoculation amount, and shaken and cultured at 37° C. and 220 rpm until an optical density 600 (OD600) was 0.4-0.6; isopropyl-β-d-thiogalactoside (IPTG) was added for induction culture, 1 mL of induced culture was transferred and centrifuged to collect bacterial cells, the bacterial cells were frozen at −20° C. for later use, and the remaining bacterial cells was collected by centrifugation at 5000 g for 10 min; 4 mL of bacterial protein extraction reagent (B-PER) was added into each gram of the above bacterial cells, each ml of B-PER contained 2 μL lysozyme and 2 μL endonuclease DNase I, the bacteria were fully resuspended, and the bacteria stood at a room temperature for 10 min-15 min; and a supernatant containing a soluble protein was collected by centrifugation at 15000 g, 5 min, and expression of a target protein was analyzed according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Prokaryotic expression conditions were optimized by changing a final concentration (0 mmol/L-1 mmol/L, induction temperature 16° C.) of the IPTG and an induction temperature (16° C., 20° C., 30° C., 37° C., final concentration of IPTG 0.5 mmol/L) to explore an optimal expression condition of the fusion protein.
(4) The bifunctional fusion protein was purified and characterized: supernatant under the optimal expression condition was sterilized by a 0.45 m sterile filter, loaded into a Ni2+-NTA affinity column, washed with a ten times column bed volume of binding buffer (TBS buffer, pondus Hydrogenii (pH) 8.0), and eluted with binding buffers containing different concentrations of imidazole (20 mmol/L, 50 mmol/L, 100 mmol/L, 200 mmol/L) to collect elution components. Expression products and purification of the bifunctional fusion protein were characterized through 12% SDS-PAGE, and the gray scale of a gel picture was adjusted according to Image J software to analyze a protein band after elution by gray scale, so as to determine purity of the purified fusion protein.
According to the above method, the purified AFB1-ScFV-AP bifunctional fusion protein was finally obtained, and had an amino acid sequence shown as SED ID NO.1.
(5) Activity of the bifunctional fusion protein was analyzed:
Alkaline phosphatase activity of the bifunctional fusion protein was determined according to a disodium p-nitrophenyl phosphate method. 20 μL of the bifunctional fusion protein or the alkaline phosphatase diluted 50, 100, 200, 400, 800 and 1000 times were each added into an ELISA plate, then 100 μL p-nitrophenylphosphate (PNPP) reagent (2 mg/mL, dissolved into 0.1 M diethanolamine buffer containing 2 mM MgCl2, pH 9.8) was added into holes, mixed evenly, and incubated at 37° C. for 10 min, 50 μL 4M NaOH solution was added to terminate reaction, absorbance at 405 nm was measured by a microplate reader, and concentrations (mM) of PNPP were calculated separately. Enzyme activity (U/mL)=PNPP concentration/reaction time*sample dilution multiple, and enzyme specific activity (U/mg)=enzyme activity (U/mL)/protein concentration (mg/mL). The alkaline phosphatase activity of the bifunctional fusion protein was evaluated by comparing the specific activity of the bifunctional fusion protein and the AP.
Antibody affinity of the bifunctional fusion protein was determined according to a biofilm interference technology. A sensor was balanced through a phosphate buffer saline (PBS) buffer for 60 s, and a biotin-labeled target mycotoxin was added for fixation and blocked through a PBST buffer (PBS buffer containing 0.02% Tween-20, pH 7.4) for 180 s. Association and dissociation of the bifunctional fusion protein and the specific nanobody with the target mycotoxin under different gradients were observed at a constant temperature of 30° C., association rate constant (Kon) values, dissociation rate constant (Koff) values and dissociation constant (KD) values (Kon value/Koff values) of the bifunctional fusion protein and the specific nanobody with the target mycotoxin were calculated according to an Octet RED96e data processing program, and antibody affinity of the bifunctional fusion protein was characterized by comparing the KD values. Kon represented a formation rate of an antigen-antibody complex, and the larger the Kon value was, the quicker antigen-antibody association was; Koff reflected stability of the formed complex, and the larger the Koff value was, the quicker complex dissociation was; and KD may reflect the size of an association capability of interaction between an antigen and an antibody, and when the KD value reaches 10-8 mol/L, it was indicated that the antigen and the antibody had extremely high affinity.
A DON-ScFV-AP bifunctional fusion protein was prepared through the method of Example 1, and had an amino acid sequence shown as SED ID NO:2.
A ZEN-ScFV-AP bifunctional fusion protein was prepared through the method of Example 1, and had an amino acid sequence shown as SED ID NO:3.
A T-2-ScFV-AP bifunctional fusion protein was prepared through the method of Example 1, and had an amino acid sequence shown as SED ID NO:4.
An FB1-ScFV-AP bifunctional fusion protein was prepared through the method of Example 1, and had an amino acid sequence shown as SED ID NO:5.
An OTA-ScFV-AP bifunctional fusion protein was prepared through the method of Example 1, and had an amino acid sequence shown as SED ID NO:6.
1. Streptavidin magnetic particles were prepared:
2. A biotin-labeled AFB1 antigen was prepared:
3. An AFB1-ScFV-AP bifunctional fusion protein working solution was prepared:
4. A sample diluent was prepared:
5. A washing solution was prepared:
6. An AFB1 standard solution was prepared:
7. A substrate solution was prepared:
8. Assembly was carried out: the above reagents were assembled into a kit and stored at 2° C.-8° C.
A schematic diagram of magnetic chemiluminescence immunoassay based on the bifunctional fusion protein for AFB1 was shown in
Methodological investigation test for test of AFB1 was carried out on the magnetic chemiluminescence immunoassay kit for AFB1 prepared in Example 7.
(1) A stability test was carried out: the magnetic chemiluminescence immunoassay kit for AFB1 was treated through an advanced stabilizer (0.01 M phosphate buffer, 0.1% bovine serum albumin solution, 0.1% proclin 300 and 1% trehalose), and stored at 37° C. after treatment for an accelerated stability test to obtain a stability result shown in Table 1. According to experience, storage at 37° C. for 1 day was equivalent to storage at 4° C. for 2 months. That is, the kit was stored at 4° C. for 12 months without affecting use.
(2) Sensitivity was analyzed:
(3) A linear relation was investigated:
(4) Specificity of the magnetic chemiluminescence immunoassay kit for AFB1 was investigated:
(1) A sample to be tested was pretreated: 5.0 g of crushed sample to be tested was weighed into a 50 mL centrifuge tube, a 25.0 mL methanol/water (70:30) solution was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for AFB1 (prepared in Example 7) was used as follows:
(3) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for AFB1 were determined:
5 g of negative brown rice, rice, corn, peanut butter, wheat and peanut oil samples were weighed separately, different amounts of AFB1 standards were added, and 7 parallels were constituted, and determined through a magnetic chemiluminescence immunoassay kit for AFB1-labeled sample extract. Recovery rates, means and relative standard deviations (RSDs) of the 7 parallels were shown in Tables 2, 3, 4, 5, 6 and 7. The recovery rate was between 79.100 and 12000, and the RSD was less than 10%.
Methodological investigation test for test of DON was carried out on the magnetic chemiluminescence immunoassay kit for DON prepared in Example 7.
(1) A stability test was carried out: the magnetic chemiluminescence immunoassay kit for DON was treated through an advanced stabilizer (0.01 M phosphate buffer, 0.100 bovine serum albumin solution, 0.1% proclin 300 and 1% trehalose), and stored at 37° C. after treatment for an accelerated stability test to obtain a stability result shown in Table 1. According to experience, storage at 37° C. for 1 day was equivalent to storage at 4° C. for 2 months. That is, the kit was stored at 4° C. for 12 months without affecting use.
(2) Sensitivity was analyzed:
(3) A linear relation was investigated:
(4) Specificity of the magnetic chemiluminescence immunoassay kit for DON was investigated:
(1) A sample to be tested was pretreated: 5.0 g of crushed sample to be tested was weighed into a 50 mL centrifuge tube, 25.0 mL water was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for DON was used as follows:
(3) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for DON were determined:
5 g of negative corn, wheat and flour samples were weighed separately, different amounts of DON standards were added, and 7 parallels were constituted, and determined through a magnetic chemiluminescence immunoassay kit for DON-labeled sample extract. Recovery rates, means and RSDs of the 7 parallels were shown in Tables 9, 10 and 11. The recovery rate was between 80% and 120%, and the RSD was less than 9%.
Reference was made to the magnetic chemiluminescence immunoassay kit for ZEN prepared in Example 7.
(1) A sample to be tested was pretreated: 5.0 g of crushed sample to be tested was weighed into a 50 mL centrifuge tube, 25.0 mL 80% acetonitrile aqueous solution was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for ZEN was used as follows:
(3) Sensitivity of the magnetic chemiluminescence immunoassay kit for ZEN was analyzed:
(4) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for ZEN were determined:
5 g of negative corn, wheat, brown rice and flour samples were weighed separately, different amounts of ZEN standards were added, and 7 parallels were constituted, and determined through a magnetic chemiluminescence immunoassay kit for ZEN-labeled sample extract. Recovery rates, means and RSDs of the 7 parallels were shown in Tables 12, 13, 14 and 15. The recovery rate was between 80% and 120%, and the RSD was less than 10%.
Reference was made to the magnetic chemiluminescence immunoassay kit for T-2 prepared in Example 7.
(1) A sample to be tested was pretreated: 5.0 g of crushed negative corn sample was weighed into a 50 mL centrifuge tube, different amounts of T-2 standards were added separately, final spike levels were 47.8 μg/Kg and 108 μg/Kg separately, 7 parallels were constituted, 25.0 mL 80% acetonitrile aqueous solution was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for T-2 was used as follows:
(3) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for T-2 were determined:
the extract of the sample to be tested obtained in step (1) was determined through the magnetic chemiluminescence immunoassay kit for T-2. Recovery rates, means and RSDs of the 7 parallels were shown in Table 16. The recovery rate was between 89.0% and 107.5%, and the RSD was less than 3%.
Reference was made to the magnetic chemiluminescence immunoassay kit for FB1 prepared in Example 7.
(1) A sample to be tested was pretreated: 5.0 g of crushed negative corn sample was weighed into a 50 mL centrifuge tube, different amounts of FB1 standards were added separately, final spike levels were 47.8 μg/Kg and 108 μg/Kg separately, and 7 parallels were constituted, 25.0 mL 80% acetonitrile aqueous solution was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for FB1 was used as follows:
(3) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for FB1 were determined:
Reference was made to the magnetic chemiluminescence immunoassay kit for OTA prepared in Example 7.
(1) A sample to be tested was pretreated: 5.0 g of crushed sample was weighed into a 50 mL centrifuge tube, 25.0 mL 80% acetonitrile aqueous solution was added, the centrifuge tube was placed on a multi-tube vortex mixer and vortexed at 2500 rpm for 5 min (or homogenized at 11000 rpm for 3 min through a high-speed homogenizer, or shaken at 200 rpm for 40 min on a shaker), and 20 μL of an extract of the sample to be tested was added into the test tube, and then centrifuged at 7000 rpm for 5 min to obtain an extract of the sample to be tested.
(2) The magnetic chemiluminescence immunoassay kit for OTA was used as follows:
(3) Sensitivity of the magnetic chemiluminescence immunoassay kit for OTA was analyzed:
(4) Accuracy and repeatability of the magnetic chemiluminescence immunoassay kit for OTA were determined:
5 g of negative corn, wheat and brown rice samples were weighed separately, different amounts of OTA standards were added, and 7 parallels were constituted, and determined through a magnetic chemiluminescence immunoassay kit for OTA-labeled sample extract. Recovery rates, means and RSDs of the 7 parallels were shown in Tables 18, 19 and 20. The recovery rate was between 80% and 120%, and the RSD was less than 8%.
Obviously, the above examples of the disclosure are only examples for clearly illustrating the disclosure, and are not intended to limit the embodiments of the disclosure. Those of ordinary skill in the pertinent field can further make other different forms of changes or variations on the basis of the above description, and all the embodiments cannot be exhausted herein. Any obvious changes or variations derived from the technical solutions of the disclosure still fall within the scope of protection of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| CN202210155141.5 | Feb 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/076761 | 2/17/2023 | WO |
