CN113063938A - High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method - Google Patents
High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
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- 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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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
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- 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
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Abstract
The invention relates to a high-sensitivity gradient semi-quantitative immunochromatographic detection test strip, which comprises a test strip and a sample cup; the test paper comprises a supporting layer, an adsorption layer and a protective layer; the adsorption layer comprises a sample pad, a combination pad, a cellulose membrane layer and a water absorption material layer in sequence; the cellulose membrane layer is provided with a detection line T1, a detection line T2 and a quality control line; the combination pad is made of glass fiber cotton adsorbed with nanometer material labeled antigen; the sample cup is a container for diluting the sample, and a target monoclonal antibody diluted by the gold-labeled protein heavy-suspension liquid is pre-fixed in the sample cup. The test paper detection method can prepare two detection lines which are not interfered with each other, and realizes gradient semi-quantification; the antibody is independently dried in the sample cup, so that folding and damage in the antibody marking process are avoided, and the using amount of the antibody can be accurately controlled; the nanoparticle-labeled artificial antigen is used as a probe, each antibody intercepted on the detection line T2 can be accurately combined with two labeled antigens, signal self-amplification is realized, and the sensitivity is higher compared with that of the traditional method.
Description
Technical Field
The invention relates to an immunochromatographic assay, in particular to a high-sensitivity gradient semi-quantitative immunochromatographic assay test strip and a detection method.
Background
The immunochromatography technology is a rapid immunoassay technology developed in recent years, and the basic principle of the immunochromatography technology is the interaction between an antibody and an antigen, and the immunochromatography detection technology takes a nitrocellulose membrane as a carrier and a labeled antigen or an antibody as a tracer. Compared with the traditional immunoassay method, the immunochromatography technology has the advantages of strong specificity, accurate detection result, simple equipment operation, quick measurement, low cost, no need of skilled technicians or expensive equipment and the like, and completely meets the requirement of instant detection. At present, immunochromatography is widely used in the fields of medicine, animal husbandry, agriculture, food safety and the like.
In the field of food safety, the small molecule antigen immunochromatographic technology based on the competitive principle is most widely applied, however, the existing detection mode usually has the problems that the sensitivity and the affinity of an antibody are good, but the sensitivity of the established immunochromatographic test paper is far less good than that of the established ELISA detection method, or the color development of the test paper detection line is weak, and the like. The main reasons for this are the following three aspects: 1. the antibody is folded in the labeling process, and excessive antibody is added for stabilizing the labeling material; 2. the efficiency of intercepting the antibody by the artificial antigen is low, and excessive labeled antibody must be added to improve the color development; 3. when the colloidal gold is used for marking the antibody, no signal amplification effect exists, and the detection instrument is required to judge the result while the fluorescent materials such as quantum dots and the like realize signal amplification, so that the detection is complicated. The existing small molecule immunochromatographic test mode cannot solve the problem, so that a feasible and sensitive test paper test mode is urgently needed, and more methods are provided for the research and development of immunochromatographic test paper.
Meanwhile, there is another problem in small molecule detection: many targets need different limit standards in different samples or need gradient semi-quantification, only one detection line is provided under naked eye observation in a traditional immunochromatographic test paper mode to realize semi-quantification, and some researchers spray a plurality of detection lines by using artificial antigens with different concentrations to realize gradient semi-quantification, but the mutual influence among the detection lines is large, and the gradient semi-quantification is difficult to realize, so that the preparation of a non-interfering gradient semi-quantitative immunochromatographic method is urgently required.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a high-sensitivity gradient semi-quantitative immunochromatography detection test strip and a detection method, wherein a colloidal gold/quantum dot and other marking materials are used for marking an artificial antigen; fixing a secondary antibody or SPA on a detection line on the nitrocellulose membrane, and preparing a quality control line by using an independent quality control (C) line system; the monoclonal antibody is diluted by the antibody protective solution and dried in the sample cup, compared with the traditional method, the detection method has higher sensitivity, and two detection lines which are not interfered with each other can be prepared, so that gradient semi-quantification is realized.
In order to achieve the purpose, the invention adopts the technical scheme that:
a high-sensitivity gradient semi-quantitative immunochromatographic test strip consists of a test paper and a sample cup; the test paper comprises a supporting layer, an adsorption layer and a protective layer, wherein the adsorption layer and the protective layer are fixed on the supporting layer; the adsorption layer comprises a sample pad, a combination pad, a cellulose membrane layer and a water absorption material layer at the handle end from the testing end in sequence; the protective layer is fixed on the sample pad, the combination pad and the water absorption material layer; the cellulose membrane layer is sequentially provided with a detection line T1, a detection line T2 and a quality control line; the combination pad is made of glass fiber cotton adsorbed with nano material labeled antigen; the sample cup is a container for diluting a sample, and a target object monoclonal antibody diluted by the gold-labeled protein heavy-suspension liquid is pre-fixed in the sample cup.
The nano material labeled antigen is an artificial antigen labeled by colloidal gold, quantum dots or silicon sphere nano material.
The artificial antigen is prepared by coupling a small molecule hapten and a carrier protein; the carrier protein coupled with the artificial antigen is bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.
The sample pad is made of glass fiber cotton, a nylon film, a polyvinylidene fluoride film or a polyester film; the water absorbing material layer is made of water absorbing filter paper; the supporting layer is made of a non-water-absorbing tough material; the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane.
The detection lines T1, T2 and quality control lines are "| |" linear blots arranged in parallel, or "decacross" -shaped blots, or "T-type blots," T-T "-shaped blots, or" support "shaped blots, or" ┤ ┤ ┤ "-shaped blots.
The detection line T1 is sprayed with 2,4-D monoclonal antibody, the detection line T2 is sprayed with goat anti-mouse IgG, rabbit anti-mouse IgG or SPA, and the quality control line is sprayed with avidin.
The artificial antigen is a 2,4-D artificial antigen; 2,4-D monoclonal antibodies diluted by gold-labeled protein resuspension are pre-fixed on the sample cup; the gold-labeled protein resuspension is 20mmol/L boric acid buffer containing 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide.
The preparation method of the sample cup comprises the following steps: diluting the 2,4-D monoclonal antibody to 10 mu g/mL by using the gold-labeled protein resuspension, adding the diluted monoclonal antibody into a sample cup, freeze-drying and sealing the sample cup, and storing the sample cup at 4 ℃ for later use.
The preparation method of the combined pad comprises the following steps:
(1) diluting the artificial antigen 2,4-D-BSA to 1mg/mL by using ultrapure water, dropwise adding the 2,4-D-BSA solution with the concentration of 1mg/mL into 100mL of colloidal gold solution according to the proportion of adding 9 mu L of 2,4-D-BSA solution into 1mL of colloidal gold solution, reacting for 30min at room temperature, 5% (v/v) casein to the final concentration of 0.5%, reacting for 10min at room temperature, centrifuging for 30min at 4 ℃ and 12000r/min, discarding the supernatant, and re-suspending the precipitate by using 25mL of gold-labeled protein heavy suspension to obtain the gold-labeled 2, 4-D-BSA; preparing a gold-labeled biotin-BSA solution by the same method, and storing at 4 ℃ for later use;
(2) and (3) during gold spraying, mixing the gold-labeled 2,4-D-BSA and the gold-labeled biotin-BSA in a mass ratio of 1:1, spraying the gold-labeled 2,4-D-BSA and the gold-labeled biotin-BSA mixed solution on glass fiber cotton by using a spraying instrument according to a mass ratio of 8 mu L/cm, and performing vacuum drying at a low temperature of 4 ℃ to prepare the binding pad.
The detection method of the immunochromatographic test strip comprises the following steps: adding 80-150 mu L of sample to be detected into a sample cup, uniformly mixing, inserting the test end of the test strip into the sample cup, reading the result for 5-10min, and when the detection line T1 and the detection line T2 are developed, indicating that the sample does not contain the object to be detected or contains the concentration lower than the detection limit of T2; when the detection line T1 is colored and the detection line T2 is not colored, the concentration of the substance to be detected in the sample is higher than the detection limit of T2 and lower than the detection limit of T1; when the detection line T1 and the detection line T2 do not develop color, the concentration of the substance to be detected in the sample is larger than the detection limit of T1; the quality control line is always colored, and when the quality control line is not colored, the operation is wrong or the test paper is invalid.
The invention has the beneficial effects that:
the test strip is prepared on the basis of a competition mode, and compared with a traditional mode, the test strip has the following advantages:
1. the sensitivity is high.
(1) The antibody is independently dried in the sample cup, the antibody is firstly combined with the antigen in the sample during detection, and the measure avoids the folding of the antibody in the process of labeling the antibody and can accurately control the dosage of the antibody, thereby increasing the sensitivity of the test paper.
(2) The nano material labeled antibody is changed into a nano material labeled artificial antigen, and the amount of the nano material labeled antigen is controlled to be sufficient, so that each monoclonal antibody intercepted on the detection line T2 can be combined with 2 labeled antigens, thereby realizing the amplification of signals and increasing the sensitivity of the test paper; and the remaining labeled antigens can be guaranteed to be intercepted and developed by the monoclonal antibody on the detection line T1, and the excessive labeled antigens do not interfere the detection of the detection line T2, so that the gradient semi-quantitative detection can be realized.
2. The test strip can provide two detection lines which are not interfered with each other, set the gradient detection range, provide more accurate detection results and enable the application of the immunochromatographic test strip to be wider.
3. Simple and fast. The test strip of the invention can be used without any other reagent and instrument, and can be operated on site. And (3) dripping 100 mu L of sample solution on the test strip sample pad or inserting the test end of the test strip below the warning line into the sample solution to be detected, and judging the detection result after about 5 minutes.
4. The result display is visual, intuitive and accurate. The test strip is imprinted with an indicator (or "ten", "< - >", "" T "," < - > "," "┤") as a positive and negative marker of the detection result. When the detection line T1 and the detection line T2 both develop color, it indicates that the sample does not contain the target or contains the target at a concentration lower than the detection limit of T2; when the detection line T1 is colored and the detection line T2 is not colored, the concentration of the target substance contained in the sample is higher than the detection limit of T2 and lower than the detection limit of T1; when the detection line T1 and the detection line T2 do not develop color, the concentration of the target contained in the sample is more than the detection limit of T1; the quality control line is always colored, and when the quality control line is not colored, the operation is wrong or the test paper is invalid. The result judgment is visual, intuitive and accurate, is simple and clear, and is not easy to cause false positive, false negative and other artificial misjudgments.
5. The cost is saved. The rapid detection test strip is used, no additional instrument and other reagents are needed, the detection can be carried out at any time and any place, the cost is low, and a large amount of expensive instruments and equipment cost can be saved.
The detection principle of the invention is as follows:
the principle of the immunochromatographic test is that a labeling material is combined with a target substance artificial antigen to form a labeling antigen, the labeling antigen is adsorbed on glass fiber cotton, one end of the labeling antigen is connected with a Nitrocellulose (NC) membrane which is fixed with a target substance monoclonal antibody (a detection line T1), a secondary antibody or SPA (a detection line T2) and an independent quality control line (a C line, such as a biotin avidin system), and the other end of the labeling antigen is connected with a sample pad. And then the test paper is manufactured and assembled together with other needed water-absorbing fibers, supporting materials, covering materials and the like according to a design process to manufacture the rapid detection test paper, and meanwhile, the target monoclonal antibody is fixed in the sample cup. When the detection sample does not contain the target or the concentration of the target is less than the detection limit of the detection line T2, free antibodies in the sample cup react with the marked artificial antigens to form a compound, the compound is intercepted by the fixed second antibody or SPA when flowing through the detection line T2, an obvious and visual band appearing due to the accumulation of the marked materials is the detection line T2, the obvious and visual band appearing by the fixed target monoclonal antibody when the excessive marked artificial antigens flow through the detection line T1 is the detection line T1, if the detection sample contains the target and the concentration of the target is more than the detection limit of the detection line T2, the target is combined with the free target monoclonal antibodies in the sample cup, because the monoclonal antibody binding site is blocked and will not react with the labeled artificial antigen any more, the target antibody will not be labeled, when captured by the immobilized secondary antibody or SPA as it flows through detection line T2, the band will no longer be displayed. When the concentration of the target object is less than the detection limit of the detection line T1, the target object antibody fixed on the detection line T1 is not completely sealed by the antigen, and an interception and marking artificial antigen display strip is displayed; when the concentration of the target substance is greater than the detection limit of the detection line T1, the target substance antibody fixed on the detection line T1 is completely sealed by the antigen, so that the artificial antigen is no longer intercepted and labeled, and a strip is not displayed; independent quality control line: for example, the complex gold-labeled biotin-BSA will be intercepted by the avidin immobilized on the membrane to show that the band is a quality control line.
Drawings
FIG. 1 is a schematic diagram of the test paper strip of the present invention.
FIG. 2 is a schematic cross-sectional view of the test strip of the present invention.
In the figure, 1 is a protective layer covering the sample pad 2 and the bonding pad 4, 2 is a sample pad, 3 is a support layer, 4 is a bonding pad, 5 is a cellulose membrane layer, 6 is a detection line (detection line T1 and detection line T2), 7 is a quality control line, 8 is a protective layer covering the water absorbent material layer, and 9 is a water absorbent material layer.
Detailed Description
The following examples are given to illustrate specific embodiments of the present invention and are not intended to represent all possible embodiments of the present invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in the examples, and those skilled in the art can implement the immunochromatography or prepare test strips described in the present invention by using other similar materials or reaction conditions according to the principle of the present invention, and these modifications are within the scope of the present invention.
Example 1
A high-sensitivity gradient semi-quantitative immunochromatographic test strip for rapidly detecting trace 2,4-D comprises a test paper and a sample cup; the test paper comprises a supporting layer, an adsorption layer and a protective layer, wherein the adsorption layer and the protective layer are fixed on the supporting layer; the adsorption layer comprises a sample pad, a combination pad, a cellulose membrane layer and a water absorption material layer at the handle end from the testing end in sequence; the protective layer is fixed on the sample pad, the combination pad and the water absorption material layer; the cellulose membrane layer is sequentially provided with a detection line T1, a detection line T2 and a quality control line; the combination pad is made of glass fiber cotton adsorbed with nano material labeled antigen; the sample cup is a container for diluting a sample, and a 2,4-D monoclonal antibody diluted by a gold-labeled protein resuspension is pre-fixed on the sample cup.
The nano material labeled antigen is 2,4-D artificial antigen labeled by colloidal gold, quantum dots or silicon sphere nano material; the artificial antigen is prepared by coupling a small molecule hapten and a carrier protein; the carrier protein coupled with the artificial antigen is bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.
The sample pad is made of glass fiber cotton, a nylon film, a polyvinylidene fluoride film or a polyester film; the water absorbing material layer is made of water absorbing filter paper; the supporting layer is made of a non-water-absorbing tough material; the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane.
The detection lines T1, T2 and quality control lines are "| |" linear blots arranged in parallel, or "decacross" -shaped blots, or "T-type blots," T-T "-shaped blots, or" support "shaped blots, or" ┤ ┤ ┤ "-shaped blots.
The detection line T1 is sprayed with 2,4-D monoclonal antibody, the detection line T2 is sprayed with SPA, and the quality control line is sprayed with avidin.
And protective films are covered on the sample pad, the combination pad and the water absorbing material layer, and a sample mark line is printed on the protective film corresponding to the junction of the sample pad and the combination pad and is 0.3-0.7cm away from one side of the sample pad.
Example 2
The preparation method of the high-sensitivity gradient semi-quantitative immunochromatographic test strip for trace 2,4-D detection comprises the following steps:
1. preparation of 2,4-D Artificial antigen (2,4-D-BSA)
Weighing 2,4-D100mg, adding 200 mu L of methanol for dissolving, adding 7mL of PBS, then leading the solution to become turbid, then dropwise adding 1mol/L NaOH solution until the solution is clear, and then adjusting the pH value of the solution to be neutral by 0.1MHCl, thus obtaining solution A; weighing 15mgBSA (bovine serum albumin) and dissolving in 300 μ LPBS to obtain solution B; slowly adding the solution A into the solution B under the stirring state at 4 ℃, then adding 100mg of carbodiimide, reacting for 12 hours under the stirring state at 4 ℃, taking out a reaction product, and performing PBS flow dialysis for 72 hours at-20 ℃ for later use.
2. Preparation of 2,4-D monoclonal antibody
The prepared 2,4-D-BSA is used for immunizing Balb/C mice with the age of 6-8 weeks at the dosage of 20 mu g of protein/200 mu L/mouse each time, 4-6 points of subcutaneous injection are injected on the back, 4 times are carried out in total, and the immunization interval time is 3 weeks. First-stage immunization, diluting the prepared 2,4-D-BSA with sterile PBS, mixing with equivalent Freund's Complete Adjuvant (FCA), and fully emulsifying; the immunization was boosted 1 time with 2,4-D-BSA diluted in sterile PBS, and with equal amounts of Freund' sMixing incomplete adjuvants (FIA), emulsifying, 4 th immunizing for 7 days, measuring serum antibody titer and inhibition titer, and screening mice with high titer and good inhibition effect as fusion mice. The fusion mice were immunized with ultra-strong power, and 2,4-D-BSA was diluted with sterile PBS to 50. mu.g protein/200. mu.L, and was injected intraperitoneally without adjuvant. Collecting blood from infraorbital sinus of the immunized mouse after 3-4 days, and separating positive serum; removing neck and killing, soaking the mouse in 75% (v/v) alcohol for 5-10min to sterilize the body surface, taking out the spleen aseptically, cutting and grinding the spleen, filtering through a 120-mesh nylon gauze, centrifuging at 1000r/min for 10min, and collecting splenocytes. Mixing the separated spleen cells and NS0 myeloma cells according to a ratio of 10:1, centrifuging at 1000r/min for 10min, discarding supernatant, slowly adding 0.7-1.0 mL of 50% polyethylene glycol 4000(v/v) into cell precipitates in a water bath at 37 ℃, adding within 1min, adding 0.1-0.3 mL in the first 30s, adding 0.2-0.4 mL in the middle 15s, and adding 15s finally; then, 15mL of serum-free 1640 medium was slowly added to stop the action of PEG, the mixture was centrifuged at 37 ℃ for 5-10min and 1000r/min for 10min to discard the supernatant, the cell pellet was resuspended in HAT (H-Hypoxanthine Hypoxanthine, A-Aminopterin, T-Thymidine Thymidine) selection medium, and the mixture was added to a 96-well cell culture plate (8-10 plates) at a concentration of 100. mu.L-200. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator. Culturing for 10-14 days, screening positive holes by using an indirect ELISA method, selecting holes with strong positive, high inhibition rate and vigorous cell growth, carrying out limited dilution and cloning for 2 times, then carrying out expanded culture, and establishing a hybridoma cell strain.
Preparing antibody in batches by in vivo induced ascites method, collecting SPF BALB/C mice, injecting 400 μ L/mouse of liquid paraffin into abdominal cavity, diluting with culture medium RMPI-1640 to concentration of 1 × 10 after 7-10 days6Each cell/mL of hybridoma cells was injected into the abdominal cavity at 500. mu.L/cell. When the abdominal cavity of the mouse is swollen and the skin is tight, ascites is collected, centrifuged at 3000r/min for 5min, and the precipitate is discarded. Then, ascites was purified by ammonium octylate method, 1mL of ascites was diluted with 4mL of acetic acid-sodium acetate buffer solution (pH 4.0), and the pH of the acetic acid-sodium acetate buffer solution of ascites was adjusted to 4.5 with 1M of NaOH solution. Then slowly stirring and adding 130 mu L of octanoic acid, stirring and reacting for 30min at room temperature, 6000r/mCentrifuging in for 30min, removing precipitate, filtering the supernatant with filter paper to remove impurities, mixing the filtrate with 10 times of concentrated Phosphate Buffer Solution (PBS) at a volume ratio of 9:1, adjusting pH to 7.4 with 1M NaOH, and cooling in ice bath. Adding ammonium sulfate powder at 0.2778g/mL, stirring at 4 deg.C for 2h, centrifuging at 6000r/min for 20min, removing supernatant, dissolving precipitate with 1mL PBS, dialyzing with PBS for 3d, centrifuging at 6000r/min for 10min, discarding precipitate, and freezing at-20 deg.C. Through detection, the prepared monoclonal antibody 1F11 can specifically react with 2,4-D and can be used for preparing 2,4-D immunochromatography test paper.
3. Preparation of gold-labeled protein and conjugate pad
Preparing colloidal gold solution by adopting a sodium citrate reduction method. Adding 99mL of ultrapure water into a clean 200mL conical flask with scales, placing the conical flask on a heating magnetic stirrer for heating and stirring, adding 1mL of 1% (w/v) chloroauric acid solution, heating to boiling, then quickly adding 1.6mL of 1% (w/v) trisodium citrate solution, continuously stirring and heating the solution to change the color through transparent-black-deep red-wine red, heating for 5min when the color does not change any more, cooling at room temperature, then using ultrapure water to fix the volume of the colloidal gold to 100mL, and storing at 4 ℃ for later use.
Diluting 2,4-D-BSA with ultrapure water to a concentration of 1mg/mL, adding 1mg/mL 2,4-D-BSA solution dropwise into 100mL colloidal gold solution according to a proportion of 9. mu.L 2,4-D-BSA solution into 1mL colloidal gold solution, reacting at room temperature for 30min, 5% (v/v) casein to a final concentration of 0.5%, reacting at room temperature for 10min, centrifuging at 4 ℃ and 12000r/min for 30min, discarding the supernatant, resuspending the precipitate with 25mL gold-labeled protein resuspension solution (20mmol/L boric acid buffer solution containing 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide), preparing gold-labeled biotin-BSA solution by the same method, and storing at 4 ℃ for later use.
And during gold spraying, mixing the gold-labeled 2,4-D-BSA and the gold-labeled biotin-BSA in a mass ratio of 1:1 to obtain a gold-labeled protein mixed solution, spraying the gold-labeled protein mixed solution onto glass fiber cotton by using a spraying instrument according to a mass ratio of 8 mu L/cm, and performing vacuum drying at a low temperature of 4 ℃ to prepare the bonding pad.
4. Assembly of test strips
Spraying avidin on a nitrocellulose membrane (NC membrane) C line position; the 2,4-D monoclonal antibody is sprayed on the NC membrane T1 line; SPA spraying on NC membrane at T2 line position, drying at 42 deg.C for 4h, adding desiccant, sealing to obtain cellulose membrane layer, and keeping. Then the sample pad, the combination pad, the cellulose membrane layer, the water absorption material layer and the protective layer are sequentially stuck on the bottom plate of the supporting layer according to the figure 1, the components are overlapped by 1-2mm, and the components are cut into the test paper strip by a cutting machine.
5. Preparation of sample cups
Diluting the 2,4-D monoclonal antibody to 10 mu g/mL by using the gold-labeled protein resuspension, adding the diluted monoclonal antibody into a sample cup, freeze-drying and sealing the sample cup, and storing the sample cup at 4 ℃ for later use.
6. Detection method of immunochromatographic test paper
Adding 80-150 mu L of sample to be detected into a sample cup, uniformly mixing, inserting a test end of the test paper into the sample cup for detection, reading the result within 5-10min, and indicating that the sample does not contain 2,4-D or contains 2,4-D with the concentration lower than the detection limit of T2 when the test paper T1 and T2 lines are colored; when the test paper T1 line is developed and the T2 line is not developed, the concentration of 2,4-D contained in the sample is more than the detection limit of T2 and less than the detection limit of T1; when the test paper T1 and the test paper T2 do not develop color, the concentration of 2,4-D contained in the sample is larger than the detection limit of T1; the C line is always colored, and when the C line is not colored, the operation is wrong or the test paper is invalid.
7. Sensitivity detection
The 2,4-D colloidal gold immunochromatographic test strip prepared above was used to detect a sample of 2,4-D standard PBS solution at a concentration of 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, or 6.25 ng/mL. The result shows that the T1 and T2 lines of the test paper strip do not develop color when the concentration of 2,4-D is more than 1000ng/mL, the T2 line of the test paper strip T1 does not develop color when the concentration of 2,4-D is more than 25ng/mL and less than 1000ng/mL, and the T1 and T2 lines develop color when the concentration of 2,4-D is more than 25ng/mL, so that the T2 line detection limit of the 2,4-D high-sensitivity gradient semi-quantitative immunochromatographic test paper strip is determined to be 25ng/mL, and the T1 line detection limit is determined to be 1000 ng/mL.
Comparative example
A2, 4-D colloidal gold immunochromatographic test strip adopting a traditional detection mode is adopted, wherein 2,4-D artificial antigen is adsorbed on a detection line on a nitrocellulose membrane, SPA is adsorbed on a quality control line, and a 2,4-D monoclonal antibody is labeled by colloidal gold and is used as a gold-labeled antibody fiber layer.
When in use: spraying the 2,4-D monoclonal antibody marked by the colloidal gold on a nitrocellulose membrane combined pad, drying for 1h at 42 ℃, and adding a drying agent for sealing for later use. Spraying SPA at the C line (quality control line) position of NC membrane at concentration of 0.2mg/mL, spraying 2,4-D artificial antigen at the T line (detection line) position of NC membrane at concentration of 0.9mg/mL, drying at 42 deg.C for 4h, adding desiccant, and sealing for use. Then sequentially adhering the NC film, the combination pad, the sample pad, the water absorption layer, the protective layer and the like on the supporting bottom plate, overlapping 1-2mm among the components, and cutting into the test paper strip by using a cutting machine.
Through sensitivity detection, the sensitivity of the colloidal gold immunochromatographic test strip in the 2,4-D traditional detection mode is 1000 ng/mL. The comparison shows that the sensitivity of the 2,4-D novel colloidal gold immunochromatographic test strip is improved by 40 times compared with that of the 2,4-D traditional colloidal gold immunochromatographic test strip, two gradient detection limits of 25ng/mL and 1000ng/mL are provided, and the gradient detection is realized.
Claims (10)
1. A high-sensitivity gradient semi-quantitative immunochromatographic assay test strip is characterized by consisting of a test strip and a sample cup; the test paper comprises a supporting layer, an adsorption layer and a protective layer, wherein the adsorption layer and the protective layer are fixed on the supporting layer; the adsorption layer comprises a sample pad, a combination pad, a cellulose membrane layer and a water absorption material layer at the handle end from the testing end in sequence; the protective layer is fixed on the sample pad, the combination pad and the water absorption material layer; the cellulose membrane layer is sequentially provided with a detection line T1, a detection line T2 and a quality control line; the combination pad is made of glass fiber cotton adsorbed with nano material labeled antigen; the sample cup is a container for diluting a sample, and a target object monoclonal antibody diluted by the gold-labeled protein heavy-suspension liquid is pre-fixed in the sample cup.
2. The immunochromatographic assay test strip of claim 1, wherein the nanomaterial-labeled antigen is an artificial antigen labeled with colloidal gold, quantum dots, or silica spheres nanomaterial.
3. The immunochromatographic assay test strip of claim 1, wherein the artificial antigen is prepared by coupling a small molecule hapten and a carrier protein; the carrier protein coupled with the artificial antigen is bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.
4. The immunochromatographic test strip of claim 2, wherein the sample pad is made of glass fiber cotton, a nylon membrane, a polyvinylidene fluoride membrane, or a polyester membrane; the water absorbing material layer is made of water absorbing filter paper; the supporting layer is made of a non-water-absorbing tough material; the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane.
5. The immunochromatographic test strip of claim 3, wherein the detection line T1, the detection line T2 and the quality control line are in parallel arranged "| |" straight-line blots, or are in parallel arranged "| | |", straight-line blots
A font arrangement print or
A font arrangement print or
A font arrangement print or
A font arrangement print or
And (5) arranging the print in a font manner.
6. The immunochromatographic test strip of claim 1, wherein the detection line T1 is coated with 2,4-D monoclonal antibody, the detection line T2 is coated with goat anti-mouse IgG, rabbit anti-mouse IgG, or SPA, and the quality control line is coated with avidin.
7. The immunochromatographic test strip of claim 1, wherein the artificial antigen is a 2,4-D artificial antigen; 2,4-D monoclonal antibodies diluted by gold-labeled protein resuspension are pre-fixed on the sample cup; the gold-labeled protein resuspension is 20mmol/L boric acid buffer containing 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide.
8. The immunochromatographic test strip of claim 7, wherein the preparation method of the sample cup is as follows: diluting the 2,4-D monoclonal antibody to 10 mu g/mL by using the gold-labeled protein resuspension, adding the diluted monoclonal antibody into a sample cup, freeze-drying and sealing the sample cup, and storing the sample cup at 4 ℃ for later use.
9. The immunochromatographic test strip of claim 7, wherein the preparation method of the conjugate pad is as follows:
(1) diluting the artificial antigen 2,4-D-BSA to 1mg/mL by using ultrapure water, dropwise adding the 2,4-D-BSA solution with the concentration of 1mg/mL into 100mL of colloidal gold solution according to the proportion of adding 9 mu L of 2,4-D-BSA solution into 1mL of colloidal gold solution, reacting for 30min at room temperature, 5% (v/v) casein to the final concentration of 0.5%, reacting for 10min at room temperature, centrifuging for 30min at 4 ℃ and 12000r/min, discarding the supernatant, and re-suspending the precipitate by using 25mL of gold-labeled protein heavy suspension to obtain the gold-labeled 2, 4-D-BSA; preparing a gold-labeled biotin-BSA solution by the same method, and storing at 4 ℃ for later use;
(2) and (3) during gold spraying, mixing the gold-labeled 2,4-D-BSA and the gold-labeled biotin-BSA in a mass ratio of 1:1, spraying the gold-labeled 2,4-D-BSA and the gold-labeled biotin-BSA mixed solution on glass fiber cotton by using a spraying instrument according to a mass ratio of 8 mu L/cm, and performing vacuum drying at a low temperature of 4 ℃ to prepare the binding pad.
10. The detection method of the immunochromatographic test strip of any one of claims 1 to 9, comprising the steps of: adding 80-150 mu L of sample to be detected into a sample cup, uniformly mixing, inserting the test end of the test strip into the sample cup, reading the result for 5-10min, and when the detection line T1 and the detection line T2 are developed, indicating that the sample does not contain the object to be detected or contains the concentration lower than the detection limit of T2; when the detection line T1 is colored and the detection line T2 is not colored, the concentration of the substance to be detected in the sample is higher than the detection limit of T2 and lower than the detection limit of T1; when the detection line T1 and the detection line T2 do not develop color, the concentration of the substance to be detected in the sample is larger than the detection limit of T1; the quality control line is always colored, and when the quality control line is not colored, the operation is wrong or the test paper is invalid.
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