CN113030467A - Self-amplification precisely-controlled indirect competition immunochromatography test strip and application thereof - Google Patents

Self-amplification precisely-controlled indirect competition immunochromatography test strip and application thereof Download PDF

Info

Publication number
CN113030467A
CN113030467A CN202110272753.8A CN202110272753A CN113030467A CN 113030467 A CN113030467 A CN 113030467A CN 202110272753 A CN202110272753 A CN 202110272753A CN 113030467 A CN113030467 A CN 113030467A
Authority
CN
China
Prior art keywords
sample
labeled
detection
gold
layer
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.)
Granted
Application number
CN202110272753.8A
Other languages
Chinese (zh)
Other versions
CN113030467B (en
Inventor
孙亚宁
杨苏珍
郭振华
金前跃
杨继飞
胡骁飞
邓瑞广
张改平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Academy of Agricultural Sciences
Original Assignee
Henan Academy of Agricultural Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Henan Academy of Agricultural Sciences filed Critical Henan Academy of Agricultural Sciences
Priority to CN202110272753.8A priority Critical patent/CN113030467B/en
Publication of CN113030467A publication Critical patent/CN113030467A/en
Application granted granted Critical
Publication of CN113030467B publication Critical patent/CN113030467B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/577—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
    • 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
    • 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
    • G01N33/588—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
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Materials Engineering (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a self-amplification and accurate control indirect competitive immunochromatography detection test strip, which 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 absorption 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 cellulose membrane layer is sequentially provided with a detection line 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 the target monoclonal antibody is pre-fixed in the sample cup. The test strip independently dries the antibody 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, and each antibody intercepted on the detection line can be accurately combined with two labeled antigens, so that signal self-amplification is realized, and the sensitivity of the test paper is improved.

Description

Self-amplification precisely-controlled indirect competition immunochromatography test strip and application thereof
Technical Field
The invention relates to an immunochromatographic assay, in particular to an indirect competitive immunochromatographic assay test strip for self-amplification and accurate control of hapten detection and application.
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 immunochromatography technology based on the competition principle is most widely applied, however, the existing detection mode usually encounters the situation that the antigen, antibody and other immune reagents can successfully establish an ELISA detection method, but cannot successfully establish immunochromatography test paper; and in many applications the sensitivity of the test paper still needs to be improved. 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 simple and sensitive test paper test mode is urgently needed, and a better tool is provided for the research and development of immunochromatographic test paper.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an indirect competitive immunochromatography detection test strip for self amplification and accurate control of hapten detection and application thereof, wherein a colloidal gold and other marking materials are used for marking artificial antigens; the detection line on the nitrocellulose membrane fixes a second antibody or SPA, and an independent quality control (C) line is selected, wherein the independent quality control (C) line is a pair of substances which can be specifically combined and do not react with any component in the test paper, and can be antigen antibody, biotin-avidin and the like; the monoclonal antibody is diluted by the gold-labeled protein resuspension and dried in a sample cup, compared with the traditional method, the detection method can accurately control the using amount of the antibody, and realizes signal amplification based on the gold-labeled antigen, so that the sensitivity of the immunochromatographic test paper is higher.
In order to achieve the purpose, the invention adopts the technical scheme that:
an indirect competitive immunochromatographic test strip with self amplification and accurate control comprises two parts, namely 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 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 the 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 a nitrocellulose membrane, a pure cellulose membrane or a carboxylated cellulose membrane; the detection lines and the quality control lines are parallel arranged straight line blots which are shaped like a Chinese character 'li', or cross arranged blots, or T-type arranged blots, T-shaped arranged blots, or ┤ ┤ -shaped arranged blots.
The detection line is sprayed with goat anti-mouse IgG, rabbit anti-mouse IgG or SPA.
The artificial antigen is a 2,4-D artificial antigen; 2,4-D monoclonal antibodies diluted by gold-labeled protein resuspension are pre-fixed in 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, re-suspending the precipitate by using 25mL of gold-labeled protein heavy suspension to obtain gold-labeled 2,4-D-BSA, preparing a gold-labeled biotin-BSA solution by using the same method, and storing for later use at 4 ℃;
(2) 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, 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 bonding pad.
The detection method of the indirect competitive immunochromatographic test strip comprises the following steps: adding 80-150 mu L of sample to be detected into a sample cup, mixing uniformly, inserting the test end of the test strip into the sample cup, reading the result within 5-10min, and indicating that the sample does not contain the object to be detected or the concentration of the object to be detected is less than the detection limit when the detection line is developed; when the detection line does not develop color, the concentration of the substance to be detected in the sample is larger than the detection limit; 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 indirect competitive immunochromatographic test strip is applied to hapten detection in the fields of food safety detection, environmental detection and illegal veterinary drug detection.
The invention has the beneficial effects that:
the test strip is prepared on the basis of a competition mode, and compared with the traditional competition mode, the test strip has the following advantages that:
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, 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 labeled artificial antigen, and the excessive labeled antigen can ensure that each intercepted monoclonal antibody is combined with 2 labeled antigens, so that the amplification of signals is realized, and the sensitivity of the test paper is increased. And excessive labeled antigen does not influence the combination of the antibody and the antigen in the sample, does not interfere SPA to intercept the monoclonal antibody, increases the color development and does not influence the sensitivity of the test paper. (3) The detection line is changed from fixing artificial antigen to fixing secondary antibody/SPA, so that the interception efficiency of the detection line is increased, and the influence of the antigenicity of the artificial antigen on the sensitivity and color development of the test paper is reduced. Therefore, the indirect competitive immunochromatographic assay test strip has the characteristics of self amplification and accurate control.
2. 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) dropwise adding 80-150 mu L of sample solution into the sample cup for reacting for 3 minutes, dropwise adding the reaction solution onto 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 5-10 minutes.
3. The result display is visual, intuitive and accurate. The test strip takes an "|" (or "ten", "" -T "," "switching", "┤") blot as a positive and negative marker of the detection result, namely, a "|" blot is displayed on the cellulose membrane, which indicates that a sample contains a target substance with a certain concentration (the visual sensitivity concentration of the test strip) or more, and two "|" blots are displayed, which indicates that the detected sample does not contain the target substance or the content is lower than a certain concentration. 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.
4. 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:
when a detection sample is added into a sample cup, when the sample is negative, the redissolved antibody in the sample cup reacts with the artificial antigen marked by the nano material to form a nano material marked antigen-antibody compound when flowing through the binding pad, the antigen-antibody compound flows through a T detection line and is intercepted by goat anti-mouse IgG, rabbit anti-mouse IgG or SPA and the like fixed on the detection line, a strip which is obvious and visual due to accumulation of nano particles is the detection line, marked biotin is intercepted by avidin when flowing through a C quality control line to form a quality control line, namely, the test paper shows that 2 strips indicate that the sample is negative; if the detection sample is positive, the antigen to be detected and the redissolved antibody are combined in the sample cup to form an antigen-antibody compound, the compound does not react with the labeled antigen when flowing through the combination pad, the antigen-antibody compound does not have a nano material label, and is intercepted by the fixed SPA when flowing through the detection line, a strip is not displayed, the labeled biotin is intercepted by the avidin when flowing through the quality control line to form a quality control line, namely, the test paper only develops color of the quality control line, and the sample is positive.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the test 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 a sample pad 2 and a 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, 7 is a quality control line, 8 is a protective layer covering a 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 technique or prepare the test strip 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 self-amplification and accurate control 2,4-D indirect competitive immunochromatography detection test strip consists of two parts, namely 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 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 gold-labeled protein resuspension is pre-fixed in 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 and the quality control lines are parallel arranged straight line blots which are shaped like a Chinese character 'li', or cross arranged blots, or T-type arranged blots, T-shaped arranged blots, or ┤ ┤ -shaped arranged blots.
The detection line is sprayed with a substance specifically bound with the monoclonal antibody of the target object: such as goat anti-mouse IgG, rabbit anti-mouse IgG or SPA.
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
A preparation method of a self-amplification and accurate control 2,4-D indirect competition immunochromatography detection test strip comprises the following steps:
1. preparation of 2,4-D Artificial antigen
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 of NaOH solution until the solution is clear, and then adjusting the pH value of the solution to be neutral by using 0.1M HCl, thus obtaining solution A; weighing 15mg of Bovine Serum Albumin (BSA) and dissolving in 300 mu LPBS to obtain a solution B after full dissolution; slowly adding the solution A into the solution B under the stirring state at 4 ℃, then adding 100mg of carbodiimide (EDC), reacting for 12 hours under the stirring state at 4 ℃, taking out a reaction product, and performing PBS flow dialysis for 72 hours to obtain 2,4-D-BSA for freezing storage 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 artificial antigen with sterile PBS, mixing with equivalent Freund's Complete Adjuvant (FCA), and fully emulsifying; the 2,4-D-BSA is diluted by sterile PBS for 1 time, mixed with equivalent Freund's Incomplete Adjuvant (FIA), fully emulsified, and used for measuring the titer and the inhibition titer of serum antibodies 7 days after 4 th immunization, and a mouse with high titer and good inhibition effect is selected as a fusion mouse. 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 body surface, taking out spleen aseptically, cutting and grinding the spleen, filtering through 120-mesh nylon gauze, centrifuging at 1000r/min for 10min, and collecting spleen cells. 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, and 1mL of ascites was diluted with 4mL of sodium acetate buffer solution (pH 4.0), and the pH of the sodium acetate buffer solution of ascites was adjusted to 4.5 with 1M NaOH solution. Then slowly stirring and adding 130 mu L of octanoic acid, stirring and reacting for 30min at room temperature, centrifuging for 30min at 6000r/min, removing precipitates, filtering supernatant with filter paper to remove impurities, mixing filtrate with 10 times of concentrated Phosphate Buffer Solution (PBS) according to the volume ratio of 9:1, namely the filtrate is in 1 time of PBS ion environment, then adjusting the pH value to 7.4 with 1M NaOH, and placing the mixture into an ice bath for cooling. 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 of colloidal gold solution according to a proportion of 9. mu.L 2,4-D-BSA solution into 1mL of 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 of 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 (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 protein 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 bonding pad.
4. Assembly of test strips
Spraying avidin on a nitrocellulose membrane (NC membrane) quality control line C line position; SPA spraying on the detection line T line position of NC membrane, drying at 42 deg.C for 4h, adding desiccant, sealing, and making into cellulose membrane layer for use. 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 a reaction 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 less than the detection limit when the detection line and the quality control line are simultaneously developed; when the detection line does not develop color and only the quality control line develops color, the concentration of the 2,4-D contained in the sample is larger than the detection limit.
7. Sensitivity detection
The 2,4-D colloidal gold immunochromatographic test strip prepared above is used for detection by taking 2,4-D standard PBS solutions with the concentrations of 3.125ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL and 50ng/mL as samples. The result shows that the test strip detection line does not develop color at 50ng/mL and 25ng/mL, and the test strip detection line develops color at 12.5ng/mL, 6.25ng/mL and 3.125ng/mL, so that the sensitivity of the novel 2,4-D colloidal gold immunochromatographic test strip is judged to be 25 ng/mL.
Comparative example 1
A2, 4-D colloidal gold immunochromatographic test strip adopting a traditional detection mode is adopted, wherein 2,4-D-BSA 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 used as a gold-labeled antibody fiber layer.
When in use: spraying the 2,4-D monoclonal antibody labeled by the colloidal gold on a nitrocellulose membrane (NC 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.
Example 3
A self-amplification and accurate control gentamicin indirect competitive immunochromatography detection 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 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 samples, and gentamicin monoclonal antibodies diluted by colloidal gold protein resuspension are pre-fixed on the sample cup.
The nano material labeled antigen is a gentamicin 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 and the quality control lines are parallel arranged straight line blots which are shaped like a Chinese character 'li', or cross arranged blots, or T-type arranged blots, T-shaped arranged blots, or ┤ ┤ -shaped arranged blots.
The detection line is sprayed with SPA which is specifically combined with the Fc end of the target antibody.
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 4
A preparation method of a self-amplifying and accurately controlled gentamicin indirect competitive immunochromatography detection test strip comprises the following steps:
1. preparation of Gentamicin Artificial antigen (GM-BSA)
Weighing 20mg of GM and 12mg of BSA, dissolving in 1.5mL of double distilled water, adding 100mg of carbodiimide, reacting for 24h at the temperature of 4 ℃ under stirring, taking out the reaction product, and performing PBS flow dialysis for 72h to obtain GM-BSA, and freezing and storing at the temperature of-20 ℃ for later use.
2. Preparation of gentamicin monoclonal antibody:
immunizing Balb/C mice with the age of 6-8 weeks with GM-BSA at the dosage of 50 mu g of protein per 200 mu L per mouse, injecting 4-6 points subcutaneously on the back, and immunizing for 4 times in total, wherein the immunization interval time is 3 weeks. Diluting GM-BSA with sterile PBS, mixing with Freund's Complete Adjuvant (FCA) of equal amount, and emulsifying; and (3) boosting immunization for 1 time, diluting GM-BSA with sterile PBS, mixing with equivalent Freund's Incomplete Adjuvant (FIA), fully emulsifying, measuring the titer and inhibition titer of serum antibodies 7 days after 4 th immunization, and screening mice with high titer and good inhibition effect as fusion mice. Performing super-strong immunity on the fusion mouse, diluting the carrier protein coupled with the gentamicin artificial antigen to 50 mu g of protein/200 mu L by using sterile PBS, and directly injecting the carrier protein into the abdominal cavity without adding an 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 body surface, taking out spleen aseptically, cutting and grinding the spleen, filtering through 120-mesh nylon gauze, centrifuging at 1000r/min for 10min, and collecting spleen cells. 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 (PEG) 4000(v/v) into cell precipitates in a 37 ℃ water bath, finishing the adding within 1min, adding 0.1-0.3 mL in the first 30s, adding 0.2-0.4 mL in the middle 15s, and finishing the adding in the last 15 s; 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, centrifuging for 30min at 6000r/min, removing precipitates, filtering supernatant with filter paper to remove impurities, mixing filtrate with 10 times of concentrated Phosphate Buffer Solution (PBS) according to the volume ratio of 9:1, namely the filtrate is in 1 time of PBS ion environment, then adjusting the pH value to 7.4 with 1M NaOH, and placing the mixture into an ice bath for cooling. Ammonium sulfate powder was added at a concentration of 0.2778g/mL, 4 deg.CStirring to react for 2h, centrifuging at 6000r/min for 20min, removing supernatant, dissolving precipitate with 1ml PBS, performing flowing dialysis with PBS for 3d, centrifuging at 6000r/min for 10min, discarding precipitate, and freezing at-20 deg.C for use. Through detection, the prepared monoclonal antibody 5E2-D7 can specifically react with gentamicin and can be used for preparing gentamicin 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.
GM-BSA was diluted with ultrapure water to a concentration of 1mg/mL, GM-BSA solution at a concentration of 1mg/mL was added dropwise to 100mL of colloidal gold solution in a ratio of 20. mu.L of GM-BSA to 1mL of colloidal gold solution, the reaction was carried out at room temperature for 30min, 5% (v/v) casein was added to a final concentration of 0.5%, the reaction was carried out at room temperature for 10min, centrifugation was carried out at 12000r/min at 4 ℃ for 30min, the supernatant was discarded, the precipitate was resuspended in 25mL of gold-labeled protein resuspension (20mmol/L of boric acid buffer containing 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide), a gold-labeled biotin-BSA solution was prepared in the same manner, and stored at 4 ℃ for further use.
And during gold spraying, mixing the gold-labeled GM-BSA and the gold-labeled biotin-BSA in a mass ratio of 1:1, spraying the mixed solution of the gold-labeled GM-BSA and the gold-labeled biotin-BSA on glass fiber cotton by using a spraying instrument according to a mass ratio of 8 mu L/cm, and drying in vacuum at a low temperature of 4 ℃ to prepare the bonding pad.
4. Assembly of test strips
Spraying avidin on a nitrocellulose membrane (NC membrane) quality control line C line position; SPA spraying on the detection line T line position of NC membrane, 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
The gentamicin monoclonal antibody is diluted to 10 mu g/mL by using the gold-labeled protein resuspension, added into a sample cup, freeze-dried and sealed, and stored at 4 ℃ for later use.
6. Detection method of immunochromatographic test paper
Adding 80-150 mu L of a sample to be detected into a sample cup, uniformly mixing, wherein the sample cup is an enzyme linked immunosorbent assay cup, dry gentamycin monoclonal antibody diluted by protein protective solution is filled in the enzyme linked immunosorbent assay cup, then inserting a test end of a test paper into a reaction cup for detection, reading the result for 5-10min, displaying a 'I' blot on a cellulose membrane, indicating that the concentration of the gentamycin contained in the sample is greater than the detection limit, and displaying two 'II' blots, indicating that the detected sample does not contain gentamycin or the content of the gentamycin is lower than the detection limit. When the detection line and the quality control line are simultaneously developed, the fact that the sample does not contain gentamicin or the concentration of the gentamicin is lower than the detection limit is indicated; when the test line of the test paper does not develop color and only the quality control line develops color, the concentration of the gentamicin contained in the sample is larger than the detection limit.
7. Sensitivity detection
The gentamicin colloidal gold immunochromatographic test strip prepared by the method is used for detecting by taking a gentamicin standard solution with the concentration of 0.39ng/mL-6.25ng/mL as a sample. The result shows that the test strip detection line does not develop color when the concentration is more than 3.13ng/mL, so that the sensitivity of the novel gentamicin colloidal gold immunochromatographic test strip is judged to be 3.13 ng/mL.
Comparative example 2
The gentamicin colloidal gold immunochromatographic test strip adopting a traditional detection mode is adopted, wherein gentamicin artificial antigen is adsorbed on a detection line on a nitrocellulose membrane, SPA is adsorbed on a quality control line, and gentamicin monoclonal antibody marked by colloidal gold is used as a gold-labeled antibody fiber layer.
When in use: spraying the colloidal gold labeled gentamicin monoclonal antibody on a nitrocellulose membrane conjugate pad, drying at 42 ℃ for 1h, adding a drying agent, and sealing for later use. Spraying SPA on the C line (quality control line) of NC membrane, spraying gentamicin artificial antigen on the T line (detection line) of NC membrane, drying at 42 deg.C for 4h, adding desiccant, and sealing. 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 traditional gentamicin detection mode is 10 ng/mL. The contrast shows that the sensitivity of the novel gentamicin colloidal gold immunochromatographic test strip is improved by 3.2 times compared with that of the traditional colloidal gold immunochromatographic test strip.

Claims (10)

1. An indirect competitive immunochromatography detection test strip with self amplification and accurate control is characterized by comprising two parts, namely 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 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 the 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 indirect competitive 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 indirect competitive immunochromatographic assay test strip of claim 1, wherein the artificial antigen is prepared by coupling a small molecule hapten with a carrier protein; the carrier protein coupled with the artificial antigen is bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.
4. The indirect competitive immunochromatographic assay test strip of claim 1, 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 a nitrocellulose membrane, a pure cellulose membrane or a carboxylated cellulose membrane; the detection lines and the quality control lines are parallel arranged straight line blots which are shaped like a Chinese character 'li', or cross arranged blots, or T-type arranged blots, T-shaped arranged blots, or ┤ ┤ -shaped arranged blots.
5. The indirect competitive immunochromatographic assay test strip of claim 4, wherein the detection line is sprayed with goat anti-mouse IgG, rabbit anti-mouse IgG or SPA.
6. The indirect competitive immunochromatographic assay test strip of claim 2, 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 in 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.
7. The indirect competitive immunochromatographic assay test strip of claim 6, 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.
8. 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, re-suspending the precipitate by using 25mL of gold-labeled protein heavy suspension to obtain gold-labeled 2,4-D-BSA, preparing a gold-labeled biotin-BSA solution by using the same method, and storing for later use at 4 ℃;
(2) 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, 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 bonding pad.
9. The detection method of the indirect competition immunochromatographic test strip according to any one of claims 1 to 8, comprising the following steps: adding 80-150 mu L of sample to be detected into a sample cup, mixing uniformly, inserting the test end of the test strip into the sample cup, reading the result within 5-10min, and indicating that the sample does not contain the object to be detected or the concentration of the object to be detected is less than the detection limit when the detection line is developed; when the detection line does not develop color, the concentration of the substance to be detected in the sample is larger than the detection limit; 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.
10. Use of the indirect competitive immunochromatographic test strip according to any one of claims 1 to 8 for hapten detection in the fields of food safety detection, environmental detection and detection of prohibited agricultural and veterinary drugs.
CN202110272753.8A 2021-03-13 2021-03-13 Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof Active CN113030467B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110272753.8A CN113030467B (en) 2021-03-13 2021-03-13 Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110272753.8A CN113030467B (en) 2021-03-13 2021-03-13 Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof

Publications (2)

Publication Number Publication Date
CN113030467A true CN113030467A (en) 2021-06-25
CN113030467B CN113030467B (en) 2024-02-02

Family

ID=76469462

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110272753.8A Active CN113030467B (en) 2021-03-13 2021-03-13 Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof

Country Status (1)

Country Link
CN (1) CN113030467B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113740534A (en) * 2021-11-04 2021-12-03 北京乐普诊断科技股份有限公司 Novel coronavirus neutralizing antibody detection card and preparation method thereof
CN114113615A (en) * 2021-12-03 2022-03-01 河南省农业科学院 Immunochromatographic test strip for screening universal monoclonal antibody and detection method
CN116297424A (en) * 2023-02-01 2023-06-23 南京市产品质量监督检验院(南京市质量发展与先进技术应用研究院) Quick detection test paper for pesticide residue on vegetables

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011057475A1 (en) * 2009-11-12 2011-05-19 上海科新生物技术股份有限公司 Test strip for liver screen test based on gold immunochromatographic assay and method of preparing the same
CN103048455A (en) * 2012-09-27 2013-04-17 江苏维赛科技生物发展有限公司 Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof
CN103412125A (en) * 2013-08-25 2013-11-27 河南科技学院 Gold-labeled test strip for rapid detection of chromium ions as well as preparation method and application thereof
CN104101707A (en) * 2013-04-09 2014-10-15 河南工业大学 2, 4-dichlorphenoxyacetic acid residue rapid detection test paper strip
CN105004863A (en) * 2015-07-08 2015-10-28 河南省农业科学院 Colloidal gold immunochromatography test paper for rapidly detecting soybean sensitizing protein beta-conglycinin, and production method thereof
WO2015188633A1 (en) * 2014-06-11 2015-12-17 陈岩松 Immunochromatography detection method and test paper
CN205015344U (en) * 2015-07-08 2016-02-03 河南省农业科学院 Short -term test soybean sensitization albumen glycinin's colloidal gold immunoassay chromatography test paper
CN105572390A (en) * 2014-10-13 2016-05-11 江苏维赛科技生物发展有限公司 Immunocolloidal gold detection card for herbicide 2,4-D and preparation method thereof
CN110058019A (en) * 2019-05-15 2019-07-26 河南省农业科学院 A kind of hog cholera antibody blocking Test paper

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011057475A1 (en) * 2009-11-12 2011-05-19 上海科新生物技术股份有限公司 Test strip for liver screen test based on gold immunochromatographic assay and method of preparing the same
CN103048455A (en) * 2012-09-27 2013-04-17 江苏维赛科技生物发展有限公司 Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof
CN104101707A (en) * 2013-04-09 2014-10-15 河南工业大学 2, 4-dichlorphenoxyacetic acid residue rapid detection test paper strip
CN103412125A (en) * 2013-08-25 2013-11-27 河南科技学院 Gold-labeled test strip for rapid detection of chromium ions as well as preparation method and application thereof
WO2015188633A1 (en) * 2014-06-11 2015-12-17 陈岩松 Immunochromatography detection method and test paper
CN105572390A (en) * 2014-10-13 2016-05-11 江苏维赛科技生物发展有限公司 Immunocolloidal gold detection card for herbicide 2,4-D and preparation method thereof
CN105004863A (en) * 2015-07-08 2015-10-28 河南省农业科学院 Colloidal gold immunochromatography test paper for rapidly detecting soybean sensitizing protein beta-conglycinin, and production method thereof
CN205015344U (en) * 2015-07-08 2016-02-03 河南省农业科学院 Short -term test soybean sensitization albumen glycinin's colloidal gold immunoassay chromatography test paper
CN110058019A (en) * 2019-05-15 2019-07-26 河南省农业科学院 A kind of hog cholera antibody blocking Test paper

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EUN-HYE LEE;YOUNG AH KIM;YONG TAE LEE;BRUCE D. HAMMOCK;HYE-SUNG LEE: "Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN", FOOD & AGRICULTURAL IMMUNOLOGY, vol. 24, no. 2 *
JASDEEP KAUR; K. VIKAS SINGH; ROBIN BORO; K. R. THAMPI; MANOJ RAJE; GRISH C. VARSHNEY; C. RAMAN SURI: "Immunochromatographic Dipstick Assay Format Using Gold Nanoparticles Labeled Protein−Hapten Conjugate for the Detection of Atrazine", ENVIRONMENTAL SCIENCE & TECHNOLOGY, vol. 41, no. 14, XP055047538, DOI: 10.1021/es070194j *
蒋蔚;刘迎春;陈永军;王权;: "莱克多巴胺胶体金免疫层析法快速检测试纸条的研制", 中国动物传染病学报, no. 06 *
郑腾;张体银;朱海;何明亮;张险朋;付辉;李文最;金虹;白泉阳;王武军;张志灯;: "H7N9禽流感病毒纳米荧光颗粒试纸条的研制", 畜牧与兽医, no. 10 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113740534A (en) * 2021-11-04 2021-12-03 北京乐普诊断科技股份有限公司 Novel coronavirus neutralizing antibody detection card and preparation method thereof
CN114113615A (en) * 2021-12-03 2022-03-01 河南省农业科学院 Immunochromatographic test strip for screening universal monoclonal antibody and detection method
CN114113615B (en) * 2021-12-03 2024-03-05 河南省农业科学院 Immunochromatography detection test strip for screening universal monoclonal antibodies and detection method
CN116297424A (en) * 2023-02-01 2023-06-23 南京市产品质量监督检验院(南京市质量发展与先进技术应用研究院) Quick detection test paper for pesticide residue on vegetables

Also Published As

Publication number Publication date
CN113030467B (en) 2024-02-02

Similar Documents

Publication Publication Date Title
CN113030467B (en) Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof
CN113063938B (en) High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method
KR100303525B1 (en) Immunologically Simple Semi-quantitative Method and Apparatus
DK160108C (en) Method and equipment for direct or indirect detection of reaction between a specific binding agent and the corresponding acceptor substance
CN101988924B (en) Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method
CN111378018B (en) Test strip for detecting novel coronavirus antibody and preparation method and application thereof
CN110068688B (en) Lactoferrin competition method nanoflower immunity detection flow chromatography detection card in cow's milk
EP3460475A1 (en) Immunochromatographic analysis device for detecting zika virus
CN109085333A (en) A kind of preparation, detection kit and the preparation method of rheumatoid factor antigen
CN103123353A (en) Immunodetection assay for mycobacterium tuberculosis complex
CN113063935B (en) Integrated self-amplifying indirect competitive immunochromatography test paper and detection method
CN109188000A (en) A kind of test strips and preparation method thereof of portable inspectiont human parathyroid hormone
CN103739675B (en) Strawberry veinbanding virus antibody and antigenic peptide, immunogen and application
CN114518447A (en) Signal enhancement type colloidal gold immunochromatographic test strip for detecting bovine parvovirus and application thereof
JP4536588B2 (en) Diagnostic instrument for adult T-cell leukemia
CN107402300B (en) A kind of method of quick identification human parathyroid
CN105753981A (en) Anti-human respiratory syncytial virus N protein antibodies and immunochromatographic kit using the same
CN102621315B (en) Colloidal gold chromatography anti-Ro52 antibody detection test paper and preparation method thereof
CN105753982B (en) The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody
CN105585633B (en) The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody
CN102621311B (en) Colloidal gold chromatography anti-SSB antibody detection test paper and preparation method thereof
CN105585635B (en) The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody
CN108957002A (en) Antibody against swine fever virus quantitative testing test paper card with double antigens sandwich and double detection lines
CN111220802B (en) Clenbuterol hydrochloride small molecule hapten high sensitivity detection test paper based on nano antibody and preparation method thereof
CN105585634B (en) The immune chromatography reagent kit of anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the application antibody

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
GR01 Patent grant
GR01 Patent grant