CN111474359B - Preparation method and application of colloidal gold immunochromatographic test strip for multi-joint detection - Google Patents
Preparation method and application of colloidal gold immunochromatographic test strip for multi-joint detection Download PDFInfo
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- CN111474359B CN111474359B CN202010320221.2A CN202010320221A CN111474359B CN 111474359 B CN111474359 B CN 111474359B CN 202010320221 A CN202010320221 A CN 202010320221A CN 111474359 B CN111474359 B CN 111474359B
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- 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
- G01N33/587—Nanoparticles
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- 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
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- 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
Abstract
The invention provides a preparation method and application of a multi-joint detection colloidal gold immunochromatographic test strip. The colloidal gold immunochromatographic test strip for multiplex detection comprises: the PVC bottom plate with viscose, along bottom plate width direction from supreme sample pad, the gold mark pad, chromatographic film and the absorption pad that sets gradually down, and adjacent component links up each other. The test strip has good sensitivity, good stability and long effective period of room-temperature storage, and provides a simpler, accurate and efficient detection product for the rapid field detection of food safety.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a preparation method and application of a multi-joint detection colloidal gold immunochromatographic test strip.
Background
Mycotoxins are a class of small molecule secondary metabolites produced by fungi. Crops are easily polluted by mycotoxin in the links of growth, harvesting, storage, transportation and the like. Some mycotoxins have carcinogenic, teratogenic, and mutagenic effects, and when contaminated with a mixture, toxicity is significantly enhanced. Mycotoxins can enter the food chain through contaminating cereals and feed or through animal-derived foods, causing serious harm to human and animal health, and have become the focus of research in the field of food safety in recent years.
The milk is rich in various nutrients such as protein, calcium, vitamins and the like, and plays an important role in human dietary structure. With the intensified and large-scale development of the modern dairy cow breeding industry, veterinary drug residues and illegal addition become one of the most important subjects for controlling the milk safety globally, so that the establishment of a rapid, effective and sensitive method for analyzing the veterinary drug residues and illegal additives in the milk is necessary for guaranteeing the milk quality safety.
CN110940811A discloses a detection card for simultaneously and quantitatively detecting two toxins in flour, which comprises a sample pad, a colloidal gold film, a nitrocellulose film and a water absorption pad; wherein the colloidal gold membrane is coated with a colloidal gold marker for detecting an antibody, and the nitrocellulose membrane is coated with a detection line T1 for vomitoxin antigen, a detection line T2 for fumonisin antigen and a quality control line C for IgG antibody.
CN110687294A discloses a detection card for rapidly and quantitatively detecting mycotoxin and application thereof, wherein the detection card comprises a bottom plate, a sample pad, a combination pad, a basement membrane and a water absorption pad, the combination pad contains a monoclonal antibody which is marked by quantum dots and used for identifying mycotoxin, a detection line and a control line are arranged on the basement membrane, and the control line is coated with an antibody of goat anti-mouse IgG; the detection line is coated with mycotoxin whole antigen.
CN110595866A discloses a pretreatment solution for detecting mycotoxins in grain and oil and a detection method thereof, wherein the pretreatment solution consists of nitric acid, sulfuric acid, copper sulfate and vanadium pentoxide, and the detection method of the mycotoxins in the grain and oil comprises the following steps: adding grain and oil to be detected into the pretreatment solution for reaction to obtain pretreated grain and oil; then adding sodium chloride and acetonitrile aqueous solution and stirring to obtain a mixed solution; filtering the mixed solution, and adding deionized water to obtain diluted filtrate; and then, enabling the diluted filtrate to pass through an immunoaffinity column, preparing a standard solution containing the mycotoxin to be detected through a chromatographic grade methanol solution to obtain a standard curve, and finally detecting the content of the mycotoxin in the grain and oil to be detected through a laser-induced fluorescence detector.
CN110488016A discloses a zearalenone-vomitoxin dual-channel immune quantitative test strip, wherein a fluorescent probe is prepared by marking fluorescent microspheres, and comprises fluorescent microspheres, namely a zearalenone monoclonal antibody, fluorescent microspheres, namely a vomitoxin monoclonal antibody, and fluorescent microspheres, namely a goat anti-rabbit secondary antibody, and the zearalenone artificial antigen, the fluorescent microspheres artificial antigen and the goat anti-mouse secondary antibody are respectively sprayed on a nitrocellulose membrane to serve as a test line T1, a test line T2 and a quality control line C to prepare the immune chromatographic test strip; and (3) simultaneously and quantitatively analyzing the zearalenone and the vomitoxin in the sample by reading the fluorescence value of a detection line on a fluorescence immunoassay analyzer by using a competitive immunoassay method.
CN110346562A discloses a fluorescence quantitative detection card of aflatoxin B1 and application thereof, and the fluorescence quantitative detection card of aflatoxin B1 comprises a back plate, a sample pad, a quenching antibody combination pad, a fluorescence reaction film and a water absorption pad. The fluorescence quantitative detection card provided by the invention utilizes the principle that gold gathered in the T region can remarkably quench the fluorescence at the T region, and can be used for detecting the content of aspergillus flavus B1 in sample matrixes such as grains and feeds.
CN109142720A discloses a nanocomposite test strip for detecting aflatoxin B1 and a preparation method thereof, and particularly discloses a nanocomposite test strip for detecting aflatoxin B1, which comprises a sample pad, a combination pad, an NC membrane and an absorption pad, and is sequentially adhered to a PVP bottom plate according to a certain assembly sequence, wherein the combination pad is coated with a nanogold-nanosilicon dioxide composite labeled aflatoxin B1 antibody, and a detection line and a control line on the NC membrane are respectively coated with an aflatoxin B1 antibody and a goat anti-mouse IgG solution.
US4017261A discloses a diagnostic test strip for the detection and semi-quantitative assessment of blood and hemoglobin in biological materials comprising an absorbent carrier impregnated with a composition comprising (a) an acidic buffer having a pH in the range 2.5-5.0, (b) (c) an agent capable of enhancing the peroxidase activity of hemoglobin, (d) a wetting agent, (e) a solid polymeric film-forming substance and (f) an amine salt of an organic hydroperoxide.
The research and comparison of the ochratoxin A non-toxic system colloidal gold test strip with the traditional colloidal gold test strip, the research and development method of the ochratoxin A non-toxic system colloidal gold test strip is introduced in the food science, 2008, No. 09, and the method comprises the steps of production of colloidal gold, preparation of a gold-labeled antibody, assembly of the non-toxic system test strip and the like. The prepared ochratoxin A non-toxic system colloidal gold test strip is compared with the traditional colloidal gold test strip, and test results show that the detection limit and detection time of the ochratoxin A non-toxic system colloidal gold test strip are the same as those of the traditional colloidal gold test strip, so that the ochratoxin A non-toxic system colloidal gold test strip is suitable for field screening of ochratoxin A.
The detection methods of mycotoxin, veterinary drug residue and illegal addition in the prior art comprise biological identification method, chemical analysis method, instrument analysis method, immunoassay method and the like. The biological identification method has low specificity; the accuracy of the chemical analysis method is low; the instrument has high analysis sensitivity and can accurately carry out quantification, but the sample pretreatment is complex and the detection instrument is expensive, so the instrument is difficult to popularize and use in basic laboratories, common merchants and the like. The immunochromatographic test strip can be used for on-site rapid detection, does not need other auxiliary instruments, can make up for the defects of complexity and time consumption of other detection methods, and is rapidly developed in the field of rapid food safety detection in recent years.
The field needs a test strip which is simple in sample pretreatment, can simultaneously carry out combined detection on the total amount of fumonisins, ochratoxin A, zearalenone, vomitoxin, T2 toxin and aflatoxin in a grain sample, and chloramphenicol, tetracycline, melamine and aflatoxin M1 in a milk sample, and a simple preparation method thereof, and can ensure that the judgment mode of the test result is not complex.
Disclosure of Invention
In order to solve the problems, the inventor provides a manufacturing process and application of a reagent strip for rapidly detecting immunochromatography colloidal gold by multi-joint detection through deep research and cooperative development. The reagent strip realizes the combined detection of various mycotoxins and illegal additives by using the competitive inhibition immunochromatography principle.
In one aspect of the present invention, a multiple detection colloidal gold immunochromatographic test strip comprises: the PVC bottom plate with viscose, along bottom plate width direction from supreme sample pad, the gold mark pad, chromatographic film and the absorption pad that sets gradually down, and adjacent component links up each other.
Preferably, the chromatographic membrane is divided into a quality control region and a detection region.
Preferably, the detection area is continuously divided into a first detection area, a second detection area or more detection areas according to the actual detection requirement.
Preferably, the chromatographic membrane quality control area is coated with quality control points, and the detection area is coated with detection points.
More preferably, the detection spots are labeled with T1-Tn, respectively, depending on the detection item.
Preferably, the detection spots are coated in the detection area in a patterned arrangement order.
According to a preferred embodiment of the invention, the quality control point is a goat anti-rabbit polyclonal antibody, and the detection point is a substance antigen-BSA conjugate to be detected.
More preferably, the antigen-BSA conjugate for each of the spots is different.
In another aspect of the invention, a method for preparing the multiple detection colloidal gold immunochromatographic test strip is provided, which comprises the following steps: arranging a PVC bottom plate with adhesive; the sample pad, the gold label pad, the chromatographic membrane and the absorption pad are sequentially arranged along the width direction of the bottom plate from bottom to top, and adjacent components are connected with each other.
In another aspect of the invention, the application of the multiple detection colloidal gold immunochromatographic test strip is provided, and the multiple detection colloidal gold immunochromatographic test strip is used for carrying out combined detection on fumonisins, ochratoxin A, zearalenone, vomitoxin, T2 toxin and aflatoxin total amount in a grain sample, as well as chloramphenicol, tetracycline, melamine and aflatoxin M1 in a milk sample.
Referring to fig. 1-3, the test strip (i.e., the test card) is from bottom to top: the device comprises a sample adding hole and an observation window, wherein the observation window is divided into a quality control area and a detection area. Wherein the quality control area comprises three control points; the detection areas are respectively coated in the detection areas according to the number of detection items and a certain arrangement mode, and are respectively marked as a detection point 1, a detection point 2, a detection point n and the like from top to bottom or from left to right; and dripping the sample liquid to be detected from the sample adding hole, starting chromatographic reaction, and sequentially flowing through the detection area and the quality control area to perform color reaction. After 10min, the test results were visually observed.
When the test strip is used for detection, a small amount of sample to be detected is extracted, and the sample is subjected to quick and simple pretreatment to obtain a sample solution to be detected; adding enough liquid to be detected into a sample adding hole of a reagent card for chromatographic reaction, wherein if a sample contains one or more substances to be detected, the substance to be detected in the sample is combined with the colloidal gold-labeled specific monoclonal antibody positioned on a gold-labeled pad in the lateral moving process, so that the combination of the gold-labeled antibody and the antigen-BSA conjugate corresponding to the substance on a chromatographic membrane detection point is inhibited, the detection point positioned in a detection area corresponding to the substance does not develop color, and the gold-labeled rabbit IgG antibody is combined with a goat anti-rabbit antibody (quality control point) coated in a chromatographic membrane quality control area for color development; on the contrary, the detection point corresponding to the substance in the detection area is developed; the quality control points were colored in all cases.
In addition, the method has simple and quick sample pretreatment, has less requirements on experimental equipment and personnel operation, and can realize the combined detection of various mycotoxins or illegally added substances by only carrying out sample pretreatment on a small amount of samples once. The prepared chromatographic detection reagent card has good sensitivity, convenient storage and carrying, good stability and storage period at room temperature of up to 12 months. The detection process is simple to operate, and the whole process can be completely finished within 30 minutes. The method is very suitable for on-site rapid detection.
Preferably, the gold-labeled pad protects the glass fiber pad.
Preferably, the glass fibers in the glass fiber mat are surface modified glass fibers, i.e. a surface modified glass fiber mat.
More preferably, the surface of the glass fiber is modified with gamma-glycidoxypropyltrimethoxysilane. That is, the glass fiber mat is a glass fiber mat surface-modified with gamma-glycidoxypropyltrimethoxysilane.
More preferably, the surface modified glass fiber mat is made by a process comprising the steps of:
(1) immersing the glass fiber mat in about 20 wt.% ammonium hydroxide solution overnight, rinsing with purified water, and then rinsing with absolute ethanol;
(2) immersing the pad treated in the step (1) into 30 wt.% of a toluene solution of gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane at room temperature, reacting for 4-8 h at 60-120 ℃, washing with ethanol to remove the solvent, and drying;
(3) reacting the silanized glass fiber mat in the step (2) with 0.5M-1.5M sodium thiosulfate for 6-12 hours at room temperature; and
(4) and (4) after the reaction in the step (3) is finished, adding 0.2-0.5M HCl, adjusting the pH value to 7-8, reacting for 1-3h at room temperature, washing with deionized water, and drying to obtain the gamma-glycidoxypropyltrimethoxysilane-modified glass fiber mat.
Preferably, the alcohol is ethanol, propanol or isopropanol.
In the modification process, sodium thiosulfate and epoxy groups react to form 2-hydroxypropyl thiosulfate, and the 2-hydroxypropyl thiosulfate and hydrochloric acid react to generate thiosulfonyl acid. The modified glass fiber membrane contained about 400-500mmol of thiosulfate and thiosulfate per square meter, so that when the immobilized colloidal gold-antibody conjugate complex was spray-coated on its surface, the thiosulfate and thiosulfate-containing surface was able to bind more uncomplexed antibody and the binding strength was stronger, and the complexed antibody passed through the modified glass fiber pad and then entered the chromatographic detection section. By the modification, a highly efficient and stable glass fiber mat modified with sulfonyl functional groups is obtained. Tests showed that the modified glass fiber mat could be reused at 8 th use with an efficiency of about 86% or more maintained.
The present inventors have also found that the use of glass fibers modified with gamma-glycidoxypropyltrimethoxysilane and sodium thiosulfate, based on the difference in isoelectric points, allows for multiple reuses without significant sensitivity reduction, the number of reuses can be increased by more than 20% with the same sensitivity reduction, and a small amount of sample is required to produce highly reproducible results.
More preferably or particularly, the preparation process of the test strip of the invention comprises the following specific steps:
(1) the composition of colloidal gold immunochromatographic assay card
The PVC bottom plate with the viscose is sequentially provided with a sample pad, a gold label pad, a chromatographic membrane and an absorption pad from bottom to top along the width direction of the bottom plate, and adjacent components are mutually connected.
(2) Preparation of chromatographic membrane:
the chromatographic membrane is divided into a quality control area and a detection area, and the detection area can be continuously divided into a first detection area, a second detection area and the like according to actual detection requirements. Respectively coating quality control points in the chromatographic membrane quality control areas; a detection area wraps a detection point; and respectively labeling with T1-Tn according to different detection items. Coated in the detection area according to a certain arrangement sequence.
Wherein the quality control point is a goat anti-rabbit polyclonal antibody, and the detection point is an antigen-BSA conjugate of a substance to be detected. The antigen-BSA conjugates corresponding to each detection spot were different.
In a preferred embodiment, the chromatographic membrane comprises a nitrocellulose membrane or a nitrocellulose membrane as a matrix.
More preferably, the nitrocellulose membrane is prepared by: (a) providing a base film comprising a polymer comprising 20 to 40 wt.% or less of a polyarylene sulfide, 10 to 20 wt.% of a polyethersulfone, 30 to 60% of a cellulose xanthate; and (b) uniformly coating the base membrane with a coating solution comprising a nitrocellulose compound of a detectable target antibody and a nitrocellulose dissolving agent, wherein the nitrocellulose compound is uniformly distributed over the entire surface of the membrane, and wherein the pore size of the membrane is about 0.2-10 μm.
The present inventors have found that cellulose nitrate is prepared by nitration of natural cellulose, in which process heterogeneous oligomeric and polymeric nitrated products are produced in a broad distribution due to partial acid digestion of the cellulose, resulting in difficulties in achieving uniformity in the manufacture of cellulose nitrate membranes, and rendering such membranes very brittle, easily ruptured and difficult to wet. The composite nitrocellulose membrane of the invention can effectively overcome the defects, the yield in the preparation process is improved by more than 50%, and the composite nitrocellulose membrane is not easy to crack in the use process.
(3) Preparation of gold label pad
And uniformly mixing the colloidal gold antibody markers, fixing the colloidal gold antibody markers on the pretreated glass fiber pad in a spraying manner, and drying, sealing and storing in a dark place.
(4) Assembling the detection card:
the components were cut to the following sizes:
PVC bottom board: 8cm x30 cm
An absorption pad: 30mmx300 mm;
chromatography membrane: 25mm × 300 mm;
gold label pad: 8mmx300 mm;
sample pad: 23mmx300 mm;
sequentially adhering on a PVC base plate, cutting into test strips with the width of 4mm by a numerical control high-speed cutting machine, placing into a plastic card shell, sealing and storing in dark place.
(5) Use of test cards
100ul (or 8-10 drops) of sample solution to be detected is added into the sample adding hole of the detection card, and timing is started. The results were read after 10min, and the rest of the time was invalid.
Preferably, the detection card is respectively from bottom to top: the device comprises a sample adding hole and an observation window, wherein the observation window is divided into a quality control area and a detection area. Wherein the quality control area comprises three control points; the detection areas are respectively coated in the detection areas according to the number of detection items and a certain arrangement mode, and are respectively marked as a detection point 1, a detection point 2, a detection point n and the like from top to bottom or from left to right; and dripping the sample liquid to be detected from the sample adding hole, starting chromatographic reaction, and sequentially flowing through the detection area and the quality control area to perform color reaction. The detection results can be visually observed after 10 min.
The colloidal gold rapid qualitative kit for six-joint toxin detection, which is prepared by the invention, can be used for detecting 6 toxins simultaneously by utilizing a small amount of samples and carrying out simple sample pretreatment. The whole detection process can be completed in 20 minutes. The requirements on experimental equipment and operators are low, and the detection result can be accurately judged and read through visual inspection. The kit has good sensitivity and stability, and the storage period at room temperature can reach 12 months. Provides a simpler, accurate and efficient detection product for the rapid field detection of food safety, and is an excellent choice for the rapid field detection.
The test strip overcomes the defects of complex sample pretreatment method, few disposable detection items, complex result judgment mode and the like in the prior art, and provides a preparation method and application of a colloidal gold immunochromatographic test strip for multi-joint detection.
Drawings
FIGS. 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7 and 1-8 show different distribution patterns of detection spots of a test strip according to the present invention, respectively;
FIGS. 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6 show different distribution patterns of detection spots, respectively, of another test strip according to the present invention;
FIG. 3 is a schematic view of a test card according to the present invention, wherein 1 is a control region, 2 is a first test region, 3 is a second test region, 4 is a chromatographic carrier, and 5 is a well.
Detailed description of the preferred embodiments
In order that the invention may be more clearly understood, reference will now be made in detail to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1. The preparation method of the colloidal gold antibody labeled compound comprises the following steps:
transferring 20ml of colloidal gold solution into 7 centrifugal tubes, respectively, and adding 0.2M K2CO3(50-100ul) adjusting the colloidal gold solution to the pH value required by labeling; diluting fumonisin monoclonal antibody, ochratoxin A monoclonal antibody, zearalenone monoclonal antibody vomitoxin monoclonal antibody, T2 monoclonal antibody aflatoxin total amount monoclonal antibody and rabbit IgG antibody with pure water to 1mg/ml working concentration, and adding into No. 1-7 centrifuge tubes respectively to obtain final concentration of 5-15 ug/ml; reacting at room temperature for 2 hours, adding 1ml of 10% BSA solution for sealing, after fully reacting for 30 minutes, centrifuging at 12000rpm and 4 ℃ for 30 minutes, removing supernatant, re-suspending the colloidal gold-antibody conjugate complex with 1ml of borate buffer solution, uniformly mixing, and storing at 4 ℃ for later use.
2. Preparation of gold label pad
2.1 soaking the gold label pad in the gold label pad treatment solution, sealing for 20min, drying overnight, sealing and storing for later use. The gold pad treatment solution contains (0.05-0.2M pH7.0-9.0) borate buffer solution, sucrose (1% -5%), and Triton-100 (0.1% -0.5%).
2.2 mixing 7 marked colloidal gold-antibody coupling compounds uniformly according to the proportion of 2:1:1:1:1: 1; and fixing the gold label pad on the pretreated gold label pad in a spraying manner, drying for 4 hours, and sealing and storing.
3. Preparation of chromatographic membranes
Diluting fumonisin monoclonal coupled antigen, ochratoxin A monoclonal coupled antigen, zearalenone monoclonal coupled antigen, vomitoxin monoclonal coupled antigen, T2 toxin monoclonal coupled antigen, aflatoxin total amount monoclonal coupled antigen and goat anti-rabbit polyclonal antibody to a working concentration (0.1-1.0mg/ml) by using 10mM PBS solution respectively; coating sheep anti-rabbit in the chromatography membrane quality control area at 50 nl/point and 5mm intervals; coating fumonisins, ochratoxin A and zearalenone in a first detection area from left to right; coating the second detection area with vomitoxin, T2 toxin and total aflatoxin from left to right; drying overnight, sealing and storing.
4. Preparation of test cards
The components were cut to the following sizes:
PVC bottom board: 8cm x30 cm
An absorption pad: 30mmx300 mm;
chromatography membrane: 25mm × 300 mm;
gold label pad: 8mmx300 mm;
sample pad: 23mmx300 mm;
sequentially adhering to the bottom plate, cutting into test strips with width of 4mm with a numerical control high-speed cutting machine, placing into a plastic card shell, sealing, and storing in dark place.
5. Preparation of sample treatment reagents
Accurately transferring 30ml of methanol reagent, dissolving in 70ml of pure water, and uniformly mixing for later use.
6. Kit performance detection
6.1 sensitivity detection
6.1.1 accurately weighing 6 parts of negative standard substance sample, respectively adding standard substance solutions with different concentrations of fumonisin, ochratoxin A, zearalenone, vomitoxin, T2 toxin and aflatoxin, and fully mixing by using a homogenizer for detection.
6.1.2 accurately weighing 1g of homogenized sample, adding 1ml of sample extractant, and fully shaking for 1 minute. Standing for layering, taking 100ul of supernatant, vertically dropping into the sample adding hole, and timing. The results were visually observed after 10 minutes.
The experimental results are as follows: when the total concentration of fumonisin, ochratoxin A, zearalenone, vomitoxin, T2 toxin and aflatoxin in the extracting solution is respectively 25, 10, 25, 10 and 5ng/mL, the colors of six detection points positioned in the first detection area and the second detection area completely disappear respectively, and the amounts of the toxins added in the corresponding samples are respectively 50, 20, 50, 20 and 10 mug/kg, namely the visual qualitative sensitivity of the detection card.
6.2 detection of specificity, repeatability, accuracy, stability
6.2.1 specific assays
Respectively preparing six toxin single solutions and six toxin mixed solutions with the total final concentrations of fumonisins, ochratoxin A, zearalenone, vomitoxin, T2 toxin and aflatoxin of 50ng/mL, 20ng/mL, 50ng/mL, 20ng/mL and 10ng/mL by using sample pretreatment solutions; the 7 parts of the solution and 100ul of the blank sample treatment solution were transferred and dropped into the test card well, and timing was started. The results were visually observed after 10 minutes.
The experimental results are as follows: as shown in fig. 1: wherein 1-1 is a negative control; 1-2 is a detection result of a fumonisin-containing solution, and 1-3 is a detection result of an ochratoxin A-containing solution; 1-4 is the detection result of the zearalenone-containing solution; 1-5 is the detection result of the solution containing vomitoxin; 1-6 is the detection result of the solution containing T2 toxin; 1-7 is the detection result of the total amount of aflatoxin; 1-8 are the detection results of the mixed solution containing 6 kinds of toxin. The blank control detection result of the six-joint toxin detection card is obviously seen by visual inspection, the quality control points and all the detection points are developed with equal development intensity; when detecting a single toxin sample liquid, only the corresponding detection points do not develop color, and the color development conditions of the other detection points are the same as those of the blank control; when the mixed toxin sample is detected, all the corresponding detection points do not develop color, and only the quality control points develop color. The reagent card is excellent in specificity.
6.2.1 repeatability and accuracy testing
6 kinds of toxin mixed solutions with the concentration 0.5 time, 1 time and 2 times of the sensitivity concentration measured in 6.1 are prepared respectively, each concentration solution is detected for 20 times respectively, and result statistics is carried out.
The experimental results are as follows: when the detection liquid is negative, the quality control point and the detection point are all developed, and the development intensity is equivalent; when the concentration of the detection solution is 0.5 times of the sensitivity, the detection point of the detection card is equivalent to or lighter than the color development of the negative control; when the concentration of the detection liquid is 1 time of sensitivity, the detection point of the detection card is not developed at all; in 20 times of repeated determination, 1 false positive result appears at 0.5 times of sensitivity, and the accuracy rate reaches 95%. The detection card has good accuracy and reproducibility.
6.2.3 stability assessment
The test cards are respectively placed in an incubator at 37 ℃ and at normal temperature, and the reagent cards are evaluated at 7 th day, 14 th day, 28 th day, 56 th day, 77 th day, 6 th month, 9 th month and 12 th month to test the color development condition of the reagent cards. After 6 months, the detection point of the detection card is clear, the competitive inhibition reaction is obvious, the visual detection point disappears completely when the detection sensitivity is detected, and the effective service cycle of the detection card can reach 12 months.
Example 2
1. The preparation method of the colloidal gold antibody labeled compound comprises the following steps:
transferring 20ml of colloidal gold solution into 5 centrifugal tubes, respectively, and adding 0.2M K2CO3(50-100ul) adjusting the colloidal gold solution to the pH value required by labeling; respectively diluting chloramphenicol monoclonal antibody, tetracycline monoclonal antibody, melamine monoclonal antibody, aflatoxin M1 monoclonal antibody, and rabbit IgG antibody with pure water to 1mg/ml working concentration, and respectively adding into No. 1-5 centrifuge tube to final concentration of 4-20 ug/ml; reacting at room temperature for 2 hours, adding 100ul of 10% BSA solution for sealing, after reacting fully for 30 minutes, centrifuging at 12000rpm and 4 ℃ for 30 minutes, removing supernatant, resuspending the colloidal gold-antibody conjugate complex with 1ml borate buffer solution, and storing at 4 ℃ for later use after mixing uniformly.
2. Preparation of gold label pad
2.1 soaking the gold label pad in the gold label pad treatment solution, sealing for 20min, drying overnight, sealing and storing for later use. The gold pad treatment solution contains (0.05-0.2M pH7.0-9.0) borate buffer solution, sucrose (1% -5%), and Triton-100 (0.1% -0.5%).
2.2 mixing 7 marked colloidal gold-antibody coupling compounds uniformly according to the proportion of 2:0.5:1:1: 0.5; and fixing the gold label pad on the pretreated gold label pad in a spraying manner, drying for 4 hours, and sealing and storing.
3. Preparation of chromatographic membranes
Respectively diluting chloramphenicol monoclonal antibody, tetracycline monoclonal antibody, melamine monoclonal antibody, aflatoxin M1 monoclonal antibody, and goat anti-rabbit polyclonal antibody to working concentration (0.1-2.0mg/ml) with 10mM PBS solution; coating sheep anti-rabbit in the chromatography membrane quality control area at 50 nl/point and 5mm intervals; coating chloramphenicol and tetracycline in a first detection area from left to right; melamine and aflatoxin M1 coat the second detection zone from left to right; drying overnight, sealing and storing.
4. Preparation of test cards
The components were cut to the following sizes:
PVC bottom board: 8cm x30 cm
An absorption pad: 30mmx300 mm;
chromatography membrane: 25mm × 300 mm;
gold label pad: 8mmx300 mm;
sample pad: 23mmx300 mm;
sequentially adhering to the bottom plate, cutting into test strips with width of 4mm with a numerical control high-speed cutting machine, placing into a plastic card shell, sealing, and storing in dark place.
5. Preparation of sample treatment reagents
Accurately moving 1ml of milk sample to be detected, and using pure water 1:1, diluting and uniformly mixing for later use.
6. Kit performance detection
6.1 sensitivity detection
6.1.1 accurately absorbing 4 samples of the negative standard substance, respectively adding standard substance solutions of chloramphenicol, tetracycline, melamine and aflatoxin M1 with different concentrations, and fully and uniformly mixing to be detected.
6.1.2 accurately sucking 1ml of the standard sample, adding 1ml of pure water, and fully shaking and uniformly mixing for 30 seconds. 100ul of the solution to be detected is vertically dropped into the sample adding hole, and timing is carried out. The results were visually observed after 10 minutes.
The experimental results are as follows: when the concentrations of chloramphenicol, tetracycline, melamine and aflatoxin M1 in the solution to be detected are respectively 0.05, 10, 25 and 0.25ng/mL, the colors of the 4 detection points positioned in the first detection area and the second detection area completely disappear respectively, and the corresponding detection substances added into the sample are respectively 0.1, 20, 50 and 0.5ng/mL, namely the visual qualitative sensitivity of the detection card.
6.2 detection of specificity, repeatability, accuracy, stability
6.2.1 specific assays
Respectively preparing four single solutions of the object to be detected and four mixed solutions of the object to be detected, wherein the final concentrations of chloramphenicol, tetracycline, melamine and aflatoxin M1 are respectively 0.1ng/mL, 20ng/mL, 50ng/mL and 0.5ng/mL by using the sample pretreatment solution; the above 5 parts of the solution and 100ul of the blank sample treatment solution were transferred and dropped into the test card well, and timing was started. The results were visually observed after 10 minutes.
The experimental results are as follows: as shown in fig. 2: wherein 2-1 is a negative control; 2-2 is the detection result of the solution containing chloramphenicol, and 2-3 is the detection result of the solution containing tetracycline; 2-4 is the detection result of the solution containing melamine; 2-5 is the detection result of the solution containing aflatoxin M1; and 2-6 are detection results of mixed solutions containing 4 substances to be detected. The blank control detection result of the milk quadruple detection card is obviously visible by visual inspection, the quality control points and all the detection points are developed with equivalent development intensity; when a single sample liquid of the object to be detected is detected, only the corresponding detection points do not develop color, and the color development conditions of the other detection points are the same as those of the blank contrast; when the mixed sample to be detected is detected, all the corresponding detection points do not develop color, and only the quality control points develop color. The reagent card is excellent in specificity.
6.2.1 repeatability and accuracy testing
4 kinds of mixed solutions of the sample to be detected with the concentration 0.5 times, 1 time and 2 times of the sensitivity concentration measured in 6.1 are prepared respectively, each solution with the concentration is detected for 20 times respectively, and result statistics is carried out.
The experimental results are as follows: when the detection liquid is negative, the quality control point and the detection point are all developed, and the development intensity is equivalent; when the concentration of the detection solution is 0.5 times of the sensitivity, the detection point of the detection card is equivalent to or lighter than the color development of the negative control; when the concentration of the detection liquid is 1 time of sensitivity, the detection point of the detection card is not developed at all; in 20 times of repeated determination, 1-time false negative result appears at 1-time sensitivity, and the accuracy rate reaches 95%. The detection card has good accuracy and reproducibility.
6.2.3 stability assessment
The test cards are respectively placed in an incubator at 37 ℃ and at normal temperature, and the reagent cards are evaluated at 7 th day, 14 th day, 28 th day, 56 th day, 77 th day, 6 th month, 9 th month and 12 th month to test the color development condition of the reagent cards. After 6 months, the detection point of the detection card is clear, the competitive inhibition reaction is obvious, the visual detection point disappears completely when the detection sensitivity is detected, and the effective service cycle of the detection card can reach 12 months.
The above embodiments clearly show that the detection card of the present invention adopts a unique dot-shaped chromatographic film coating technique, which has obvious advantages in the field of multi-link detection compared with the conventional linear coating method. The demand of the point-shaped coating on the raw materials is greatly reduced, the cost of the raw materials is greatly reduced, and the number of detection points which can be coated in a limited chromatographic membrane color development area is also obviously increased due to the small volume and the tight distribution of the detection points, and can reach dozens or even dozens of times. By using the coating technology, the number of detection items which can be borne by the detection card is obviously increased under the condition that the conditions allow, and the method is an innovation of the joint inspection card manufacturing process.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to make and use the invention. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. All citations referred to herein are incorporated herein by reference to the extent that no inconsistency is made.
Claims (10)
1. A colloidal gold immunochromatographic test strip for multiplex detection comprises: the PVC base plate with the adhesive comprises a sample pad, a gold label pad, a chromatographic membrane and an absorption pad which are sequentially arranged along the width direction of the base plate from bottom to top, wherein adjacent components are mutually connected; wherein
The gold-labeled pad is a glass fiber pad, the glass fiber in the glass fiber pad is surface-modified glass fiber, the surface of the glass fiber is modified by gamma-glycidoxypropyltrimethoxysilane, and the surface-modified glass fiber pad is prepared by the following steps:
(1) immersing the glass fiber mat in about 20 wt.% ammonium hydroxide solution overnight, rinsing with purified water, and then rinsing with absolute ethanol;
(2) immersing the pad treated in the step (1) into 30 wt.% of a toluene solution of gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane at room temperature, reacting for 4-8 h at 60-120 ℃, washing with ethanol to remove the solvent, and drying;
(3) reacting the silanized glass fiber mat in the step (2) with 0.5M-1.5M sodium thiosulfate for 6-12 hours at room temperature; and
(4) and (4) after the reaction in the step (3) is finished, adding 0.2-0.5M HCl, adjusting the pH value to 7-8, reacting for 1-3h at room temperature, washing with deionized water, and drying to obtain the gamma-glycidoxypropyltrimethoxysilane-modified glass fiber mat.
2. The multiple detection colloidal gold immunochromatographic strip according to claim 1, wherein the chromatographic membrane is divided into a quality control region and a detection region.
3. The multiple detection colloidal gold immunochromatographic test strip according to claim 2, wherein the detection area is continuously divided into a first detection area, a second detection area or more according to actual detection requirements.
4. The multiple detection colloidal gold immunochromatographic strip in accordance with claim 3, wherein a control point is coated on the control area of the chromatographic membrane and a detection point is coated on the detection area, respectively.
5. The multiple detection colloidal gold immunochromatographic strip according to claim 4, wherein the detection spots are labeled with T1-Tn respectively according to different detection items.
6. The multiple detection colloidal gold immunochromatographic strip according to claim 5, wherein the detection spots are coated in the detection area in a patterned arrangement order.
7. A multi-connected detection colloidal gold immunochromatographic strip according to any one of claims 4 to 6, wherein the quality control point is a goat anti-rabbit polyclonal antibody, and the detection point is a substance antigen-BSA conjugate to be detected.
8. The multiple detection colloidal gold immunochromatographic strip according to claim 7, wherein the antigen-BSA conjugate for each detection spot is different.
9. The method for preparing the multiple detection colloidal gold immunochromatographic test strip according to any one of claims 1 to 8, which comprises the following steps: arranging a PVC bottom plate with adhesive; the sample pad, the gold label pad, the chromatographic membrane and the absorption pad are sequentially arranged along the width direction of the bottom plate from bottom to top, and adjacent components are connected with each other.
10. The use of the concatenated colloidal gold immunochromatographic test strip for detection according to any one of claims 1 to 8 or the concatenated colloidal gold immunochromatographic test strip prepared by the method according to claim 9, for combined detection of fumonisins, ochratoxin a, zearalenone, vomitoxin, T2 toxin, and total aflatoxins in grain samples, and chloramphenicol, tetracycline, melamine, and aflatoxin M1 in milk samples.
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| CN104749358A (en) * | 2014-05-14 | 2015-07-01 | 陈岩松 | Semiquantitative immunochromatographic test method and test paper |
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