CN111961128A - Amoxicillin complete antigen and preparation method thereof, and reagent strip and preparation method thereof - Google Patents
Amoxicillin complete antigen and preparation method thereof, and reagent strip and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an amoxicillin complete antigen and a preparation method thereof, and a reagent strip and a preparation method thereof. In the amoxicillin complete antigen, the antigenic determinant of the amoxicillin molecule can be fully exposed and has a relatively complete spatial conformation, so that the amoxicillin complete antigen provided by the invention has relatively good immunogenicity and can be used for immunoassay. The colloidal gold immunochromatographic assay reagent strip for detecting amoxicillin and the preparation method thereof have the advantages of higher amoxicillin detection sensitivity, short detection time, and simple, economic and convenient use.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to an amoxicillin complete antigen and a preparation method thereof, and a reagent strip for detecting amoxicillin and a preparation method thereof.
Background
The immunoassay is a trace analysis method based on specific recognition and reversible binding reaction between an antigen and an antibody, and is suitable for detection of macromolecular compounds (such as proteins, nucleic acids and bacteria) and measurement of small molecular compounds (such as hormones and drugs). The key to establishing an immunoassay method for small molecule compounds is the ability to prepare antibodies with high affinity and high selectivity for small molecule compounds. Most small molecule compounds have molecular weight less than 1000, and are not immunogenic, i.e., lack of T cell epitope and cannot directly induce animal body to produce specific antibody. However, the small molecule compound can be combined with the macromolecular carrier by a proper chemical modification method to generate a small molecule compound-macromolecular carrier coupling substance, namely a complete antigen, which can indirectly induce the proliferation and differentiation of B cells by means of T cell epitopes to generate specific antibodies.
Amoxicillin (Melatonin, MLT) is a small molecular substance with the chemical name of N-acetyl-5-methoxy tryptamine, the molecular weight of which is only 232.28D, and the molecular weight of which is not immunogenic, and is used for immunization, and the amoxicillin-protein carrier coupled complete antigen is formed by combining with a protein macromolecular carrier before the amoxicillin-protein carrier coupled complete antigen is immunogenic. However, the preparation of the amoxicillin-protein carrier coupled complete antigen cannot affect the spatial conformation of amoxicillin, and needs to maintain the effectiveness of amoxicillin antigenic determinants (such as 5-methoxy, N-alkyl side chains) to obtain an amoxicillin antibody with high potency and strong specificity. Therefore, how to prepare complete antigens with good immunogenicity is a difficult point in establishing an immunoassay method for determining amoxicillin.
Disclosure of Invention
The invention provides an amoxicillin complete antigen and a preparation method thereof, and a reagent strip and a preparation method thereof, aiming at solving the problems.
According to a first aspect of the embodiments of the present application, there is provided an amoxicillin complete antigen, which has a structural formula:
wherein R is a carrier protein and n is 5～20, is a natural number.
According to a second aspect of the embodiments of the present application, there is provided a method for preparing an amoxicillin complete antigen, comprising the following steps:
providing amoxicillin, the structural formula of which is:
dissolving amoxicillin in an N, N-dimethylformamide solution, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and N-hydroxysuccinimide to obtain a first reaction solution, putting a magnetic stirrer, sealing a beaker, wrapping an outer layer with tinfoil paper to avoid light, stirring at room temperature for 10-16 hours to react, and obtaining a second reaction solution, wherein in the first reaction solution, the concentration of the amoxicillin hapten is 0.05mol/L, and the molar concentrations of the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and the N-hydroxysuccinimide are respectively 1-2 times of the molar concentration of the amoxicillin hapten;
preparing a phosphate buffer solution of the carrier protein;
dropwise adding the second reaction solution into the phosphate buffer solution of the carrier protein, stirring and reacting for 10-16 hours at 0-4 ℃ in the dark to obtain a mixed solution containing the amoxicillin complete antigen, dialyzing the mixed solution containing the amoxicillin complete antigen, and centrifuging to obtain a supernatant solution containing the amoxicillin complete antigen, wherein the structural formula of the amoxicillin complete antigen is shown in the specification
In the formula, R is carrier protein, and n is a natural number of 5-20.
According to a third aspect of the embodiments of the present application, there is provided a colloidal gold immunochromatographic reagent strip for detecting amoxicillin, comprising a back plate, a sample pad, a gold-labeled pad, a reaction pad and a water absorption pad, wherein the sample pad, the gold-labeled pad, the reaction pad and the water absorption pad are sequentially disposed on the back plate; the reaction pad is provided with a detection line and a quality control line, the detection line is coated with an amoxicillin complete antigen, the quality control line is coated with an antibody of goat anti-mouse IgG, and the gold-labeled pad is sprayed with an amoxicillin antibody gold-labeled compound.
According to a fourth aspect of the embodiments of the present application, there is provided a method for preparing a colloidal gold immunochromatographic reagent strip for detecting amoxicillin, comprising the following steps:
preparing an amoxicillin antibody, preparing colloidal gold and preparing a gold-labeled pad;
setting a detection line and a quality control line;
and assembling the reagent strip.
The technical scheme provided by the embodiment of the application can have the following beneficial effects:
the amoxicillin complete antigen provided by the invention comprises an amoxicillin molecule and a carrier protein, wherein the amoxicillin molecule and the carrier protein are connected through a connecting arm, and in the amoxicillin complete antigen, an antigenic determinant of the amoxicillin molecule can be fully exposed and has a relatively complete spatial conformation, so that the amoxicillin complete antigen provided by the invention has relatively good immunogenicity.
The colloidal gold immunochromatographic reagent strip for detecting amoxicillin provided by the invention can be used for the immunodetection of amoxicillin, the detection sensitivity is greatly improved, all positive samples of amoxicillin can be rapidly screened out through the colloidal gold reagent strip, and samples larger than 2-4ug/kg can be detected.
The colloidal gold immunochromatographic reagent strip for detecting amoxicillin provided by the invention has short detection time; no special instrument is needed; the operation is simple and convenient, the operator does not need to be trained, and the detection cost is low; in a word, the detection reagent strip is simple, economical and convenient to use.
The preparation method of the colloidal gold immunochromatographic reagent strip for detecting amoxicillin provided by the invention is simple and easy to implement, the adopted test paper is conventional test paper, the adopted antibody can be prepared according to the method provided by the invention, the large-scale production can be realized, and the production cost is low.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic diagram of the structure of a colloidal gold immunochromatographic reagent strip for detecting amoxicillin prepared in the example of the present invention.
Description of reference numerals:
10. a back plate; 20. a sample pad; 30. a gold label pad; 40. a reaction pad; 41. detecting lines; 42. a quality control line; 50. An absorbent pad.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
The reagents and biomaterials in the examples described below are commercially available without specific reference.
The invention discloses a preparation method of an amoxicillin complete antigen, which comprises the steps of S101-103.
S101, providing amoxicillin, wherein the structural formula is as follows:
s102, dissolving amoxicillin in a N, N-dimethylformamide solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to obtain a first reaction solution, placing a magnetic stirrer, sealing a beaker, wrapping an outer layer with tinfoil paper to avoid light, stirring at room temperature for 10-16 hours to react, and obtaining a second reaction solution, wherein in the first reaction solution, the concentration of the amoxicillin hapten is 0.05mol/L, and the molar concentrations of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide are respectively 1-2 times of the molar concentration of the amoxicillin hapten.
S103, c, phosphate buffer solution for preparing carrier protein, wherein the molar concentration of the carrier protein is 1X 10-3mol/L, and the molar concentration of the phosphate is 0.01 mol/L.
S104, dropwise adding the second reaction solution obtained in the step (b) into the phosphate buffer solution of the carrier protein prepared in the step (c), stirring and reacting for 10-16 hours at 0-4 ℃ in a dark place to obtain a mixed solution containing the amoxicillin complete antigen, dialyzing the mixed solution containing the amoxicillin complete antigen, and centrifuging to obtain a supernatant solution containing the amoxicillin complete antigen.
The structural formula of the amoxicillin complete antigen (AMX-BSA conjugate) is shown as follows:
wherein R is BSA protein, and n is a natural number of (5-20).
In an alternative embodiment, the carrier protein is any one of globulin, bovine serum albumin, chicken ovalbumin, keyhole limpet hemocyanin, rabbit serum albumin, human serum albumin, thyroglobulin, fibrinogen, rabbit gamma globulin, and chicken gamma globulin.
Referring to fig. 1, the present invention further provides a colloidal gold immunochromatographic reagent strip for detecting amoxicillin, which comprises a back plate 10, a sample pad 20, a gold-labeled pad 30, a reaction pad 40 and a water absorption pad 50. Wherein sample pad 20, gold mark pad 30, reaction pad 40 and absorb water pad 50 set gradually on backplate 10, and sample pad 20 and absorb water pad 50 set up respectively in two tip of backplate 10, and reaction pad 40 sets up in the middle part of backplate 10, and the lower surface that is close to the tip of reaction pad 40 of gold mark pad 30 is laminated with the upper surface of reaction pad 40, and the upper surface that is close to the tip of sample pad 20 on the gold mark pad 30 is laminated with the lower surface of sample pad 20. The upper surface of one end of the reaction pad 40, which is far away from the gold mark pad 30, is attached to the upper surface of the water absorption pad 50, so that the liquid to be detected fully overflows to the reaction pad 40 through the structure and is finally absorbed by the water absorption pad 50; the reaction pad 40 is provided with a detection line 41 and a quality control line 42, the detection line 41 is coated with an amoxicillin complete antigen, the quality control line 42 is coated with an antibody of an amoxicillin antibody-gold-labeled complex, and the gold-labeled pad 30 is sprayed with the amoxicillin antibody-gold-labeled complex. Wherein the structural formula of the amoxicillin complete antigen (AMX-BSA conjugate) is shown as follows:
in the formula, R is carrier protein, and n is a natural number of 5-20.
The invention also discloses a preparation method of the colloidal gold immunochromatographic reagent strip for detecting amoxicillin, which comprises the steps of S101-S103.
S101, preparing an amoxicillin antibody, preparing colloidal gold, and preparing a gold-labeled pad.
Specifically, the preparation of the antibody takes an amoxicillin complete antigen as an immunogen, and the amoxicillin antibody is prepared by adopting a conventional method. The preparation of the colloidal gold is to prepare the colloidal gold with the particle size of 25-40 nm by adopting a trisodium citrate reduction method.
The preparation of the gold-labeled pad comprises the steps of dipping the glass fiber with the treatment solution, drying, spraying the amoxicillin antibody gold-labeled compound on the pretreated glass fiber in the amount of 0.5-4 mu l/cm, drying at 25-30 ℃ to obtain the gold-labeled pad, and placing the gold-labeled pad in the environment of 2-8 ℃ for later use.
The preparation of the amoxicillin antibody gold-labeled compound comprises the steps of preparing colloidal gold with the particle size of 25-40 nm by an original method;
and preparing an amoxicillin antibody gold-labeled compound, namely adjusting the pH value of colloidal gold to 7.0-9.0, slowly adding the amoxicillin antibody according to the amount of adding 4-25 mu g of protein into each milliliter of colloidal gold solution under the stirring condition, standing for 10-30 min, then adding bovine serum albumin BSA to the final concentration of 0.5-5%, standing for 10-30 min, centrifuging, discarding the supernatant, washing the precipitate for 2-3 times by using a washing solution, resuspending the precipitate by using a resuspension solution with one tenth of the initial volume for the last time, and standing for later use at 4 ℃.
And S102, setting a detection line and a quality control line.
The detection line is set up by configuring the amoxicillin complete antigen with the coating buffer solution to be 0.5mg/mL, and marking the amoxicillin complete antigen on the pretreated reaction pad by using a film marking instrument with the dosage of 0.8 mu l/cm.
The quality control line is set by diluting goat anti-mouse IgG to 0.5-1mg/mL with coating buffer solution, and drawing the quality control line on the pretreated reaction pad with a film drawing instrument at the dosage of 0.8 μ l/cm.
The buffer solution for coating the membrane may be borate, carbonate, phosphate, Tris-HCl or Tris-phosphate, acetate, barbiturate, etc., and the purpose of the buffer solution is to provide a pH and ionic strength for coating and firmly coating the protein on the NC membrane, wherein the pH value of the buffer solution is generally in the range of about 6-9.5, preferably in the range of neutral buffer of 6.5-7.5, and most preferably in the range of 7.0-7.4. The buffer is preferably phosphate. Sequentially adding 8.0g of NaCI, 8.2 g of KH2PO40.2g of Na2HPO4.12H2O, 2.9g of KCI and 0.2g of the reagent into a quantitative container, adding a proper amount of distilled water for dissolving, then fixing the volume to 1000mL, adjusting the pH value to 7.4, sterilizing by high-pressure sterilization of zhidao for 112Kpa for 20min, cooling, and storing in a refrigerator at 4 ℃ for later use.
S103, assembling the reagent strip.
Assembling a back plate, a sample pad, a water absorption pad, a processed gold label pad and a processed reaction pad to obtain a colloidal gold immunochromatographic reagent strip for detecting amoxicillin, wherein the colloidal gold immunochromatographic reagent strip for detecting amoxicillin comprises the back plate, the back plate is provided with the sample pad, the gold label pad, the reaction pad and the water absorption pad, the sample pad and the water absorption pad are respectively positioned at two ends of the back plate, the reaction pad is positioned in the middle of the back plate, the two ends of the gold label pad are respectively arranged on the lower surface of one end of the sample pad and the upper surface of one end of the reaction pad, the other end of the reaction pad is arranged on the lower surface of one end of the water absorption pad, the reaction pad is provided with a detection line and a quality control line, wherein the detection line is coated with an amoxicillin complete antigen, and the quality control line is coated with an antibody of an anti-amoxicillin antibody-gold label complex, and the gold-labeled pad is sprayed with an amoxicillin antibody gold-labeled compound.
In some alternative embodiments, the reagent strip may be installed in a casing, the reaction pad is exposed to allow a user to observe the reaction of the reaction pad from the outside, and the casing corresponding to the sample pad is opened to allow the user to insert the sample onto the sample pad through the opening.
The material of the back plate is a PVC plate, the material of the sample pad is glass fiber, and the material of the water absorption pad is water absorption paper. When assembling, the indoor temperature should be controlled at 25-37 deg.C and humidity 20-30%.
In an alternative embodiment, the amoxicillin antibody gold-labeled complex comprises an amoxicillin antibody and colloidal gold nanoparticles, the amoxicillin antibody gold-labeled complex having the ability to recognize and specifically bind to amoxicillin. The amoxicillin antibody and the colloidal gold nanoparticles form the amoxicillin antibody gold-labeled complex in an electrostatic combination mode. The amoxicillin antibody is a monoclonal antibody or a polyclonal antibody, the amoxicillin antibody has the capacity of identifying and specifically binding amoxicillin, the polyclonal antibody is any one of a mouse source, a horse source, a sheep source, a rabbit source and a guinea pig source, and the monoclonal antibody is a mouse source or a rabbit source.
In an alternative embodiment, the amoxicillin antibody is a monoclonal antibody and the method of making comprises steps S201-S204.
S201, animal immunization.
The conjugate AMX-BSA provided by the embodiment of the invention is used as an immune antigen to immunize Balb/c mice by 100 mu g/mouse. Mixing immunogen and equivalent Freund complete adjuvant to prepare emulsifier during first-time immunization, injecting subcutaneously at neck and back at multiple points, taking the same dose of immunogen and equivalent Freund incomplete adjuvant at intervals of 2 weeks, mixing and emulsifying, continuing third-time immunization, taking blood from tail after one week of third-time immunization, detecting serum titer and inhibition, performing tail vein impact immunization for 25/one time when the requirement is to be detected, and taking splenocytes after 3 days.
S202, cell fusion and cloning.
Taking splenocytes of the immune Balb/c mice, and carrying out the following steps: 1 ratio was fused with SP2/0 myeloma cells, and positive wells were screened by measuring cell supernatants using an indirect competitive ELISA. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
S203, freezing and restoring cells.
Preparing hybridoma cells in logarithmic growth phase into 5 xl 06 cell suspension with freezing medium, subpackaging in freezing tubes, and storing in liquid nitrogen for a long time. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
S204, preparing and purifying the monoclonal antibody.
By adopting an in vivo induction method, 0.5ml of Freund's incomplete adjuvant is injected into the abdominal cavity of a Balb/c mouse aged for 8 weeks, 5 xl 06 hybridoma cells are injected into the abdominal cavity after 7 days, and ascites is collected after 7 days. Purifying ascites with affinity chromatographic column, measuring protein concentration with ultraviolet spectrophotometer, and storing at-20 deg.c. The titer of the purified antibody is determined by an ELISA method (1: 128000), and the result shows that the purified antibody has higher specificity and sensitivity to amoxicillin.
In an alternative embodiment, the amoxicillin antibody is a polyclonal antibody and the method of preparation comprises steps S301-S302.
S301, animal immunization.
The conjugate MLT-BSA provided by the embodiment of the invention is used as an immunizing antigen to immunize Balb/c mice of 8 weeks old, and the weight is 1 mg/kg. Mixing the immunogen and equivalent Freund's complete adjuvant to prepare an emulsifier during first immunization, performing subcutaneous multi-point injection on the neck and back, mixing and emulsifying the immunogen and equivalent Freund's incomplete adjuvant at an interval of 2 weeks, and performing booster immunization once for 5 times.
S302, collecting antiserum and purifying polyclonal antibody.
Blood is collected 7 days after the last immunization, the titer of the antibody of the serum is measured, the abdominal artery is used for collecting the whole blood and collecting the serum, the purified polyclonal antibody is obtained by the separation of an affinity chromatography column, the titer (1: 10000) of the purified antibody is measured by an ELISA method, and the purified antibody shows higher specificity and sensitivity to amoxicillin.
In an alternative embodiment, the method for preparing colloidal gold includes steps S401-S403.
S401, dissolving 1g of chloroauric acid (HauCL4) in 100g of ultrapure water to prepare a 1% chloroauric acid solution 4C, and standing for later use.
S402, dissolving 1g of trisodium citrate dihydrate into 100g of ultrapure water to prepare a 1% trisodium citrate dihydrate solution 4C, and standing for later use.
S403, adding 100ml of ultrapure water into a clean triangular flask, placing the flask on a heated magnetic stirrer, stirring and heating the flask to boiling, then adding 1ml of 1% chloroauric acid, quickly adding 1.7ml of 1% trisodium citrate dihydrate solution after the flask is boiled, continuing heating and stirring for 15 minutes after the color is changed into wine red, and then turning off heating and stirring. Naturally cooling the prepared colloidal gold for later use; the particle size of the prepared colloidal gold is 25-40 nm, and the maximum absorption peak is 521 nm.
In an alternative embodiment, the preparation method of the amoxicillin antibody gold-labeled complex comprises the steps S501-S503.
S501, searching the optimal protein concentration marked by the colloidal gold.
Taking 8 polystyrene micro reaction tubes, adding 30ul of triple distilled water into each hole, adding 30ul of amoxicillin antibody prepared in example IV into the first hole, diluting the amoxicillin antibody to the last hole in a double ratio manner, adding 125ul of Ph8.0 colloidal gold solution into each hole, standing the mixture at room temperature for 15min, adding 125ul of 10% NaCl solution into each hole, observing the change of each hole, and taking the hole without color change as the minimum protein concentration (5ug/ml-10 ug/ml).
S502, adjusting the pH value of colloidal gold to 8.0, slowly adding the amoxicillin antibody according to the dosage of adding 6 mu g of protein into each milliliter of colloidal gold solution under the stirring condition, standing for 30min, then adding bovine serum albumin BSA to the final concentration of 1%, standing for 30min, centrifuging for 30min at 4 ℃ under 12000 rpm, discarding the supernatant, washing the precipitate for 2 times by using a washing solution, re-suspending the precipitate by using a tenth of the initial volume of a resuspension solution at the last time to obtain an amoxicillin antibody gold-labeled compound, and standing for later use at 4 ℃.
Wherein the heavy suspension, i.e. the preservation solution, is borate, carbonate, phosphate, Tris-HCl or Tris-phosphate, acetate, barbital, etc. containing 0.2-1% of macromolecular protein, the macromolecular protein may be bovine serum albumin, PEG20000, casein, etc., the buffer solution is preferably a phosphate buffer solution, and more preferably 0.5% BSA phosphate buffer solution with ph 7.4.
In addition, the preparation method of the colloidal gold immunochromatographic reagent strip for detecting amoxicillin provided in this embodiment is only one of the modes of applying the amoxicillin complete antigen, the amoxicillin antibody gold-labeled complex and the antibody against amoxicillin antibody gold-labeled complex provided by the present invention, and other applications, especially the application to the immunoassay of amoxicillin, should be considered as the protection scope of the embodiments of the present invention; in addition, the colloidal gold immunochromatographic reagent strip for detecting amoxicillin provided by the invention can be packaged, and comprises the following steps: and (3) assembling 1 part of the cut test paper in the prepared test paper card, enabling the sample adding window to correspond to the sample pad of the test paper, and enabling the result display window to correspond to the detection area and the control area. And packaging the dried powder, a package of drying agent, a specification and a sample injector in an outer packaging bag, and storing the packaged powder in a dark place at 4-25 ℃.
The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive various equivalent modifications or substitutions within the technical scope of the present invention, and these modifications or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. An amoxicillin complete antigen, which is characterized in that the structural formula is as follows:
wherein R is a carrier protein, and n is a natural number of 5-20.
2. An amoxicillin complete antigen according to claim 1, characterized in that the carrier protein is any of globulin, bovine serum albumin, chicken ovalbumin, keyhole limpet hemocyanin, rabbit serum albumin, human serum albumin, thyroglobulin, fibrinogen, rabbit gamma globulin and chicken gamma globulin.
3. The preparation method of the amoxicillin complete antigen is characterized by comprising the following steps:
providing amoxicillin, the structural formula of which is:
dissolving amoxicillin in an N, N-dimethylformamide solution, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and N-hydroxysuccinimide to obtain a first reaction solution, putting a magnetic stirrer, sealing a beaker, wrapping an outer layer with tinfoil paper to avoid light, stirring at room temperature for 10-16 hours to react, and obtaining a second reaction solution, wherein in the first reaction solution, the concentration of the amoxicillin hapten is 0.05mol/L, and the molar concentrations of the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and the N-hydroxysuccinimide are respectively 1-2 times of the molar concentration of the amoxicillin hapten;
preparing a phosphate buffer solution of the carrier protein;
dropwise adding the second reaction solution into the phosphate buffer solution of the carrier protein, stirring and reacting for 10-16 hours at 0-4 ℃ in the dark to obtain a mixed solution containing the amoxicillin complete antigen, dialyzing the mixed solution containing the amoxicillin complete antigen, and centrifuging to obtain a supernatant solution containing the amoxicillin complete antigen, wherein the structural formula of the amoxicillin complete antigen is shown in the specification
In the formula, R is carrier protein, and n is a natural number of 5-20.
4. A colloidal gold immunochromatographic reagent strip for detecting amoxicillin is characterized by comprising a back plate, a sample pad, a gold-labeled pad, a reaction pad and a water absorption pad, wherein the sample pad, the gold-labeled pad, the reaction pad and the water absorption pad are sequentially arranged on the back plate; the reaction pad is provided with a detection line and a quality control line, the detection line is coated with an amoxicillin complete antigen, the quality control line is coated with an antibody of an anti-amoxicillin antibody gold-labeled complex, and the gold-labeled pad is sprayed with the amoxicillin antibody gold-labeled complex.
5. The colloidal gold immunochromatographic reagent strip for detecting amoxicillin according to claim 4, characterized in that the amoxicillin antibody gold-labeled complex comprises amoxicillin antibodies and colloidal gold nanoparticles, and has the ability to recognize and specifically bind amoxicillin.
6. The colloidal gold immunochromatographic reagent strip for detecting amoxicillin according to claim 5, characterized in that the amoxicillin antibody and colloidal gold nanoparticles form the amoxicillin antibody-gold labeled complex by means of electrostatic binding.
7. The colloidal gold immunochromatographic reagent strip for detecting amoxicillin according to claim 6, characterized in that the amoxicillin antibody is a monoclonal antibody or a polyclonal antibody, and the amoxicillin antibody has the ability to recognize and specifically bind to amoxicillin.
8. The colloidal gold immunochromatographic reagent strip for detecting amoxicillin according to claim 7, wherein the polyclonal antibody is any one of mouse, horse, sheep, rabbit or guinea pig, and the monoclonal antibody is mouse or rabbit.
9. A preparation method of a colloidal gold immunochromatographic reagent strip for detecting amoxicillin is characterized by comprising the following steps:
preparing an amoxicillin antibody, preparing colloidal gold and preparing a gold-labeled pad;
setting a detection line and a quality control line;
and assembling the reagent strip.
10. The method for preparing a colloidal gold immunochromatographic reagent strip for detecting amoxicillin according to claim 9, wherein the preparation of the gold-labeled pad further comprises the preparation of a gold-labeled complex of an amoxicillin antibody.
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