CN115166231A - Side-stream immunochromatographic method for citrinin - Google Patents
Side-stream immunochromatographic method for citrinin Download PDFInfo
- Publication number
- CN115166231A CN115166231A CN202111419021.3A CN202111419021A CN115166231A CN 115166231 A CN115166231 A CN 115166231A CN 202111419021 A CN202111419021 A CN 202111419021A CN 115166231 A CN115166231 A CN 115166231A
- Authority
- CN
- China
- Prior art keywords
- citrinin
- mixing
- catalytic reaction
- lateral flow
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a side flow immunochromatographic method of citrinin, which comprises the following steps: preparing a fluorescent probe; mixing the fluorescent probe with a standard solution and a sample solution respectively, and inserting the fluorescent probe into a lateral flow immunochromatography test strip respectively; the lateral flow immunochromatographic test strip comprises a chromatographic membrane, a sample pad, absorbent paper and a bottom plate; the chromatographic film is attached to the middle part of the bottom plate, and the absorbent paper and the sample pad are respectively attached to two ends of the bottom plate; the T line of the chromatographic membrane comprises citrinin coated antigen with the concentration of 0.7 mg/mL-0.9 mg/mL, and the C line of the chromatographic membrane comprises goat anti-mouse secondary antibody with the concentration of 1.4 mg/mL-1.6 mg/mL. The detection result obtained by the lateral flow immunochromatographic method has high sensitivity, accuracy and precision, and also has good specificity.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a side-stream immunochromatographic method for citrinin.
Background
The fermented grains and the distilled grains are mainly formed by mixing sorghum, corn, rice, wheat and the like and are byproducts generated in the fermentation and production processes of wine products. In recent years, with the vigorous development of the wine industry, the yield of the byproducts is increased correspondingly. Because of high nutritive value, high yield and relatively low price, the feed is widely applied to various fields of animal feed, industrial and agricultural production and the like. However, the fermentation process contains abundant amino acids, proteins, trace elements and the like, and is easily polluted by citrinin, so that certain health hazards and economic losses are caused.
Citrinin (CIT) is a mycotoxin produced by three types of molds, penicillium, monascus and aspergillus, and is commonly found in fermented red yeast rice products, but has also been found to be present in grains, fruits, vegetables and feeds in recent years. The main target organs of citrinin are kidney and liver, and exposure to citrinin can cause genetic toxicity, teratogenicity and carcinogenicity, thus causing damage to liver, kidney, heart, reproductive system and the like, affecting the function and growth condition of animals and further causing potential damage to human health.
The traditional citrinin determination methods include instrumental analysis and immunoassay. Common instrumental analysis methods mainly include high performance liquid chromatography and chromatography-mass spectrometry. The instrumental analysis method is used as a conventional mycotoxin detection method, has high sensitivity and accurate and reliable result, but needs to carry out complicated pretreatment on a sample, is expensive and precise, has high detection cost and maintenance cost, and cannot meet the requirement of rapid screening and detection of batch samples. In conventional immunoassays, such as lateral flow immunochromatography, colloidal gold is generally used as a label, which has a low antibody adsorption amount and a low optical signal, and limits the sensitivity of the lateral flow immunochromatography.
Disclosure of Invention
Based on the method, the invention provides the side flow immunochromatographic method of the citrinin, and the detection result obtained by the method has high sensitivity, accuracy and precision and also has good specificity.
The invention is realized by the following technical scheme.
A method for performing lateral flow immunochromatography on citrinin comprises the following steps:
mixing the fluorescent microspheres, 2- (N-morpholine) ethanesulfonic acid buffer solution and phosphate buffer solution for the first time, mixing the materials after the first mixing with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and carrying out the first catalytic reaction; performing first centrifugation on the material after the first catalytic reaction, and mixing the precipitate with a coupling buffer solution for the second time; mixing the materials after the second mixing with the first antibody of the citrinin to carry out a second catalytic reaction; mixing the material after the second catalytic reaction with bovine serum albumin to perform a third catalytic reaction; centrifuging the material after the third catalytic reaction for the second time, mixing the precipitate with a heavy suspension of fluorescent microspheres, and preparing a fluorescent probe;
mixing the fluorescent probe with a standard solution and a sample solution respectively, and inserting the fluorescent probe into a lateral flow immunochromatography test strip respectively;
the lateral flow immunochromatographic test strip comprises a chromatographic membrane, a sample pad, absorbent paper and a bottom plate; the chromatographic membrane is attached to the middle part of the bottom plate, and the water-absorbing paper and the sample pad are respectively attached to two ends of the bottom plate; the T line of the chromatographic membrane comprises citrinin coated antigen with the concentration of 0.7-0.9 mg/mL, and the C line of the chromatographic membrane comprises goat anti-mouse secondary antibody with the concentration of 1.4-1.6 mg/mL.
In one embodiment, the pH value of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 6.5-7.5; and/or
The pH value of the phosphate buffer solution is 7.2-8.0.
In one embodiment, the dosage of the fluorescent microspheres is 7-9 μ L; and/or
The fluorescence properties of the fluorescent microspheres include: the excitation wavelength is 580-660; the emission wavelength is 605-690.
In one embodiment, the concentration of the first antibody to citrinin is 3.0mg/mL to 5.0mg/mL.
In one embodiment, the coupling buffer is selected from borate buffer with a concentration of 0.04mol/L to 0.06mol/L.
In one embodiment, the preparation of the sample pad comprises the following steps:
mixing the blood filtering membrane with the buffer treatment solution, and drying;
the buffer treatment liquid comprises components of bovine serum albumin, proclin-300, polyvinylpyrrolidone, tween 20 and borate buffer liquid.
In one embodiment, in the buffer treatment solution, the volume fraction of tween 20 is 0.1% to 1.0%.
In one embodiment, the temperature of the first catalytic reaction is 20 ℃ to 30 ℃; the time of the first catalytic reaction is 15 min-25 min; and/or
The temperature of the second catalytic reaction is 20-30 ℃; the time of the second catalytic reaction is 25 min-35 min; and/or
The temperature of the third catalytic reaction is 20-30 ℃; the time of the third catalytic reaction is 25 min-35 min.
In one embodiment, the temperature of the first centrifugation is 2-6 ℃, and the rotating speed of the first centrifugation is 13000-15000 rpm; and/or
The temperature of the second centrifugation is 2-6 ℃, and the rotating speed of the second centrifugation is 13000-15000 rpm.
In one embodiment, the preparation of the sample solution comprises the following steps:
mixing the sample, methanol and phosphate buffer solution, centrifuging and taking liquid.
Compared with the prior art, the lateral flow immunochromatographic method for citrinin has the following beneficial effects:
the fluorescence microsphere is used as a marker in the lateral flow immunochromatographic method for citrinin, so that the fluorescent probe is successfully activated, higher fluorescence intensity and competitive reaction inhibition rate are ensured, and the concentration of the envelope antigen and the concentration of the second antibody on the chromatographic membrane are limited, thereby obviously improving the sensitivity and the fluorescence development intensity of the detection method. In addition, the side flow immunochromatographic assay method for citrinin has high accuracy and precision, and the result is accurate and reliable.
Furthermore, the side flow immunochromatographic assay method for citrinin is low in cost, simple, convenient and quick in detection process, and suitable for quick screening and detection of batch samples.
Drawings
FIG. 1 is a sensitivity calibration curve provided by an embodiment of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the accompanying examples. The preferred embodiments of the present invention are given in the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise. In the description of the present invention, "a plurality" means at least one, e.g., one, two, etc., unless specifically limited otherwise.
The words "preferably," "more preferably," and the like, in the present disclosure mean embodiments of the disclosure that may, in some instances, provide certain benefits. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a side flow immunochromatographic method of citrinin, which comprises the following steps:
mixing fluorescent microspheres, 2- (N-morpholine) ethanesulfonic acid buffer solution and phosphate buffer solution for the first time, mixing the materials after the first mixing with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and carrying out a first catalytic reaction; performing first centrifugation on the material after the first catalytic reaction, and mixing the precipitate and the coupling buffer solution for the second time; mixing the materials after the second mixing with the first antibody of the citrinin to carry out a second catalytic reaction; mixing the material after the second catalytic reaction with bovine serum albumin to perform a third catalytic reaction; centrifuging the material after the third catalytic reaction for the second time, taking the precipitate, and mixing the precipitate with the fluorescent microsphere heavy suspension to prepare a fluorescent probe;
respectively mixing the fluorescent probe with a standard solution and a sample solution, and inserting the fluorescent probe into a lateral flow immunochromatography test strip;
the lateral flow immunochromatographic test strip comprises a chromatographic membrane, a sample pad, absorbent paper and a bottom plate; the T line of the chromatographic membrane comprises citrinin coated antigen with the concentration of 0.7-0.9 mg/mL, and the C line of the chromatographic membrane comprises goat anti-mouse secondary antibody with the concentration of 1.4-1.6 mg/mL.
An immunochromatography lateral flow assay (LFIA) method is based on antigen-antibody recognition and labeling technology, and enables components in a substance to be detected to be separated under capillary force. LFIA is mainly composed of a sample pad, a chromatographic membrane, a water absorption pad and a support plate. The chromatographic membrane is a key part of the reaction, and two or more detection (test, T) lines and quality control (C) lines which are sprayed and printed with different bioactive substances (such as antigens or antibodies) are fixed on the chromatographic membrane and are used for intercepting labeled immune complexes and visually displaying the detection result. In a competitive reaction mode, a T line is sprayed with CIT coated antigen, and a C line is sprayed with specific goat-anti-mouse secondary antibody. The detection target in the sample and the coating antigen on the T line compete together for binding to the binding site on the corresponding primary antibody. For a negative sample, the primary antibody marked with the fluorescent chromogenic substance reacts with the coated antigen on the T line and the secondary antibody on the C line respectively to present fluorescent chromogenic; for positive samples, the target substance in the sample will preferentially bind to the corresponding primary antibody, resulting in a decrease in the amount of primary antibody bound to the T-line, and thus a corresponding decrease in fluorescence development. The excess primary antibody still bound to the secondary antibody at line C and developed color, confirming the effectiveness of the strip detection.
The concentration of the coating antigen and the secondary antibody on the chromatographic membrane has great influence on the sensitivity and the fluorescence development intensity of the method, when the concentration is higher, the development is too deep, the sensitivity of the method is reduced, otherwise, when the coating concentration is too low, the fluorescence development is weaker and difficult to identify.
Further, the fluorescent microsphere is a kind of nano-particle capable of emitting light. In particular, fluorescent microspheres are polystyrene nanoparticles embedded with a fluorescent dye that absorbs light energy and produces emitted light under a range of ultraviolet excitation light.
It is understood that, in the present invention, the fluorescent microsphere resuspension refers to dissolving the fluorescent microspheres bound with the antibody in a certain ratio in the corresponding solution for further detection.
It is understood that the concentration of the citrinin-coated antigen in the present invention includes, but is not limited to, 0.7mg/mL, 0.75mg/mL, 0.8mg/mL, 0.85mg/mL, 0.9mg/mL. Preferably, the concentration of the citrinin-coated antigen is 0.8mg/mL.
It is understood that, in the present invention, the concentration of the goat anti-mouse secondary antibody includes, but is not limited to, 1.4mg/mL, 1.45mg/mL, 1.5mg/mL, 1.55mg/mL, 1.6mg/mL. Preferably, the concentration of the goat anti-mouse secondary antibody is 1.5mg/mL.
In a specific example, a first antibody to citrinin, derived from immunization of BALB/c mice with CIT-BSA, was added to the antibody dilution. Preparing an antibody diluent: 2.7g of disodium hydrogen phosphate dodecahydrate, 0.6g of sodium dihydrogen phosphate dihydrate, 16g of sodium chloride and 300 mu L of preservative Proclin, and the volume is adjusted to 1L by using deionized water.
In one specific example, the citrinin coating antigen is derived from CIT-OVA and added to the coating antigen dilution. Coating antigen diluent preparation: 1.59g of sodium carbonate and 2.93g of sodium bicarbonate, and the volume is adjusted to 1L by deionized water.
In one specific example, the goat anti-mouse secondary antibody is derived from the dilution of a goat anti-mouse enzyme-labeled secondary antibody with a secondary antibody diluent configured as follows: 2.7g of disodium hydrogen phosphate dodecahydrate, 0.6g of sodium dihydrogen phosphate dihydrate, 16g of sodium chloride and 200 mu L of a preservative Proclin, and the volume is adjusted to 1L by deionized water.
In a specific example, bovine serum albumin is derived from 50mL of fetal bovine serum albumin, 20g of sucrose, 2g of casein, 5.8g of disodium hydrogen phosphate dodecahydrate and 0.6g of sodium dihydrogen phosphate dihydrate, and the volume is adjusted to 1L by deionized water.
In a specific example, the chromatographic membrane is an NC membrane.
In one particular example, the pH of the 2- (N-morpholino) ethanesulfonic acid buffer is from 6.5 to 7.5. Specifically, the pH of the 2- (N-morpholine) ethanesulfonic acid buffer includes, but is not limited to, 6.5, 7.0, 7.5.
In a specific example, the pH of the phosphate buffer is 7.2 to 8.0. Specifically, the pH of the phosphate buffer includes, but is not limited to, 7.2, 7.5, 8.0.
In a specific example, the fluorescent microspheres are used in an amount of 7 μ L to 9 μ L. More specifically, the fluorescent microspheres are 0.2% solids.
In one particular example, the fluorescence properties of the fluorescent microspheres include: the excitation wavelength is 580-660; the emission wavelength is 605-690.
In a specific example, the concentration of the first antibody to citrinin is from 3.0mg/mL to 5.0mg/mL. It is understood that the concentration of the first antibody to citrinin includes, but is not limited to, 3.0mg/mL, 3.1mg/mL, 3.2mg/mL, 3.3mg/mL, 3.4mg/mL, 3.5mg/mL, 3.6mg/mL, 3.7mg/mL, 3.8mg/mL, 3.9mg/mL, 4.0mg/mL, 4.1mg/mL, 4.2mg/mL, 4.3mg/mL, 4.4mg/mL, 4.5mg/mL, 4.6mg/mL, 4.7mg/mL, 4.8mg/mL, 4.9mg/mL, 5.0mg/mL.
In a specific example, the amount of the first antibody to citrinin is 0.8. Mu.L to 1.2. Mu.L.
In a specific example, the coupling buffer is selected from borate buffers with a concentration of 0.04mol/L to 0.06mol/L.
It is understood that in the present invention, the concentration of borate buffer includes, but is not limited to, 0.04mol/L, 0.045mol/L, 0.046mol/L, 0.047mol/L, 0.048mol/L, 0.049mol/L, 0.05mol/L, 0.051mol/L, 0.052mol/L, 0.053mol/L, 0.054mol/L, 0.055mol/L, 0.06mol/L.
In one specific example, the preparation of the sample pad comprises the steps of:
mixing the blood filtering membrane with the buffer treatment solution, and drying;
wherein, the buffer treatment liquid comprises bovine serum albumin, proclin-300, polyvinylpyrrolidone, tween 20 and borate buffer solution.
In a specific example, the volume fraction of Tween-20 in the buffer treatment solution is 0.1-1.0%. It is understood that the volume fraction of Tween-20 in the buffer treatment solution includes, but is not limited to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.
In a more specific example, the buffer treatment fluid composition is as follows: 0.5% BSA, 0.03% Proclin-300, 0.3% PVP, 0.5% Tween-20, dissolved in 0.02mol/L BB.
In a specific example, the temperature of the first catalytic reaction is 20 ℃ to 30 ℃; the time of the first catalytic reaction is 15 min-25 min.
It is understood that, in the present invention, the temperature of the first catalytic reaction includes, but is not limited to, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, and 30 deg.C.
In one specific example, the temperature of the second catalytic reaction is 20 ℃ to 30 ℃; the time of the second catalytic reaction is 25 min-35 min.
It is understood that, in the present invention, the temperature of the second catalytic reaction includes, but is not limited to, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, and 30 deg.C.
In a specific example, the temperature of the third catalytic reaction is 20 ℃ to 30 ℃; the time of the third catalytic reaction is 25 min-35 min.
It is understood that, in the present invention, the temperature of the third catalytic reaction includes, but is not limited to, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, and 30 deg.C.
In a specific example, the temperature of the first centrifugation is 2 ℃ to 6 ℃, and the rotation speed of the first centrifugation is 13000rpm to 15000rpm.
It is understood that, in the present invention, the temperature of the first centrifugation includes, but is not limited to, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, and 6 deg.C. The rotation speed of the first centrifugation includes, but is not limited to, 13000rpm, 13500rpm, 14000rpm, 14500rpm, 15000rpm.
In a specific example, the temperature of the second centrifugation is 2 ℃ to 6 ℃, and the rotation speed of the second centrifugation is 13000rpm to 15000rpm.
It is understood that, in the present invention, the temperature of the second centrifugation includes, but is not limited to, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, and 6 deg.C. The rotation speed of the second centrifugation includes, but is not limited to, 13000rpm, 13500rpm, 14000rpm, 14500rpm, 15000rpm.
In one specific example, the preparation of the sample solution comprises the steps of:
mixing the sample, methanol and phosphate buffer solution, centrifuging and taking liquid.
In a specific example, the assembly of the test strip comprises the following steps:
the test strip consists of a sample pad, an NC membrane, absorbent paper and a PVC bottom plate, and is adhered to the PVC bottom plate from the testing end to the handle end. The coating antigen CIT and the goat anti-mouse IgG secondary antibody are sprayed on the T line and the C line of an NC membrane respectively with proper concentration, and the distance between the T line and the C line is 10mm. And (5) drying the NC film after spraying. The sample pad is soaked in the treatment solution and then dried. And (3) attaching the NC film to the middle part of the PVC base plate, respectively attaching the absorbent paper and the sample pad to two ends of the PVC base plate, and forming 2mm overlap on the NC film. And cutting the assembled test strip into test strips with the width of 3.5mm by using a cutting machine, and putting the test strips into a sealing bag and adding a drying agent for storage.
The method for lateral flow immunochromatography of citrinin of the present invention is described in further detail below with reference to specific examples. The starting materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
The embodiment provides a lateral flow immunochromatographic method for citrinin, which specifically comprises the following steps:
1. reagent material
2. Instrumentation and equipment
3. Solution system
4. Detection process
(1) Respectively adding 1mL of 0.5mol/L MES buffer solution with the pH value of 5.0-7.0 and 1mL of 0.01mol/L PB buffer solution with the pH value of 7.0-8.0 into a 1.5mL centrifuge tube, respectively adding 8 mu L of fluorescent microspheres, and uniformly mixing by oscillation;
(2) Adding 20 μ L of 0.5mg/mL EDC and 24 μ L of 0.5mg/mL NHS into each tube, mixing, shaking, and performing catalytic reaction for 20min (200rpm, 25 deg.C) with constant temperature shaking table;
(3) Centrifuging at 14000rpm and 15min at 4 ℃, discarding the supernatant, adding 1mL of coupling buffer (0.05 mol/L BB, pH = 8.0), and ultrasonically redissolving;
(4) Adding 1 μ L of CIT primary antibody, and catalyzing for 30min (200rpm, 25 deg.C) with constant temperature shaker;
(5) Blocking was performed by adding 30. Mu.L of 20% BSA and catalyzing for 30min (200rpm, 25 ℃ C.) using a constant temperature shaker;
(6) The mixture was centrifuged at 14000rpm and 15min at 4 ℃. At this time, the successfully coupled fluorescent probe sinks to the bottom of the test tube;
(7) Discarding the supernatant, redissolving with 200 μ L of prepared fluorescent microsphere resuspension, suspending with ultrasound, and keeping at 4 deg.C;
(8) And 5 mu L of fluorescent probe is put in the micropore of the ELISA plate.
(9) The blood filtering membrane is used as a sample pad, the sample pad treatment solution adopts buffer treatment solution to soak the blood filtering membrane, and the buffer treatment solution comprises the following components: 0.5% BSA, 0.03% Proclin-300, 0.3% PVP, 0.5% Tween-20, dissolved in 0.02mol/L BB. The mixture is dried by blowing at 37 ℃ for 8h.
(10) The test strip consists of a sample pad, an NC membrane, absorbent paper and a PVC bottom plate. From the testing end to the handle end, and is thus glued to the PVC base plate. Coating antigen CIT with the concentration of 0.8mg/mL and goat anti-mouse IgG secondary antibody with the concentration of 1.5mg/mL are respectively sprayed on a T line and a C line of an NC membrane at the speed of 0.8 mu L/cm, and the distance between the T line and the C line is 10mm. After spraying, the NC membrane is dried in an oven at 37 ℃ for about 12 hours. The sample pad was soaked in the treatment solution and then dried in an oven at 37 ℃ for about 12 hours. The sample pad was cut to about 16mm width with a paper cutter and the absorbent paper was cut to about 18mm width. Attaching the NC film to the middle part of the PVC base plate, respectively attaching the water-absorbing paper and the sample pad to two ends of the PVC base plate, and forming an overlap of about 2mm on the NC film. And cutting the assembled test strip into test strips with the width of 3.5mm by using a cutting machine, and placing the test strips in a sealing bag to be added with a drying agent for storage.
(11) 1g of the sample was weighed into a 15mL centrifuge tube, and 1mL of methanol-0.02 mol/L PB (70%: 30%, v/v, pH = 7.4) solution was added for extraction. Shaking with oscillator for 5min, centrifuging at 1000rpm for 10min, collecting supernatant, and storing at 4 deg.C. Before test paper strip detection, diluting the vinasse sample extracting solution by 5 times by using 0.2mol/L PB buffer solution, and uniformly mixing for further detection.
(12) And (3) putting 120 mu L of standard solution or sample solution to be detected into the enzyme-labeled micropore, slightly sucking and beating for a plurality of times, stirring and uniformly mixing for 30s by using a gun head (taking care to avoid generating bubbles), fully dissolving the fluorescent probe attached to the bottom of the enzyme-labeled micropore, and incubating for 3min at room temperature. The prepared test strip is vertically inserted into the enzyme-labeled micropore and reacts for about 15min at room temperature. After the reaction, the strip was removed and the sample pad was discarded to terminate the reaction.
The detection method provided in example 1 was evaluated:
1. sensitivity of the probe
Taking 1g of sample in a centrifuge tube, adding 0, 25, 50, 75 and 100ppb of standard substance respectively, and taking an average value by three times of concentration measurement in the same processing steps as the pretreatment. The fluorescent color intensity of the T line/C line when detecting a negative sample is defined as B 0 (ii) a The intensity of fluorescence development of the T line/C line after addition of the series of standards was defined as B. Taking log of the concentration of the standard substance as an abscissa and B/B 0 The values are plotted as ordinate and a standard curve is established. The vanishing value (cut-off-value) is defined as the lowest concentration of the detection target at which the T-line fluorescence development is completely vanished. Half inhibition rate (IC 50) is one of the important parameters for evaluating the sensitivity of a method. In a competition reaction, IC 50 The concentration of the inhibitor is defined as the concentration corresponding to 50% of the binding rate of the antigen and antibody. According to the standard curve, at 50% B/B 0 Is an IC 50 To 80% of B/B 0 (IC 20 ) The quantitative limit of detection (qLOD). The standard curve is shown in FIG. 1, and the extinction value is 50 μ g/kg, IC 50 ＝31.1μg/kg，IC 20 ＝15.5μg/kg。
2. Specificity of
The specificity of the method was evaluated as Cross Reactivity (CR). Other mycotoxins with similar structures to the target to be tested, such as OTA, AFB1, OTA and ZEN, were taken, and the standard was diluted to a series of concentrations at 0.2mol/L PB to establish a standard curve, and its IC50 value was calculated. With IC of the object 50 IC of values and other structural analogues 50 The ratio of the values was used to calculate the cross-reactivity. The results are shown in Table 1.
TABLE 1 CIT and other mycotoxin Cross-reactivity rates
3. Accuracy and precision
And adding three CIT standard substances with known concentrations, namely low, medium and high, into the blank sample, and performing sample pretreatment and detection according to the method. The addition Recovery (Recovery) and Coefficient of Variation (CV) were calculated to evaluate the accuracy and precision of the method. The test was repeated 6 times for each concentration on the same day, every 3 days, and the measurement was repeated 6 times. The results are shown in tables 2 and 3.
TABLE 2 CIT recovery test results (n = 6)
TABLE 3 CIT accuracy and precision experimental results (n = 6)
4. Actual sample detection
Taking 20 parts of alcoholic product fermentation process sample, respectively adopting LFIA method and HPLC-FLD method (lambda) ex ＝350nm,λ em =500 nm), the consistency of the method was evaluated, and the test was repeated six times per sample. For HPLC detection, sample pre-treatment was referred to AOAC office Method 2005.08 and partially revised. And purifying the sample by adopting an immunoaffinity column, and performing photochemical post-column derivatization detection. The result shows that the detection result of the LFIA method is consistent with the HPLC-FLD (R) 2 ＞0.99)。
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, which is convenient for specific and detailed understanding of the technical solutions of the present invention, but the present invention should not be construed as being limited to the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present patent should be subject to the content of the appended claims, and the description can be used to interpret the content of the claims.
Claims (10)
1. A lateral flow immunochromatographic method for citrinin is characterized by comprising the following steps:
mixing the fluorescent microspheres, 2- (N-morpholine) ethanesulfonic acid buffer solution and phosphate buffer solution for the first time, mixing the materials after the first mixing with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and carrying out the first catalytic reaction; performing first centrifugation on the material after the first catalytic reaction, and mixing the precipitate and the coupling buffer solution for the second time; mixing the materials after the second mixing with the first antibody of the citrinin to carry out a second catalytic reaction; mixing the material after the second catalytic reaction with bovine serum albumin to perform a third catalytic reaction; centrifuging the material after the third catalytic reaction for the second time, taking the precipitate, and mixing the precipitate with the fluorescent microsphere heavy suspension to prepare a fluorescent probe;
mixing the fluorescent probe with a standard solution and a sample solution respectively, and inserting the fluorescent probe into a lateral flow immunochromatography test strip respectively;
the lateral flow immunochromatographic test strip comprises a chromatographic membrane, a sample pad, absorbent paper and a bottom plate; the chromatographic film is attached to the middle part of the bottom plate, and the absorbent paper and the sample pad are respectively attached to two ends of the bottom plate; the T line of the chromatographic membrane comprises citrinin coated antigen with the concentration of 0.7-0.9 mg/mL, and the C line of the chromatographic membrane comprises goat anti-mouse secondary antibody with the concentration of 1.4-1.6 mg/mL.
2. The method for the lateral flow immunochromatography of citrinin according to claim 1, wherein the pH of said 2- (N-morpholine) ethanesulfonic acid buffer is 6.5 to 7.5; and/or
The pH value of the phosphate buffer solution is 7.2-8.0.
3. The method for lateral flow immunochromatography of citrinin according to claim 1, wherein the amount of said fluorescent microspheres is 7-9 μ L; and/or
The fluorescence properties of the fluorescent microspheres include: the excitation wavelength is 580-660; the emission wavelength is 605-690.
4. The method of claim 1, wherein the concentration of the first antibody against citrinin is from 3.0mg/mL to 5.0mg/mL.
5. The method for the lateral flow immunochromatography of citrinin according to claim 1, wherein said coupling buffer is selected from borate buffer with a concentration of 0.04mol/L to 0.06mol/L.
6. The method of lateral flow immunochromatography of citrinin according to claim 1, wherein said preparation of said sample pad comprises the steps of:
mixing the blood filtering membrane with the buffer treatment solution, and drying;
the buffer treatment liquid comprises components of bovine serum albumin, proclin-300, polyvinylpyrrolidone, tween 20 and borate buffer liquid.
7. The method of lateral flow immunochromatography of citrinin according to claim 6, wherein the volume fraction of Tween 20 in the buffer treatment solution is 0.1% to 1.0%.
8. The method for lateral flow immunochromatography of citrinin according to any one of claims 1 to 7, wherein the temperature of the first catalytic reaction is 20 ℃ to 30 ℃; the time of the first catalytic reaction is 15 min-25 min; and/or
The temperature of the second catalytic reaction is 20-30 ℃; the time of the second catalytic reaction is 25 min-35 min; and/or
The temperature of the third catalytic reaction is 20-30 ℃; the time of the third catalytic reaction is 25 min-35 min.
9. The method for the lateral flow immunochromatography of citrinin according to any one of claims 1 to 7, wherein the temperature of the first centrifugation is 2 ℃ to 6 ℃, and the rotation speed of the first centrifugation is 13000rpm to 15000rpm; and/or
The temperature of the second centrifugation is 2-6 ℃, and the rotating speed of the second centrifugation is 13000-15000 rpm.
10. The method for the lateral flow immunochromatography of citrinin according to any one of claims 1 to 7, wherein the preparation of said sample solution comprises the steps of:
mixing the sample, methanol and phosphate buffer solution, centrifuging and taking liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111419021.3A CN115166231A (en) | 2021-11-26 | 2021-11-26 | Side-stream immunochromatographic method for citrinin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111419021.3A CN115166231A (en) | 2021-11-26 | 2021-11-26 | Side-stream immunochromatographic method for citrinin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115166231A true CN115166231A (en) | 2022-10-11 |
Family
ID=83475895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111419021.3A Pending CN115166231A (en) | 2021-11-26 | 2021-11-26 | Side-stream immunochromatographic method for citrinin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115166231A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603823A (en) * | 2003-09-30 | 2005-04-06 | 江西中德生物工程有限公司 | Fabrication method and application for citrinin immune chromatography detection test paper |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN103823065A (en) * | 2014-03-13 | 2014-05-28 | 福建卫生职业技术学院 | Mycotoxin detecting method based on cytometric bead array |
| CN105891494A (en) * | 2016-06-14 | 2016-08-24 | 焦作百奥泰科生物科技有限公司 | Fluorescence immunochromatography test paper for detecting citrinin |
| WO2021093885A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin b1 and sterigmatocystin |
| CN113533731A (en) * | 2021-06-13 | 2021-10-22 | 黑龙江谱尼测试科技有限公司 | Time-resolved fluorescence immunochromatography method for simultaneously detecting aflatoxin B1 and gibberellin ketene toxin in corn |
-
2021
- 2021-11-26 CN CN202111419021.3A patent/CN115166231A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603823A (en) * | 2003-09-30 | 2005-04-06 | 江西中德生物工程有限公司 | Fabrication method and application for citrinin immune chromatography detection test paper |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN103823065A (en) * | 2014-03-13 | 2014-05-28 | 福建卫生职业技术学院 | Mycotoxin detecting method based on cytometric bead array |
| CN105891494A (en) * | 2016-06-14 | 2016-08-24 | 焦作百奥泰科生物科技有限公司 | Fluorescence immunochromatography test paper for detecting citrinin |
| WO2021093885A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin b1 and sterigmatocystin |
| CN113533731A (en) * | 2021-06-13 | 2021-10-22 | 黑龙江谱尼测试科技有限公司 | Time-resolved fluorescence immunochromatography method for simultaneously detecting aflatoxin B1 and gibberellin ketene toxin in corn |
Non-Patent Citations (6)
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rubtsova et al. | Chemiluminescent biosensors based on porous supports with immobilized peroxidase | |
| CN103674935B (en) | A kind of method of measuring gibberellin based on hybridization chain reaction signal amplification technique | |
| CN105067811A (en) | T-2 toxin detecting product based on fluorescent microsphere immunochromatography and preparation method thereof | |
| WO2022104534A1 (en) | Kit for chemiluminescence immunoassay, and preparation method therefor and application thereof | |
| CN103823065A (en) | Mycotoxin detecting method based on cytometric bead array | |
| WO2017206800A1 (en) | Centrifugal chromatography immunoassay method | |
| CN111089956A (en) | Fluorescent microsphere immunochromatography test strip for triple quantitative detection of fusarium toxin, and preparation method and application thereof | |
| JPWO2019138898A1 (en) | Immunochromatographic test piece and measurement kit and measurement method | |
| US5401636A (en) | Enhanced sensitivity agglutination assays multivalent ligands | |
| CN113567666A (en) | Fluorescent microsphere labeled immunochromatography novel coronavirus detection test strip and preparation method and application thereof | |
| CN110596396B (en) | Method for detecting protein, test strip and kit | |
| CN109444423A (en) | Tetraodotoxin immunofluorescence Rapid detection test strip and preparation method and detection method | |
| CN106896094B (en) | Method for simultaneously detecting CLE, RAC and SBL and special paper chip thereof | |
| JPS61286755A (en) | Solid phase immunity testing method and composition using light-emitting fine particle | |
| CN106568967B (en) | A kind of sensitive detection method for ochratoxin A | |
| CN115166231A (en) | Side-stream immunochromatographic method for citrinin | |
| CN104387467A (en) | Detection kit and detection paper for Beta-adrenergic receptor stimulant multiresidue detection | |
| WO2023207166A1 (en) | Five-linked fluorescence immunoassay quantitative test strip for simultaneously testing various mycotoxins and heavy metal cd in cereals | |
| CN110596378A (en) | Multichannel universal chromatography method for detecting small molecules, test strip and kit | |
| CN208172009U (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip | |
| CN110988339A (en) | Time-resolved immune quantitative test strip for detecting aflatoxin M1 in milk | |
| CN106645686B (en) | One kind being directed to fumonisin B1Sensitive detection method | |
| CN105315165B (en) | The preparation method and its application in quantum dot immune fluorescent kit of Ractopamine haptens and antigen | |
| CN112180080B (en) | Small molecule triple immunochromatography detection method, test strip and kit | |
| CN108241059B (en) | Colloidal gold rapid test card and kit for detecting fumonisins, and method for detecting fumonisins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |