CN113030467B - Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof - Google Patents

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

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CN113030467B
CN113030467B CN202110272753.8A CN202110272753A CN113030467B CN 113030467 B CN113030467 B CN 113030467B CN 202110272753 A CN202110272753 A CN 202110272753A CN 113030467 B CN113030467 B CN 113030467B
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gold
detection
layer
labeled
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CN113030467A (en
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孙亚宁
杨苏珍
郭振华
金前跃
杨继飞
胡骁飞
邓瑞广
张改平
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Henan Academy of Agricultural Sciences
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Abstract

The invention relates to a self-amplifying and precisely controlled indirect competitive immunochromatography detection test strip, which consists of two parts, namely test paper and a sample cup; the test paper comprises a supporting layer, an adsorption layer and a protective layer which are fixed on the supporting layer; the adsorption layer is sequentially a sample pad, a bonding pad, a cellulose membrane layer and a water absorbing material layer at the handle end from the test end; a detection line and a quality control line are sequentially arranged on the cellulose film layer; the bonding 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 monoclonal antibody is pre-immobilized in the sample cup. The test strip independently dries the antibody in the sample cup, avoids folding and damage in the process of labeling the antibody, and can accurately control the dosage of the antibody; the nanoparticle labeled artificial antigen is used as a probe, each antibody intercepted on the detection line can be accurately combined with two labeled antigens, and the self-amplification of signals is realized, so that the sensitivity of the test paper is improved.

Description

Self-amplification precisely-controlled indirect competitive immunochromatography test strip and application thereof
Technical Field
The invention relates to an immunochromatography detection method, in particular to a self-amplifying and precisely-controlled indirect competitive immunochromatography detection test strip for hapten detection and application thereof.
Background
The immunochromatography technology is a rapid immunoassay technology developed in recent years, and the basic principle of the immunochromatography technology is that an interaction between an antibody and an antigen is a chromatography detection technology taking a nitrocellulose membrane as a carrier and marking the antigen or the 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. Currently, immunochromatography has been 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 immune reagents such as antigens, antibodies and the like, so that an ELISA detection method can be successfully established, but an immunochromatography test paper cannot be successfully established; and in many applications the test paper sensitivity still needs to be improved. The following three main reasons are: 1. the antibodies fold during the labelling process and an excess of antibodies is added to stabilize the labelling material; 2. the efficiency of intercepting the antibody by the artificial antigen is low, and excessive labeled antibody is required to be added to improve the color development; 3. when the colloidal gold marks the antibody, no signal amplification function is achieved, and when the signal amplification is achieved by fluorescent materials such as quantum dots, the detection instrument is required to judge the result, so that the detection is complicated. The existing small molecule immunochromatography detection mode cannot solve the problem, so that a simple and sensitive test paper detection mode is urgently needed, and a better tool is provided for research and development of immunochromatography test paper.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a self-amplifying and precisely-controlled indirect competitive immunochromatography detection test strip for hapten detection and application thereof, wherein artificial antigens are marked by marking materials such as colloidal gold; the detection line on the nitrocellulose membrane is fixed with a secondary antibody or SPA, and an independent quality control (C) line is selected from a pair of substances which do not react with any component in the test paper and can be specifically combined, and can be antigen-antibody, biotin-avidin and the like; the monoclonal antibody is diluted by gold-labeled protein heavy suspension and dried in a sample cup, and compared with the traditional method, the detection method can accurately control the dosage of the antibody, and realize signal amplification based on gold-labeled antigen, so that the sensitivity of the immunochromatography test paper is higher.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
an indirect competitive immunochromatography detection test strip with 'self-amplification' and 'accurate control' consists of two parts, namely 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 is sequentially a sample pad, a bonding pad, a cellulose membrane layer and a water absorbing material layer at the handle end from the test end; the protective layer is fixed on the sample pad, the bonding pad and the water absorbing material layer; a detection line and a quality control line are sequentially arranged on the cellulose film layer; the bonding 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 monoclonal antibody diluted by the gold-labeled protein heavy suspension is pre-fixed in the sample cup.
The nano material marked antigen is artificial antigen marked 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, nylon membrane, polyvinylidene fluoride membrane or polyester membrane; the water-absorbing material layer is made of water-absorbing filter paper; the supporting layer is made of a tough material which does not absorb water; the cellulose film layer is made of nitrocellulose film, pure cellulose film or carboxylated cellulose film; the detection line and the quality control line are parallel arranged ' II ' -shaped linear print, or ' ten ' -shaped print, or ' T-type print, or ' ┤ ┤ ' -shaped print.
And 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; the sample cup is internally pre-fixed with a 2,4-D monoclonal antibody diluted by gold-labeled protein heavy suspension; the gold-labeled protein heavy suspension is 20mmol/L boric acid buffer solution, which contains 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 2,4-D monoclonal antibody to 10 mug/mL with gold-labeled protein heavy suspension, adding into a sample cup, freeze drying, sealing, and preserving at 4 ℃ for standby.
The preparation method of the bonding pad comprises the following steps:
(1) Diluting artificial antigen 2,4-D-BSA to 1mg/mL by using ultrapure water, adding 1mL of colloidal gold solution into 2,4-D-BSA solution with the concentration of 1mg/mL according to the proportion of 9 mu L of 2,4-D-BSA solution, dropwise adding the 2,4-D-BSA solution with the concentration of 1mg/mL into 100mL of colloidal gold solution, reacting for 30min at room temperature, centrifuging for 30min at 12000r/min at room temperature, 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 gold-labeled biotin-BSA solution by adopting the same method, and preserving at 4 ℃ for standby;
(2) When spraying gold, mixing gold-labeled 2,4-D-BSA and gold-labeled biotin-BSA according to a mass ratio of 1:1, and spraying the gold-labeled 2,4-D-BSA and gold-labeled biotin-BSA mixed solution on glass fiber cotton according to a mass ratio of 8 mu L/cm by using a spraying instrument, and carrying out vacuum drying at a low temperature of 4 ℃ to prepare the bonding pad.
The detection method of the indirect competitive immunochromatography 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 detection test strip into the sample cup, reading the result for 5-10min, and indicating that the sample does not contain the sample to be detected or contains concentration smaller than the detection limit when the detection line develops color; when the detection line does not develop color, the concentration of the object to be detected in the sample is larger than the detection limit; the quality control line always develops color, and when the quality control line does not develop color, the operation is wrong or the test paper fails.
The indirect competitive immunochromatographic test strip is applied to hapten detection in the fields of food safety detection, environment detection and illegal agricultural and veterinary drug detection.
The invention has the beneficial effects that:
the test strip is prepared based on a competition mode, and has the following advantages compared with the traditional competition mode:
1. the sensitivity is high. (1) The antibody is dried independently in the sample cup, the antibody is firstly combined with the antigen in the sample, and the measure avoids 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 the excessive labeled antigen does not influence the combination of the antibody and the antigen in the sample, does not interfere with the SPA interception of the monoclonal antibody, and does not influence the sensitivity of the test paper while increasing the color development. (3) The detection line is changed from the fixed artificial antigen to the fixed secondary antibody/SPA, so that the interception efficiency of the detection line is increased, and the influence of antigenicity of the artificial antigen on the sensitivity and color development of the test paper is reduced. Therefore, the indirect competitive immunochromatography detection test strip has the characteristics of self amplification and accurate control.
2. Simple and rapid. The test strip of the invention can be used for field operation without any other reagents and instruments. And (3) dripping 80-150 mu L of sample solution into a sample cup for reaction for 3 minutes, dripping the reaction solution onto a sample pad of the test strip or inserting the test strip below a test end warning line into the sample solution to be tested, and judging the detection result about 5-10 minutes.
3. The result display is visual, intuitive and accurate. The test strip shows "," T "," "┤") marks as positive and negative marks of the detection result, i.e. a "|" print is shown on the cellulose membrane, the sample contains a certain concentration (the concentration of the visual sensitivity of the test paper) and more than the target object, two 'II' -shaped marks are displayed, and the sample to be detected does not contain the target object or has the content lower than a certain concentration. The result judgment is visual, visual and accurate, simple and clear, and is not easy to cause artificial misjudgment such as false positive and false negative.
4. The cost is saved. The rapid detection test paper strip is used, no instrument or other reagent is needed, the detection can be carried out anytime and anywhere, 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, and the sample is negative, the re-dissolved antibody in the sample cup reacts with the artificial antigen marked by the nano material to form a nano material marked antigen-antibody complex when the nano material passes through a binding pad, the nano material marked antigen-antibody complex is intercepted by goat anti-mouse IgG, rabbit anti-mouse IgG or SPA and the like fixed on a detection line when the nano material passes through a T detection line, the nano particles are enriched to generate obvious and visual strips which are the detection line, and the marked biotin is intercepted by avidin to form a quality control line when the marked biotin passes through a C quality control line, namely 2 strips are displayed by test paper to indicate that the sample is negative; if the detected sample is positive, the antigen to be detected in the sample cup is combined with the redissolved antibody to form an antigen-antibody complex, the complex does not react with the labeled antigen when flowing through the combining pad, the antigen-antibody complex is not provided with a nano material label, the antigen-antibody complex is intercepted by the fixed SPA when flowing through the detection line and does not display a strip, the labeled biotin is intercepted by the avidin when flowing through the quality control line to form the quality control line, namely, the test paper only develops color by the quality control line, and the positive sample is indicated.
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 a test strip according to 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 supporting layer, 4 is a bonding pad, 5 is a cellulose film layer, 6 is a detection line, 7 is a quality control line, 8 is a protective layer covering a water absorbing material layer, and 9 is a water absorbing material layer.
Detailed Description
The following description of the specific embodiments of the invention is further defined by reference to examples, which are intended to be illustrative only and are not intended to represent all of the possible aspects of the invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in these examples, and those skilled in the art can implement the immunochromatography techniques described in the present invention or prepare test strips using other similar materials or reaction conditions according to the principles of the present invention, and all such modifications are within the scope of the present invention.
Example 1
A2, 4-D indirect competition immunochromatography detection test strip with 'self-amplification' and 'accurate control' 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 is sequentially a sample pad, a bonding pad, a cellulose membrane layer and a water absorbing material layer at the handle end from the test end; the protective layer is fixed on the sample pad, the bonding pad and the water absorbing material layer; a detection line and a quality control line are sequentially arranged on the cellulose film layer; the bonding pad is made of glass fiber cotton adsorbed with nano material labeled antigen; the sample cup is a container for diluting a sample, and 2,4-D monoclonal antibody diluted by gold-labeled protein heavy suspension is pre-fixed in the sample cup.
The nano material marked antigen is a 2,4-D artificial antigen marked by colloidal gold, quantum dots or silicon sphere nano materials; 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, nylon membrane, polyvinylidene fluoride membrane or polyester membrane; the water-absorbing material layer is made of water-absorbing filter paper; the supporting layer is made of a tough material which does not absorb water; the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane.
The detection line and the quality control line are parallel arranged ' II ' -shaped linear print, or ' ten ' -shaped print, or ' T-type print, or ' ┤ ┤ ' -shaped print.
The detection line is sprayed with a substance specifically combined with a target monoclonal antibody: such as goat anti-mouse IgG, rabbit anti-mouse IgG or SPA, etc.
The sample pad, the bonding pad and the water absorbing material layer are covered with protective films, and sample mark lines are printed at positions, which are 0.3 cm to 0.7cm away from one side of the sample pad, on the protective films corresponding to the junction of the sample pad and the bonding pad.
Example 2
A preparation method of a self-amplifying and precisely controlled 2,4-D indirect competitive immunochromatography detection test strip comprises the following steps:
1. preparation of 2,4-D artificial antigen
Weighing 100mg of 2,4-D, adding 200 mu L of methanol for dissolution, adding 7mL of PBS, then adding 1mol/LNaOH solution dropwise until the solution is clear, and then adjusting the pH value of the solution to be neutral by 0.1M HCl, wherein the solution is A solution; 15mg of Bovine Serum Albumin (BSA) was weighed and dissolved in 300. Mu.LPBS, and after sufficient dissolution, this was solution B; slowly adding the solution A into the solution B under the stirring condition of 4 ℃, adding 100mg of carbodiimide (EDC), reacting for 12 hours under the stirring condition of 4 ℃, taking out the reaction product, and performing PBS (phosphate buffer solution) flow dialysis for 72 hours to obtain 2,4-D-BSA, and freezing at-20 ℃ for later use.
2. Preparation of 2,4-D monoclonal antibodies
Balb/C mice aged 6-8 weeks were immunized with 20. Mu.g protein/200. Mu.L/dose of each prepared 2,4-D-BSA, injected subcutaneously on the back at 4-6 spots, and immunized for 4 total times at 3 weeks intervals. Firstly, diluting the prepared 2,4-D artificial antigen with sterile PBS, mixing with equivalent Freund's Complete Adjuvant (FCA), and fully emulsifying; boosting for 1 time, diluting 2,4-D-BSA with sterile PBS, mixing with equivalent Freund's Incomplete Adjuvant (FIA), fully emulsifying, measuring serum antibody titer and inhibition titer after 7 days of 4 th immunization, screening high-titer mice with good inhibition effect, and taking the mice as fusion mice. The fusion mice were hyperimmunized and 2,4-D-BSA was diluted to 50. Mu.g protein/200. Mu.L with sterile PBS and injected intraperitoneally without adjuvant. Collecting blood from the infraorbital sinus of the immunized mice after 3-4 days, and separating positive serum; removing neck to kill, soaking the body surface of the mouse in 75% (v/v) alcohol for 5-10min, sterilizing, taking spleen aseptically, shearing and grinding the spleen, filtering with 120 mesh nylon gauze, centrifuging at 1000r/min for 10min, and collecting spleen cells. Mixing the separated spleen cells with NS0 myeloma cells according to the proportion of 10:1, centrifuging at 1000r/min for 10min, discarding the supernatant, slowly adding 0.7-1.0 mL of 50% polyethylene glycol 4000 (v/v) into the cell sediment in a water bath at 37 ℃, adding 0.1-0.3 mL for the first 30s, adding 0.2-0.4 mL for the middle 15s, and adding the cell sediment for the last 15 s; then 15mL of serum-free 1640 medium is slowly added to stop the action of PEG, water bath is carried out at 37 ℃ for 5-10min, centrifugation is carried out at 1000r/min for 10min, the supernatant is discarded, the cell sediment is resuspended in HAT (H-hypoxantine Hypoxanthine, A-Aminopterin, T-Thymidine Thymidine) selection medium and added into 96-well cell culture plates (8-10 blocks), 100 mu L-200 mu L/well is placed at 37 ℃ and 5% CO 2 Culturing in an incubator. Culturing for 10-14 days, screening positive holes by using an indirect ELISA method, selecting holes with strong positivity, high inhibition rate and vigorous cell growth for 2 times of limiting dilution cloning, and then performing expanded culture to establish hybridoma cell strains.
In-vivo ascites induction method for preparing antibody in batches, taking SPF-grade BALB/C mice, injecting 400 mu L/mouse liquid paraffin into abdominal cavity, diluting with culture medium RMPI-1640 to 1×10 concentration after 7-10 days 6 Individual cellsThe hybridoma cells were injected into the abdominal cavity at 500. Mu.L/mL. When the abdominal cavity of the mouse is enlarged and the skin is tensed, adopting ascites, centrifuging for 5min at 3000r/min, and discarding the sediment. Then purifying the ascites by an ammonium octoate sulfate method, adding 4mL of sodium acetate buffer solution (pH 4.0) into 1mL of the ascites to dilute the ascites, and adjusting the pH value of the sodium acetate buffer solution of the ascites to 4.5 by using 1M NaOH solution. Then adding 130 mu L of octanoic acid by slow stirring, stirring at room temperature for reaction for 30min, centrifuging for 30min at 600 r/min, removing precipitate, filtering the supernatant by using filter paper to remove impurities, mixing the filtrate with 10 times of concentrated Phosphate Buffer (PBS) according to the volume ratio of 9:1, namely, placing the filtrate in a 1 time PBS ion environment, then adjusting the pH value to 7.4 by using 1M NaOH, and cooling in an ice bath. Adding ammonium sulfate powder according to 0.2778g/mL concentration, stirring and reacting at 4 ℃ for 2h, centrifuging at 6000r/min for 20min, removing supernatant, dissolving precipitate by 1mLPBS, dialyzing by PBS for 3d, centrifuging at 6000r/min for 10min, discarding the precipitate, and freezing at-20 ℃ for later use. 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 proteins and conjugate pads
The colloidal gold solution is prepared by adopting a sodium citrate reduction method. 99mL of ultrapure water is added into a clean 200mL conical flask with scales, the conical flask is placed on a heating magnetic stirrer for heating and stirring, 1mL of 1% (w/v) chloroauric acid solution is added, heating is carried out until boiling, then 1.6mL of 1% (w/v) trisodium citrate solution is rapidly added, stirring is continued, the color of the heated solution is changed through transparent-black-dark red-wine red, heating is carried out for 5min when the color is not changed, cooling is carried out at room temperature, and then the colloidal gold is fixed to 100mL by using ultrapure water and is stored at 4 ℃ for standby.
2,4-D-BSA was diluted with ultrapure water to a concentration of 1mg/mL, 2,4-D-BSA solution was added dropwise to 100mL of the colloidal gold solution in a ratio of 9. Mu.L of 2,4-D-BSA solution to 1mL of the colloidal gold solution, 30min of reaction at room temperature, 5% (v/v) of casein to a final concentration of 0.5%, 10min of reaction at room temperature, 12000r/min of centrifugation at 12000r/min, and the supernatant was discarded, and the precipitate was resuspended with 25mL of gold-labeled protein suspension (20 mmol/L of BSA containing 1% (w/v) of BSA, 3% (w/v) of trehalose and 0.03% (w/v) of sodium azide), and the gold-labeled biotin-BSA solution was prepared in the same manner and stored at 4℃for use. When spraying gold, mixing gold-labeled 2,4-D-BSA and gold-labeled biotin-BSA according to the mass ratio of 1:1, spraying gold-labeled protein on glass fiber cotton according to the mass ratio of 8 mu L/cm by using a spraying instrument, and carrying out vacuum drying at a low temperature of 4 ℃ to prepare the bonding pad.
4. Assembly of test strips
Spraying avidin on the C line position of a quality control line of a nitrocellulose membrane (NC membrane); SPA is sprayed on the position of a detection line T of the NC film, dried for 4 hours at 42 ℃, and sealed by adding a drying agent to prepare a cellulose film layer for standby. And then sequentially adhering the sample pad, the bonding pad, the cellulose film layer, the water absorbing material layer and the protective layer on the bottom plate of the supporting layer according to the specification shown in figure 1, overlapping the components by 1-2mm, and cutting into test strips by a cutting machine.
5. Preparation of sample cup
Diluting 2,4-D monoclonal antibody to 10 mug/mL with gold-labeled protein heavy suspension, adding into a sample cup, freeze drying, sealing, and preserving at 4 ℃ for standby.
6. Detection method of immunochromatography test paper
Adding 80-150 mu L of sample to be detected into a sample cup, uniformly mixing, inserting a test paper testing end into a reaction cup for detection, reading a result after 5-10min, and indicating that the sample does not contain 2,4-D or contains 2,4-D and the concentration of the 2,4-D is less than the detection limit when the detection line and the quality control line develop simultaneously; when the detection line does not develop color, 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 standard PBS solution with the concentration of 3.125ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL and 50ng/mL is used as a sample, and the prepared 2,4-D colloidal gold immunochromatographic test strip is used for detection. The results show that the test strip detection lines are not developed at 50ng/mL and 25ng/mL, and are developed 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 determined to be 25ng/mL.
Comparative example 1
The 2,4-D colloidal gold immunochromatographic test strip with a traditional detection mode is adopted, wherein a detection line on a nitrocellulose membrane is adsorbed with 2,4-D-BSA, a quality control line is adsorbed with SPA, and a colloidal gold labeled 2,4-D monoclonal antibody is used as a gold-labeled antibody fiber layer.
When in use, the utility model is characterized in that: spraying the colloidal gold labeled 2,4-D monoclonal antibody on a nitrocellulose membrane (NC membrane) bonding pad, drying at 42 ℃ for 1h, and adding a drying agent for sealing for later use. SPA is sprayed at the position of a C line (quality control line) of an NC membrane according to the concentration of 0.2mg/mL, 2,4-D artificial antigen is sprayed at the position of a T line (detection line) of the NC membrane according to the concentration of 0.9mg/mL, and the NC membrane is dried for 4 hours at 42 ℃, and is sealed by adding a drying agent for standby. Then sequentially adhering NC film, bonding pad, sample pad, water-absorbing layer, and protective layer on the support base plate, overlapping each component by 1-2mm, and cutting into test paper strip by cutting machine.
The sensitivity of the colloidal gold immunochromatographic test strip in the 2,4-D traditional detection mode is 1000ng/mL after sensitivity detection. Compared with the traditional 2,4-D colloidal gold immunochromatographic test strip, the sensitivity of the novel 2,4-D colloidal gold immunochromatographic test strip is improved by 40 times.
Example 3
A gentamicin indirect competition immunochromatography detection test strip with 'self-amplification' and 'accurate control' consists of two parts, namely 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 is sequentially a sample pad, a bonding pad, a cellulose membrane layer and a water absorbing material layer at the handle end from the test end; the protective layer is fixed on the sample pad, the bonding pad and the water absorbing material layer; a detection line and a quality control line are sequentially arranged on the cellulose film layer; the bonding 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 gentamicin monoclonal antibody diluted by the gold-labeled protein heavy suspension is pre-fixed on the container.
The nano material marked antigen is gentamicin artificial antigen marked 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, nylon membrane, polyvinylidene fluoride membrane or polyester membrane; the water-absorbing material layer is made of water-absorbing filter paper; the supporting layer is made of a tough material which does not absorb water; the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane.
The detection line and the quality control line are parallel arranged ' II ' -shaped linear print, or ' ten ' -shaped print, or ' T-type print, or ' ┤ ┤ ' -shaped print.
And the detection line is sprayed with SPA which is specifically combined with the Fc end of the target antibody.
The sample pad, the bonding pad and the water absorbing material layer are covered with protective films, and sample mark lines are printed at positions, which are 0.3 cm to 0.7cm away from one side of the sample pad, on the protective films corresponding to the junction of the sample pad and the bonding pad.
Example 4
A preparation method of a gentamicin indirect competitive immunochromatography detection test strip with 'self-amplification' and 'accurate control' 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 24 hours at 4 ℃ under stirring, taking out the reaction product, and dialyzing with PBS for 72 hours to obtain GM-BSA, and freezing at-20 ℃ for later use.
2. Preparation of gentamicin monoclonal antibody:
Balb/C mice aged 6-8 weeks were immunized with 50. Mu.g protein/200. Mu.L/dose of GM-BSA, injected subcutaneously on the back at 4-6 spots, and immunized 4 times at 3 weeks intervals. First, diluting GM-BSA with sterile PBS, mixing with equivalent Freund's Complete Adjuvant (FCA), and emulsifying; the method comprises the steps of boosting for 1 time, diluting GM-BSA by sterile PBS, mixing with equivalent Freund's Incomplete Adjuvant (FIA), fully emulsifying, measuring serum antibody titer and inhibition titer after 7 days of 4 th immunization, screening the mice with high titer and good inhibition effect as fusion mice. Super-immunizing fused mouse, diluting carrier protein coupled with artificial gentamicin antigen with sterile PBSReleased to 50. Mu.g protein/200. Mu.L and injected directly intraperitoneally without adjuvant. Collecting blood from the infraorbital sinus of the immunized mice after 3-4 days, and separating positive serum; removing neck to kill, soaking the body surface of the mouse in 75% (v/v) alcohol for 5-10min, sterilizing, taking spleen aseptically, shearing and grinding the spleen, filtering with 120 mesh nylon gauze, centrifuging at 1000r/min for 10min, and collecting spleen cells. Mixing the separated spleen cells with NS0 myeloma cells according to the ratio of 10:1, centrifuging at 1000r/min for 10min, discarding the supernatant, slowly adding 0.7-1.0 mL of 50% polyethylene glycol (PEG) 4000 (v/v) into the cell sediment in a water bath at 37 ℃, adding 0.1-0.3 mL for the first 30s, adding 0.2-0.4 mL for the middle 15s, and adding the cell sediment for the last 15 s; then 15mL of serum-free 1640 medium is slowly added to stop the action of PEG, water bath is carried out at 37 ℃ for 5-10min, centrifugation is carried out at 1000r/min for 10min, the supernatant is discarded, the cell sediment is resuspended in HAT (H-hypoxantine Hypoxanthine, A-Aminopterin, T-Thymidine Thymidine) selection medium and added into 96-well cell culture plates (8-10 blocks), 100 mu L-200 mu L/well is placed at 37 ℃ and 5% CO 2 Culturing in an incubator. Culturing for 10-14 days, screening positive holes by using an indirect ELISA method, selecting holes with strong positivity, high inhibition rate and vigorous cell growth for 2 times of limiting dilution cloning, and then performing expanded culture to establish hybridoma cell strains.
In-vivo ascites induction method for preparing antibody in batches, taking SPF-grade BALB/C mice, injecting 400 mu L/mouse liquid paraffin into abdominal cavity, diluting with culture medium RMPI-1640 to 1×10 concentration after 7-10 days 6 Individual cells/mL of hybridoma cells were injected into the abdominal cavity at 500 μl/mL. When the abdominal cavity of the mouse is enlarged and the skin is tensed, adopting ascites, centrifuging for 5min at 3000r/min, and discarding the sediment. Then purifying the ascites by an ammonium octoate sulfate method, adding 4mL of acetic acid-sodium acetate buffer solution (pH 4.0) into 1mL of the ascites to dilute the ascites, and adjusting the pH value of the acetic acid-sodium acetate buffer solution of the ascites to 4.5 by using 1M NaOH solution. Then adding 130 mu L of octanoic acid with slow stirring, stirring at room temperature for reaction for 30min, centrifuging for 30min at 600 r/min, removing precipitate, filtering supernatant with filter paper to remove impurities, mixing filtrate with 10 times of concentrated Phosphate Buffer (PBS) at volume ratio of 9:1, namely, the filtrate is in 1 time PBS ion environment, adjusting pH to 7.4 with 1M NaOH, and placing into ice bathAnd (5) cooling. Adding ammonium sulfate powder according to 0.2778g/mL concentration, stirring and reacting at 4 ℃ for 2h, centrifuging at 6000r/min for 20min, removing supernatant, dissolving precipitate by 1mLPBS, dialyzing by PBS for 3d, centrifuging at 6000r/min for 10min, discarding the precipitate, and freezing at-20 ℃ for later 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 proteins and conjugate pads
The colloidal gold solution is prepared by adopting a sodium citrate reduction method. 99mL of ultrapure water is added into a clean 200mL conical flask with scales, the conical flask is placed on a heating magnetic stirrer for heating and stirring, 1mL of 1% (w/v) chloroauric acid solution is added, heating is carried out until boiling, then 1.6mL of 1% (w/v) trisodium citrate solution is rapidly added, stirring is continued, the color of the heated solution is changed through transparent-black-dark red-wine red, heating is carried out for 5min when the color is not changed, cooling is carried out at room temperature, and then the colloidal gold is fixed to 100mL by using ultrapure water and is stored at 4 ℃ for standby.
GM-BSA was diluted with ultrapure water to a concentration of 1mg/mL, the GM-BSA solution having a concentration of 1mg/mL was added dropwise to the 100mL colloidal gold solution in a ratio of 20. Mu.L of GM-BSA to 1mL colloidal gold solution, reacted at room temperature for 30min,5% (v/v) casein was added to a final concentration of 0.5%, reacted at room temperature for 10min, centrifuged at 4℃for 12000r/min for 30min, the supernatant was discarded, and the precipitate was resuspended with 25mL gold-labeled protein resuspension (20 mmol/L boric acid buffer containing 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide), and the gold-labeled biotin-BSA solution was prepared in the same manner and stored at 4℃for use.
When spraying gold, mixing gold-labeled GM-BSA and gold-labeled biotin-BSA according to the mass ratio of 1:1, and spraying the gold-labeled GM-BSA and gold-labeled biotin-BSA mixed solution on glass fiber cotton according to the mass ratio of 8 mu L/cm by using a spraying instrument, and carrying out vacuum drying at a low temperature of 4 ℃ to prepare the bonding pad.
4. Assembly of test strips
Spraying avidin on the C line position of a quality control line of a nitrocellulose membrane (NC membrane); SPA is sprayed on the position of a detection line T of the NC film, dried for 4 hours at 42 ℃, and sealed by adding a drying agent to form a cellulose film layer for standby. And then sequentially adhering the sample pad, the bonding pad, the cellulose film layer, the water absorbing material layer and the protective layer on the bottom plate of the supporting layer according to the specification shown in figure 1, overlapping the components by 1-2mm, and cutting into test strips by a cutting machine.
5. Preparation of sample cup
Diluting gentamicin monoclonal antibody to 10 mug/mL with gold-labeled protein heavy suspension, adding into a sample cup, freeze-drying and sealing, and preserving at 4 ℃ for standby.
6. Detection method of immunochromatography test paper
Adding 80-150 mu L of sample to be detected into a sample cup, uniformly mixing, wherein the sample cup is an ELISA cup, a dried gentamicin monoclonal antibody diluted by a protein protection solution is filled in the ELISA cup, then a test paper test end is inserted into a reaction cup for detection, the result is read out for 5-10min, a 'print' is displayed on a cellulose film, the concentration of gentamicin in the sample is larger than the detection limit, and two 'print' are displayed, the content of gentamicin in the sample to be detected is not smaller than the detection limit. When the detection line and the quality control line develop simultaneously, the concentration of gentamicin in the sample is lower than the detection limit; when the test paper detection line does not develop color, only the quality control line develops color, the concentration of 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 detection by taking gentamicin standard substance 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 gentamycin colloidal gold immunochromatographic test strip is determined to be 3.13ng/mL.
Comparative example 2
The gentamicin colloidal gold immunochromatography test strip adopts a traditional detection mode, wherein a gentamicin artificial antigen is adsorbed by a detection line on a nitrocellulose membrane, SPA is adsorbed by a quality control line, a gentamicin monoclonal antibody is marked by colloidal gold, and the gentamicin monoclonal antibody is used as a gold-labeled antibody fiber layer.
When in use, the utility model is characterized in that: spraying colloidal gold labeled gentamicin monoclonal antibody on nitrocellulose membrane bonding pad, drying at 42 deg.C for 1 hr, adding desiccant, and sealing. SPA is sprayed on the C line (quality control line) position of the NC film, gentamicin artificial antigen is sprayed on the T line (detection line) position of the NC film, and the NC film is dried for 4 hours at 42 ℃, and is sealed by adding a drying agent for standby. Then sequentially adhering NC film, bonding pad, sample pad, water-absorbing layer, and protective layer on the support base plate, overlapping each component by 1-2mm, and cutting into test paper strip by cutting machine.
Through sensitivity detection, the sensitivity of the colloidal gold immunochromatographic test strip in a gentamicin traditional detection mode is 10ng/mL. Compared with the traditional colloidal gold immunochromatography test strip, the sensitivity of the novel colloidal gold immunochromatography test strip for gentamicin is improved by 3.2 times.

Claims (8)

1. An indirect competitive immunochromatography detection test strip is characterized by comprising two parts of 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 is sequentially a sample pad, a bonding pad, a cellulose membrane layer and a water absorbing material layer at the handle end from the test end; the protective layer is fixed on the sample pad, the bonding pad and the water absorbing material layer; a detection line and a quality control line are sequentially arranged on the cellulose film layer; the bonding 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 monoclonal antibody diluted by the gold-labeled protein heavy suspension is pre-fixed in the sample cup; the nano material marked antigen is artificial antigen marked by colloidal gold, quantum dots or silicon sphere nano material; SPA is sprayed on the detection line; the gold-labeled protein heavy suspension is 20mmol/L boric acid buffer solution, which contains 1% (w/v) BSA, 3% (w/v) trehalose and 0.03% (w/v) sodium azide.
2. The indirect competitive immunochromatographic 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.
3. The indirect competitive immunochromatographic test strip of claim 1, wherein the sample pad is made of glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane or polyester membrane; the water-absorbing material layer is made of water-absorbing filter paper; the supporting layer is made of a tough material which does not absorb water; the cellulose film layer is made of nitrocellulose film, pure cellulose film or carboxylated cellulose film; the detection line and the quality control line are parallel arranged ' II ' -shaped linear print, or ' ten ' -shaped print, or ' T-type print, or ' ┤ ┤ ' -shaped print.
4. The indirect competitive immunochromatographic test strip of claim 1, wherein the artificial antigen is a 2,4-D artificial antigen; the sample cup is internally pre-fixed with a 2,4-D monoclonal antibody diluted by gold-labeled protein heavy suspension.
5. The indirect competitive immunochromatographic test strip of claim 4, wherein the sample cup is prepared by the following steps: diluting the 2,4-D monoclonal antibody to 10 mug/mL by using gold-labeled protein heavy suspension, adding the 2,4-D monoclonal antibody into a sample cup, freeze-drying and sealing the sample cup, and preserving the sample cup at 4 ℃ for later use.
6. The indirect competitive immunochromatographic test strip of claim 5, wherein the binding pad is prepared by the following steps:
(1) Diluting artificial antigen 2,4-D-BSA to 1mg/mL by using ultrapure water, adding 1mL colloidal gold solution into the 2,4-D-BSA solution according to the proportion of 9 mu L of 2,4-D-BSA solution, dropwise adding the 2,4-D-BSA solution with the concentration of 1mg/mL into 100mL colloidal gold solution, reacting at room temperature for 30min, centrifuging at 12000r/min for 30min at room temperature for 0.5% with 5% (v/v) casein, discarding supernatant, re-suspending the precipitate by using 25mL gold-labeled protein heavy suspension to obtain gold-labeled 2,4-D-BSA solution, preparing gold-labeled biotin-BSA solution by adopting the same method, and preserving at 4 ℃ for standby;
(2) When spraying gold, mixing gold-labeled 2,4-D-BSA and gold-labeled biotin-BSA according to a mass ratio of 1:1, and spraying the gold-labeled 2,4-D-BSA and gold-labeled biotin-BSA mixed solution on glass fiber cotton according to 8 mu L/cm by a spraying instrument, and performing vacuum drying at a low temperature of 4 ℃ to prepare the bonding pad.
7. A method of detecting an indirect competitive immunochromatographic test strip according to any one of claims 1 to 6, 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 detection test strip into the sample cup, reading the result for 5-10min, and indicating that the sample does not contain the sample to be detected or contains concentration smaller than the detection limit when the detection line develops color; when the detection line does not develop color, the concentration of the object to be detected in the sample is larger than the detection limit; the quality control line always develops color, and when the quality control line does not develop color, the operation is wrong or the test paper fails.
8. Use of the indirect competitive immunochromatographic test strip according to any one of claims 1 to 6 for hapten detection in the fields of food safety detection, environmental detection and illicit agricultural and veterinary drug detection.
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