Patent Grant
12276661
This application includes a Sequence Listing in the ASCII text file in .txt format that is electronically submitted via EFS-Web on Aug. 9, 2024. The ASCII text file contains a sequence listing entitled “0123389183US9UpdatedSequenceListing.txt” created on Aug. 9, 2024 and is 3,895 bytes in size. The Sequence Listing contained in this 0123389183US9UpdatedSequenceListing.txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The application specifically relates to a time-resolved fluorescence immunochromatography kit for simultaneously detecting mixed pollution of aflatoxin and carbaryl, and a preparation method and application thereof.
Aflatoxin is a kind of main mycotoxin pollutants with high toxicity and wide pollution, and is divided as a kind of carcinogen by World Health Organization. The toxicity of aflatoxin is manifested in carcinogenesis, mutagenesis and teratogenesis in animals, and cause of DNA damage and immunosuppression. Aflatoxin pollution may occur at various stages of plantation, harvest and storage, transportation, consumption, etc., the polluted products mainly are cereals and nuts, and aflatoxin is also detected in other products such as meat, egg, and milk. Carbaryl is a kind of carbamates pesticide widely used in crops such as vegetables, fruits, cereals, etc., and has a good control effect on pests; however because of its abuse and prohibited use in agricultural production, carbaryl poses a significant threat to people's food safety, thus China established the maximum concentration of 0.5-1 μg/mL for carbaryl in cereals, and 1 μg/mL in vegetable. Aflatoxin and carbaryl in cereals tend to result in simultaneous pollution, there is a need to develop a convenient and efficient test technology for simultaneously detecting the two kinds of pollutants.
At present, the test methods for aflatoxin and carbaryl mainly consist of liquid chromatography, gas chromatography-mass spectrometry method, liquid chromatography-mass spectrometry method, etc. These methods have good stability, high sensitivity, and good accuracy, but pretreatment step is complicated, and sample test cost is high. Immunochromatographic method overcomes the above-described shortcomings, and is based on antigen-antibody specific reaction, wherein antigen is fixed by cellulose nitrate, and in chromatography process, free target substance and antigen on a test line competitively bind with labeled antibody, and content of the target substance in the sample is calculated on the basis of the amount of labeled substance binding onto the test line.
The problem to be solved by the application is to provide a time-resolved fluorescence immunochromatography kit which may simultaneously detect mixed pollution of aflatoxin and carbaryl, and a preparation method and application thereof. The time-resolved fluorescence immunochromatography kit may be used in synchronous detection of contents of aflatoxin and carbaryl in a sample, and has the characteristics of simple and fast operation, and high sensitivity.
In order to solve the above-described technical problems, a technical scheme adopted by the present application is as below:
A time-resolved fluorescence immunochromatography kit for simultaneously detecting mixed pollution of aflatoxin and carbaryl, wherein the immunochromatographic time-resolved fluorescence immunochromatography kit comprises an immunochromatographic time-resolved fluorescent test strip, and a sample reaction vial containing a europium-labeled anti-aflatoxin monoclonal antibody freeze-dried product and a europium-labeled anti-carbaryl monoclonal antibody freeze-dried product, wherein the immunochromatographic time-resolved fluorescent test strip comprises a paperboard, an absorbent pad, a test pad and a sample pad are successively stuck onto one side of the paperboard from top to bottom, adjacent pads are overlapped and connected at junctions, the test pad uses a nitrocellulose membrane as a base pad, quality control line and test lines are arranged transversely on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, the test lines are located below the quality control line, the number of the test lines is 2, and the test lines are respectively coated with an aflatoxin-bovine serum albumin conjugate and a carbaryl-ovalbumin conjugate; the anti-carbaryl monoclonal antibody is secreted by a hybridoma cell strain Jnw1D2 with an accession number of CCTCC NO. C201654. The hybridoma cell strain was stored/deposited under the terms of the Budapest Treaty on Mar. 29, 2016 at China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, China, one of recognized International Depository Authorities (IDAs), and has an accession number of CCTCC NO: C201654. The hybridoma cell strain Jnw1D2 will be irrevocably and without restriction or condition released to the public upon the issuance of a patent would satisfy the deposit requirement made herein.
According to the above-described technical scheme, the europium-labeled anti-carbaryl monoclonal antibody is prepared according to the following method: subjecting an anti-carbaryl monoclonal antibody and an activated europium-labeling reagent to an oscillatory reaction in a boric acid buffer solution for 2 hours or more, performing centrifuging to remove supernatant, and performing sealing to obtain a target product; and the europium-labeled anti-aflatoxin monoclonal antibody is prepared according to the following method: subjecting an anti-aflatoxin monoclonal antibody and an activated europium-labeling reagent to an oscillatory reaction in a boric acid buffer solution for 2 hours or more, performing centrifuging to remove supernatant, and performing sealing to obtain a target product.
According to the above-described technical scheme, an activation method of the europium-labeling reagent comprises the steps: taking the europium labeling reagent, subjecting the europium labeling reagent to ultrasonic dispersion in a boric acid buffer solution, then slowly adding a carbodiimide solution, performing oscillating activation, performing centrifuging to remove supernatant, and performing redissolving by using a boric acid buffer solution for later use, wherein the activation time is 15-30 min.
According to the above-described technical scheme, a sealing fluid used in the sealing is a boric acid buffer solution containing 0.5-1% BSA.
According to the above-described technical scheme, the mass ratio of the europium-labeling reagent (on the basis of effective amount) to the aflatoxin monoclonal antibody is 1:(0.001-0.1), the mass ratio of the europium-labeling reagent (on the basis of effective amount) to the anti-carbaryl monoclonal antibody is 1:(0.001-0.1).
According to above-described technical scheme, a freeze-dried product of the europium-labeled anti-aflatoxin monoclonal antibody is prepared by adding the europium-labeled monoclonal antibody into an antibody protective liquid and then performing freeze-drying;
According to the above-described technical scheme, the absorbent pad in the immunochromatographic time-resolved fluorescent test strip has a length of 15-35 mm and a width of 3-5 mm; the sample pad has a length of 12-18 mm and a width of 2-5 mm, and the overlapping length of each adjacent pads is 1-3 mm; in the immunochromatographic time-resolved fluorescent test strip, the spacing between the test line near the quality control line on the test pad and a top edge of the nitrocellulose membrane is 6-15 mm, the spacing between every two adjacent test lines is 1.5-4.5 mm, and the spacing between the test line near the quality control line and the quality control line is 4-10 mm; and the sample reaction vial is a 1-5 mL bayonet bottle.
According to the above-described technical scheme, in the immunochromatographic time-resolved fluorescent test strip and, the coating amount of the aflatoxin-bovine serum albumin conjugate for every centimeter of the test lines on the test pad is 0.4-0.8 μg, and the coating amount of the carbaryl-ovalbumin conjugate for every centimeter of the test lines is 0.8-1.0 μg; and the content of the europium-labeled anti-aflatoxin monoclonal antibody freeze-dried product in the sample reaction vial is 0.1-0.3 μg, and the content of the europium-labeled anti-carbaryl monoclonal antibody freeze-dried product in the sample reaction vial is 0.2-0.4 μg.
According to the above-described technical scheme, a preparation method for the time-resolved fluorescent test strip is as follows:
According to the above-described technical scheme, every 10 mL of a coating buffer solution used in the preparation of an aflatoxin-bovine serum albumin conjugate coating liquid and a carbaryl-ovalbumin conjugate coating liquid in the preparation of the immunochromatographic time-resolved fluorescent test strip contains bovine serum albumin 0.1 g, sodium azide 0.002 g, sodium chloride 0.08 g, disodium hydrogen phosphate dodecahydrate 0.029 g, potassium chloride 0.002 g, and potassium dihydrogen phosphate 0.002 g;
Application of above-described immunochromatographic time-resolved fluorescence immunochromatography kit in content test of aflatoxin and carbaryl comprises the steps of: pretreating a sample to be detected to obtain a solution of the sample to be detected, adding the solution of the sample to be detected into a sample reaction vial, performing uniform mixing, inserting the time-resolved fluorescence test strip, performing a reaction for 6 min at a temperature of 37° C., and then performing testing by a time-resolved fluorescence tester to obtain a ratio of fluorescence intensity of test lines (T) to fluorescence intensity of the quality control line (C) on the immunochromatographic time-resolved fluorescence test strip; and acquiring contents of aflatoxin and carbaryl in the solution of the sample to be tested base on the respective relationship curves of the ratio (T/C) of the fluorescence intensity of the test lines to the fluorescence intensity of the quality control line on the immunochromatographic time-resolved fluorescent test strip with concentrations of aflatoxin and carbaryl, and finally performing conversion to obtain the contents of aflatoxin and carbaryl in the sample to be tested.
According to the above-described technical scheme, the respective relationship curves of the ratio (T/C) of the fluorescence intensity of the test lines to the fluorescence intensity of the quality control line of the immunochromatographic time-resolved fluorescent test strip with the concentrations of aflatoxin and carbaryl are obtained by using the following method:
Beneficial effects of the present application are as below:
Screening of Hybridoma Cell Strain Jnw1D2
1. Animal Immunization
Six 6 week-old BALB/c mice are purchased, bovine serum albumin (BSA) is coupled with 6-(1-naphthoxyformamide)-hexanoic acid (CNH) to obtain carbaryl complete antigen CNH-BSA, and the mice are immunized. During the first time of immunization, the carbaryl complete antigen and an equal volume of Freund's complete adjuvant are emulsified and then injected subcutaneously into multiple points of the nape of the mice. The second time of immunization is carried out three weeks later. A Freund's incomplete adjuvant and an equal volume of fumonisin complete antigen are emulsified and then injected subcutaneously into multiple points of the nape of the mice. The third time of immunization and the fourth time of immunization are conducted two weeks after the last time of immunization separately, in the same immunization manner as the second time of immunization. The same dose is adopted for four times of immunization, 100 μg per mouse only. On the 7th day after the third time of immunization, blood is collected from the caudal vein of the mice and serum is separated. The serum titer of the mice is monitored through indirect ELISA, the sensitivity of the serum of the mice is measured through indirect competitive ELISA, a mouse corresponding to the serum with higher titer and sensitivity is selected for the final booster immunization, and the immune dose is 2 times the previous dose.
2. Cell Fusion
Three days after the booster immunization, cell fusion is carried out with a conventional method using 50% (by weight) polyethylene glycol, namely PEG (molecular weight being 1450) as a fusion agent. Specifically, under an aseptic condition, the mice to be fused are killed by breaking the neck, spleen cells are isolated and mixed with murine myeloma cells SP2/0 at a ratio of 5:1, and the mixed cells are washed with an RPMI-1640 basal medium and centrifuged at 1200 rpm for 5 min. A supernatant is discarded, draining is conducted, 1 mL of PEG is added for fusion of 1 min, the RPMI-1640 basal medium is slowly added, centrifugation is conducted, a supernatant is discarded, a precipitation is fusion cells, resuspension is conducted with 20 mL of complete medium, suspended cells are added to 80 mL of semi-solid medium, and the mixture is added to a 6-well cell culture plate after uniform mixing at 2 mL/well, and then placed in a 37° C. CO2 incubator for culture.
the cell complete medium containing 1% HAT contains 20% (volume percentage) fetal calf serum, 75% (volume percentage) RPMI-1640 basal medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) Penicillin-Streptomycin (10000 units per milliliter of penicillin and 10000 μg per milliliter of streptomycin), 2% (volume percentage) growth factor (HFCS) and 1% (weight percentage) hypoxanthine-aminopterin-thymidine (i.e., HAT) and methylcellulose (purchased from Sigma-Aldrich company).
3. Screening and Cloning of Cell Strain
2-3 weeks after the cell fusion, when cell colonies grow to megascopic, colonies are picked out from the culture medium by a micropipette, transferred to a 96-well cell culture plate and cultivated by using HAT liquid, and when the cells grow to ⅔ of bottom of the well, culture supernatant is sucked out to conduct a test. A two-step screening method is employed: a first step adopts an indirect ELISA method to screen out the positive wells resistant to carbaryl but not resistant to a carrier protein BSA; and a second step adopts an indirect competitive ELISA method to detect the positive wells screened out in the first step, carbaryl is used as a competition agent, the wells with high absorbance and sensitivity are chosen (high absorbance means a well in which the competition agent is 0 namely final determined value of the positive control well is high, the high sensitivity means that the concentration of the competition agent when inhibition rate being 50% (i.e., IC50 value) is small), a subcloning is conducted by using limited dilution method, after the subcloning a test is conducted by using the two-step method, as such the subcloning is repeated 4-5 times, and then a hybridoma cell strain Jnw1D2 is obtained, and was stored/deposited under the terms of the Budapest Treaty at China Center for Type Culture Collection (CCTCC) on Mar. 29, 2016, with an accession preservation number of CCTCC NO: C201654.
4. Sequence Determination of Antibody Variable Region of Anti-Carbaryl Monoclonal Antibody Hybridoma Cell Strain Jnw1D2.
The obtained gene sequence results: the length of the coding gene sequence of the heavy chain variable region is 339 bp, the sequence is shown in SEQ ID NO: 1, it is deduced from the obtained gene sequence that the heavy chain variable region coded by the gene sequence is composed of 113 amino acids, and the sequence is shown in SEQ ID NO: 3; and the length of the coding gene sequence of the light chain variable region is 315 bp, the sequence is shown in SEQ ID NO: 2, it is deduced from the obtained gene sequence that the light chain variable region coded by the gene sequence is composed of 105 amino acids, and the sequence is shown in SEQ ID NO: 4.
5. Preparation, Purification, Subtypes and Characterization of Anti-Carbaryl Monoclonal Antibody
The obtained anti-carbaryl monoclonal antibody hybridoma cell strain Jnw1D2 is injected into a BALB/c mouse previously treated with a Freund's incomplete adjuvant, the ascites of the mouse is collected, and the antibody is purified with an octanoic acid-ammonium sulfate method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4° C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 30-35 μL, and the mixture is mixed at room temperature for 30-60 min and stands at 4° C. for 2 h or more. Then centrifugation is conducted at 4° C. and 12000 r/min for 30 min or more, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, ammonium sulfate is slowly added in an ice bath till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4° C. for 2 h or more, then centrifugation is conducted at 4° C. and 12000 r/min for 30 min or more, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites with a pH of 7.4, placed in a dialysis bag and dialyzed with 0.01 mol/L PBS for two days, then dialysis is conducted with PB for two days, a protein solution in the dialysis bag is taken out and centrifuged, a supernatant is collected, a precipitate is discarded, and then the product is pre-frozen at −70° C. and then lyophilized in a lyophilizer. Lyophilized powder is collected, thus obtaining the purified anti-carbaryl monoclonal antibody;
the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride and 0.2 g of potassium dihydrogen phosphate to 100 mL.
Subtype of the anti-carbaryl monoclonal antibody secreted by the hybridoma cell strain Jnw1D2 identified by a commercially available subtype identification kit is IgG2b. The titer of the antibody determined by Enzyme Linked Immunosorbent Assay (ELISA) can reach 1.6×104. 50% inhibition concentration IC50 against carbaryl is 0.668 ng/kg, and no cross reaction with Carbofuran, Aldicarb, Methomyl, Metolcarb, Propoxur, Pirimicarb, Dimethacarb, Methiocarb, etc. presents (cross reaction rate <0.001). Through literature retrieval and comparison, the specificity exceeds the reported antibody.
An anti-aflatoxin monoclonal antibody is secreted by a hybridoma cell strain 1C11 with an accession number of CCTCC NO. C201013. The hybridoma cell strain 1C11 was stored/deposited under the terms of the Budapest Treaty in CCTCC in Wuhan University, Wuhan, China, on Jul. 13, 2010 and has an accession number of CCTCC NO: C201013. The hybridoma cell strain 1C11 will be irrevocably and without restriction or condition released to the public upon the issuance of a patent would satisfy the deposit requirement made herein. The anti-aflatoxin monoclonal antibody is prepared in advance specifically according to the method reported in the patent with an authorization number of CN201010245095.5. The preparation method comprises the steps of: injecting a BALB/c mouse treated in advance by Freund's incomplete adjuvant with the obtained hybridoma cell strain 1C11, collecting ascites of the mouse, and performing purifying treatment to obtain the anti-aflatoxin monoclonal antibody. The purifying method is an octanoic acid-ammonium sulfate method, which comprises the specific operations of: filtering the mouse ascites with a double layer filter paper, centrifuging the filtered ascites at 4° C. and 12000 r/min for 15 minutes or more, sucking supernatant, mixing the supernatant with 4-fold volumes of acetate buffer, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid for every milliliter ascites is 30-35 μL, conducting mixing at room temperature for 30-60 minutes, permitting standing at 4° C. for 2 hours or more, then conducting centrifuging at 4° C. and 12000 r/min for 30 minutes or more, discarding precipitates, filtering the obtained supernatant with double-layer filter paper, then adding phosphate buffer in a volume 1/10 of the volume of filtrate, and with a molar concentration of 0.1 mol/L and pH value 7.4, adjusting the pH value of the mixed liquid to 7 with 2 mol/L sodium hydroxide solution, pre-cooling the mixed liquid at 4.4° C., slowly adding ammonium sulfate until the final concentration of ammonium sulfate is 0.277 g/mL, permitting standing at 4° C. for 2 hours or more, then performing centrifuging at 4° C. and 12000 r/min for 30 minutes or more, discarding supernatant, re-suspending the obtained precipitate with 0.01 mol/L phosphate buffer with a volume 1/10 of the volume of original ascites, placing the re-suspended solution in a dialysis bag, performing dialysis with pure water, placing the fully dialyzed protein solution in a −70° C. refrigerator for refrigeration, then performing freeze-drying by a vacuum freeze drier, collecting the freeze-dried powder to obtain a purified anti-aflatoxin monoclonal antibody, and placing the antibody in a −20° C. refrigerator for later use; and
A time-resolved fluorescence fluorescence immunochromatography kit for quantitatively detecting aflatoxin and carbaryl, comprising a fluorescent test strip, a europium-labeled anti-aflatoxin monoclonal antibody, a europium-labeled carbaryl monoclonal antibody, and a sample reaction vial; the fluorescent test strip is as shown in
Acquisition of the Fluorescent Test Strip:
800 μL of 0.2 mol/L boric acid buffer solution (pH 8.18) is taken, 200 μL of a europium-labeling reagent (particle size 100 nm, solid content 1%) is added, and shaking for uniform mixing is carried out. Ultrasound treatment is carried out for 3 seconds. 40 μL of 15 mg/mL EDC solution is added, and shaking for uniform mixing is carried out for 15 minutes. Centrifuging at 13000 r/min and 10° C. is carried out for 10 minutes, the supernatant is removed, 1 mL of the boric acid buffer solution is added for re-dissolving, shaking for uniform mixing is carried out, and ultrasound treatment is carried out for 3 seconds. 20 μg of the anti-aflatoxin monoclonal antibody is added, uniform mixing is carried out, and shaking table treatment at 250 r/min and 25° C. is carried out overnight. Centrifuging is carried out again, the supernatant is removed, 1 mL of the boric acid buffer solution containing 0.5% BSA is added for re-dissolving, shaking for uniform mixing is carried out, ultrasound treatment is carried out for 10 minutes, and shaking table treatment at 25° C. is carried out for 2 hours to obtain a target product namely the europium-labeled anti-aflatoxin monoclonal antibody. The above-described europium-labeling reagent can be purchased from but not limited to Shanghai Youni Biotechnology Co., Ltd.
Acquisition of the Europium-Labeled Anti-Carbaryl Monoclonal Antibody:
800 μL of 0.2 mol/L boric acid buffer solution (pH 8.18) is taken, 200 μL of a europium-labeling reagent (particle size 100 nm, solid content 1%) is added, and shaking for uniform mixing is carried out. Ultrasound treatment is carried out for 3 seconds. 40 μL of 15 mg/mL EDC solution is added, and shaking for uniform mixing is carried out for 15 minutes. Centrifuging at 13000 r/min and 10° C. is carried out for 10 minutes, the supernatant is removed, 1 mL of the boric acid buffer solution is added for re-dissolving, shaking for uniform mixing is carried out, and ultrasound treatment is carried out for 3 seconds. 20 μg of the anti-carbaryl monoclonal antibody is added, uniform mixing is carried out, and shaking table treatment at 250 r/min and 25° C. is carried out overnight. Centrifuging is carried out again, the supernatant is removed, 1 mL of the boric acid buffer solution containing 0.5% BSA is added for re-dissolving, shaking for uniform mixing is carried out, ultrasound treatment is carried out for 10 minutes, and shaking table treatment at 25° C. is carried out for 2 hours to obtain a target product namely the europium-labeled anti-aflatoxin monoclonal antibody. The above-described europium-labeling reagent can be purchased from but not limited to Shanghai Youni Biotechnology Co., Ltd.
Acquisition of the sample reaction vial: the above-described europium-labeled anti-aflatoxin monoclonal antibody at a ratio of 1:300 (V:V), and the above-described europium-labeled anti-carbaryl monoclonal antibody at a ratio of 1:250 (V:V) are added to a certain volume of antibody protective liquid, freeze-drying in a freeze-drier is performed, 0.1 μg of the europium-labeled anti-aflatoxin monoclonal antibody freeze-dried product and 0.3 μg of the europium-labeled anti-carbaryl monoclonal antibody freeze-dried product are taken, and added in a sample reaction vial. The antibody protective liquid contains 0.01 vt % TWEEN® 20, 0.5 wt % sucrose and 1 wt % bovine serum albumin (BSA).
Application of the Above-Described Immunochromatographic Time-Resolved Fluorescent Test Strip in Quantitative Test of Aflatoxin and Carbaryl:
1. Establishment of relationship curves of ratio (T/C) of the fluorescence intensity of the test lines to the fluorescence intensity of the quality control line on the immunochromatographic time-resolved fluorescent test strip with the concentrations of aflatoxin and carbaryl:
2. Labeled recovery experiment of aflatoxin and carbaryl contents in cereal sample:
25 g of a cereal sample is taken, and 20 mL of 80% methanol water solution is added, a homogenization extraction is conducted for 5 minutes, standing and filtering with double-layer filter paper are carried out, and the filtrate is collected, and diluted at a ratio of 1:1. Aflatoxin (carbaryl) standard substance is respectively and accurately added at 0.005 ng/ml, 0.01 ng/ml and 0.1 ng/ml (0.05 ng/ml, 0.1 ng/ml and 1 ng/ml), 300 μL of the above-described test liquid sample to be tested is taken and added into a sample reaction vial containing the europium-labeled monoclonal antibody, uniform mixing is carried out, one end of the fluorescent test strip sample pad is inserted into the reaction vial, a reaction is carried out at 37° C. for 10 minutes, and the fluorescent test strip is detected by a test instrument. The test instrument is time-resolved fluoroimmunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. A ratio (T/C) of time-resolved fluorescence intensity of test lines (T) to time-resolved fluorescence intensity of the quality control line (C) on each fluorescence test strip is obtained; and then by substituting the ratio (T/C) into the above-described relationship curves of ratio (T/C) of the time-resolved fluorescence intensity of the test lines to the time-resolved fluorescence intensity of the quality control line on the fluorescent test strip with the standard substance concentrations, the concentrations of aflatoxin and carbaryl in the sample solution are obtained, and then the contents of aflatoxin and carbaryl in the sample can be calculated on the basis of dilution ratio. The labeled recovery rates of aflatoxin in cereal sample are successively: 106.5%, 90.2%, and 86.1%; and the labeled recovery rates of carbaryl are successively: 104.1%, 89.5%, and 83.7%.
A time-resolved fluorescence immunochromatography kit for quantitatively detecting aflatoxin and carbaryl, comprising a fluorescent test strip, a europium-labeled anti-aflatoxin monoclonal antibody, a europium-labeled carbaryl monoclonal antibody, and a sample reaction vial; the fluorescent test strip comprises a paperboard, an absorbent pad, a test pad and a sample pad are successively stuck onto one side of the paperboard from top to bottom, adjacent pads are overlapped and connected at the junctions, and the overlapping length is 2 mm; the absorbent pad in the immunochromatographic time-resolved fluorescent test strip has a length of 18 mm and a width of 3 mm; the test pad has a length of 25 mm and a width of 3 mm; the sample pad has a length of 15 mm and a width of 3 mm, and the overlapping length of each adjacent pads is 2 mm; and the test pad uses the nitrocellulose membrane as a base pad, a quality control line and two test lines are arranged transversely on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, the coating amount is 0.6 μg/cm, the test lines are located below the quality control line, the number of the test lines is 2, the test lines are respectively coated with an aflatoxin-bovine serum albumin conjugate and a carbaryl-ovalbumin conjugate, the coating amounts are respectively 0.6 μg/cm and 0.6 μg/cm, the spacing between the test line near the quality control line and a top edge of the nitrocellulose membrane is 12 mm, the spacing between the quality control line and the first test line is 3 mm, and the spacing between the two test lines is 3 mm.
0.2 μg of a europium-labeled anti-aflatoxin monoclonal antibody freeze-dried product and 0.2 μg of a europium-labeled anti-carbaryl monoclonal antibody freeze-dried product are put into the sample reaction vial. The antibody protective liquid is water solution of 0.1 vt % (volume fraction) TWEEN® 20, 1 wt % sucrose and 0.5 wt % bovine serum albumin (BSA); in the preparation of the anti-aflatoxin monoclonal antibody, the mass ratio of the europium-labeling reagent to the aflatoxin monoclonal antibody (on the basis of effective amount) is 1:0.1, and in preparation of the anti-carbaryl monoclonal antibody, the mass ratio of the europium-labeling reagent to the carbarylantibody (on the basis of effective amount) is 1:0.1.
Labeled recovery experiment of aflatoxin and carbaryl contents in nut sample: 5 g of a nut sample is taken, and 20 mL of 80% methanol water solution is added, a homogenization extraction is conducted for 5 minutes, standing and filtering with double-layer filter paper are carried out, and the filtrate is collected, and diluted at a ratio of 1:1. Aflatoxin (carbaryl) standard substance is respectively and accurately added at 0.005 ng/ml, 0.01 ng/ml and 0.1 ng/ml (0.05 ng/mL, 0.1 ng/mL and 1 ng/mL), 300 μL of the above-described test liquid sample to be tested is taken and added into a sample reaction vial containing the europium-labeled monoclonal antibody, uniform mixing is carried out, one end of the fluorescent test strip sample pad is inserted into the reaction vial, a reaction is carried out at 37° C. for 10 minutes, and the fluorescent test strip is detected by a test instrument. The test instrument is time-resolved fluoroimmunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. A ratio (T/C) of time-resolved fluorescence intensity of test lines (T) to time-resolved fluorescence intensity of the quality control line (C) on each fluorescence test strip is obtained; and then by substituting the ratio (T/C) into the above-described relationship curves of ratio (T/C) of the time-resolved fluorescence intensity of the test lines to the time-resolved fluorescence intensity of the quality control line on the fluorescent test strip with the standard substance concentrations, the concentrations of aflatoxin and carbaryl in the sample solution are obtained, and then the contents of aflatoxin and carbaryl in the sample can be calculated on the basis of dilution ratio. The determined aflatoxin labeled recovery rates in the nut sample are successively: 103.0%, 92.0%, and 85.4%; and the carbaryl labeled recovery rates are successively: 108.5%, 91.5%, and 80.9%.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201611268788.X | Dec 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/119440 | 12/28/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/121677 | 7/5/2018 | WO | A |
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| 1523356 | Aug 2004 | CN |
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| 103278630 | Sep 2013 | CN |
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| 105486858 | Apr 2016 | CN |
| 105652005 | Jun 2016 | CN |
| 106918704 | Jul 2017 | CN |
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| Number | Date | Country | |
|---|---|---|---|
| 20210132058 A1 | May 2021 | US |
