Benzoic acid hapten, benzoic acid complete antigen, preparation method thereof and reagent strip
阅读说明：本技术 苯甲酸半抗原、苯甲酸完全抗原及其制备方法与试剂条 (Benzoic acid hapten, benzoic acid complete antigen, preparation method thereof and reagent strip ) 是由 张吉 张伟涛 张金超 宁波 于 2020-05-28 设计创作，主要内容包括：本发明公开了一种苯甲酸半抗原、苯甲酸完全抗原及其制备方法与试剂条。该苯甲酸完全抗原中,其苯甲酸分子的抗原决定簇能充分暴露,且具有较完整的空间构象,因此,本发明提供的苯甲酸完全抗原具有较好的免疫原性,可用于免疫分析。(The invention discloses a benzoic acid hapten, a benzoic acid complete antigen, a preparation method thereof and a reagent strip. In the benzoic acid complete antigen, the antigenic determinant of the benzoic acid molecule can be fully exposed and has complete spatial conformation, so that the benzoic acid complete antigen provided by the invention has better immunogenicity and can be used for immunoassay.)
1. A benzoic acid hapten having the formula:
2. a preparation method of a benzoic acid complete antigen is characterized by comprising the following steps:
providing benzoic acid;
dissolving the benzoic acid in a proper amount of distilled water, adjusting the pH value to 1-2, and stirring in an ice bath at the temperature of 4 ℃ for 10 minutes to obtain a reaction solution;
slowly adding the precooled 1M sodium nitrite solution into the reaction solution, and continuing to react for 30 minutes at the temperature of 4 ℃ to obtain the benzoic acid hapten as claimed in claim 1.
3. A benzoic acid complete antigen, characterized in that it has the structural formula:
in the structural formula, R is carrier protein, and n is a natural number of 5-20.
4. A preparation method of a benzoic acid complete antigen is characterized by comprising the following steps:
preserving the benzoic acid hapten as defined in claim 1 at 0-4 ℃ to obtain a first reaction solution;
preparing a phosphate buffer solution of the carrier protein;
dropwise adding the first reaction solution into the phosphate buffer solution of the carrier protein, and stirring at 0-4 ℃ to obtain a mixed solution containing a benzoic acid complete antigen;
and dialyzing the mixed solution, and centrifuging to obtain a supernatant solution containing the benzoic acid complete antigen.
5. The method of preparing a benzoic acid complete antigen as claimed in claim 4, wherein 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.
6. The colloidal gold immunochromatographic reagent strip for detecting benzoic acid is characterized by comprising a back plate, a sample pad, a gold label pad, a reaction pad and a water absorption pad, wherein the sample pad, the gold label 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 a benzoic acid complete antigen, the quality control line is coated with an antibody of an anti-benzoic acid antibody gold-labeled compound, the gold-labeled pad is sprayed with the benzoic acid antibody gold-labeled compound, and the structural formula of the benzoic acid complete antigen is shown in the specification
7. The colloidal gold immunochromatographic reagent strip for detecting benzoic acid according to claim 6, wherein the benzoic acid antibody gold-labeled complex comprises a benzoic acid antibody and colloidal gold nanoparticles, and the benzoic acid antibody is a monoclonal antibody or a polyclonal antibody.
8. The reagent strip for immunochromatography of benzoic acid detection according to claim 7, wherein the benzoic acid antibody-gold labeled complex is formed by electrostatically binding the benzoic acid antibody and the colloidal gold nanoparticles.
9. The colloidal gold immunochromatographic reagent strip for detecting benzoic acid according to claim 6, wherein the antibody of the benzoic acid antibody gold-labeled complex coated on the quality control line is a monoclonal antibody or a polyclonal antibody.
10. The colloidal gold immunochromatographic reagent strip for detecting benzoic acid according to claim 7 or 9, wherein the polyclonal antibody is any one of mouse, horse, sheep, rabbit and guinea pig, and the monoclonal antibody is mouse or rabbit.
Technical Field
The invention relates to the field of biomedicine, in particular to a benzoic acid hapten, a benzoic acid complete antigen, a preparation method thereof and a reagent strip.
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.
Benzoic acid (MLT) is a small molecule substance with chemical name of N-acetyl-5-methoxy tryptamine, molecular weight of 232.28D only, and is not immunogenic, and is used for immunization, and must be combined with a protein macromolecular carrier to form a complete antigen of benzoic acid-protein carrier coupling. However, the preparation of benzoic acid-protein carrier conjugated complete antigen cannot affect the steric conformation of benzoic acid, and the effectiveness of benzoic acid antigenic determinants (such as 5-methoxy, N-alkyl side chains) needs to be maintained to obtain high-titer and high-specificity benzoic acid antibody. Therefore, how to prepare complete antigens with good immunogenicity is a difficult point to establish an immunoassay method for measuring benzoic acid.
Disclosure of Invention
The invention provides a benzoic acid hapten and aims to solve the problems.
According to a first aspect of embodiments herein, there is provided a benzoic acid hapten of the formula:
according to a second aspect of the embodiments of the present application, there is provided a method for preparing a benzoic acid complete antigen, comprising the steps of:
providing benzoic acid;
dissolving the benzoic acid in a proper amount of distilled water, adjusting the pH value to 1-2, and stirring in an ice bath at the temperature of 4 ℃ for 10 minutes to obtain a reaction solution;
slowly adding the precooled 1M sodium nitrite solution into the reaction solution, and continuing to react for 30 minutes at the temperature of 4 ℃ to obtain the benzoic acid hapten as claimed in claim 1.
According to a third aspect of the embodiments herein, there is provided a benzoic acid complete antigen having the formula:
in the structural formula, R is carrier protein, n is 5～20, is a natural number.
According to a fourth aspect of the embodiments of the present application, there is provided a method for preparing a benzoic acid complete antigen, comprising the steps of:
preserving the benzoic acid hapten as defined in claim 1 at 0-4 ℃ to obtain a first reaction solution;
preparing a phosphate buffer solution of the carrier protein;
dropwise adding the first reaction solution into the phosphate buffer solution of the carrier protein, and stirring at 0-4 ℃ to obtain a mixed solution containing a benzoic acid complete antigen;
and dialyzing the mixed solution, and centrifuging to obtain a supernatant solution containing the benzoic acid complete antigen.
According to a fourth aspect of the embodiments of the present application, there is provided a colloidal gold immunochromatographic reagent strip for detecting benzoic acid, 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 a benzoic acid complete antigen, the quality control line is coated with an antibody of an anti-benzoic acid antibody gold-labeled compound, the gold-labeled pad is sprayed with the benzoic acid antibody gold-labeled compound, and the structural formula of the benzoic acid complete antigen is shown in the specification
The technical scheme provided by the embodiment of the application can have the following beneficial effects: the benzoic acid complete antigen provided by the invention comprises benzoic acid molecules and carrier protein, wherein the benzoic acid molecules and the carrier protein are connected through a connecting arm, and in the benzoic acid complete antigen, antigenic determinants of the benzoic acid molecules can be fully exposed and have complete spatial conformation, so that the benzoic acid complete antigen provided by the invention has better immunogenicity.
The colloidal gold immunochromatographic reagent strip for detecting benzoic acid provided by the invention can be used for immunoassay of benzoic acid, the detection sensitivity is greatly improved, and all positive samples of benzoic acid can be rapidly screened out through the colloidal gold kit.
The colloidal gold immunochromatographic reagent strip for detecting benzoic acid provided by the invention has short detection time; no special instrument is needed; the operation is simple, the operator does not need training, and the detection cost is low. The test paper is conventional test paper, the antibody can be prepared according to the method provided by the invention, large-scale production can be carried out, 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 structural diagram of a colloidal gold immunochromatographic reagent strip for detecting benzoic acid according to an embodiment 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.
The reagents and biomaterials in the examples described below are commercially available without specific reference.
The invention discloses a benzoic acid hapten and a preparation method thereof, wherein the structural formula of the benzoic acid hapten is
The preparation method of the benzoic acid hapten comprises the steps of S101-S103.
S101, providing benzoic acid. Specifically, the structural formula of the benzoic acid is shown as
S102, dissolving the benzoic acid in a proper amount of distilled water, adjusting the pH value to 1-2, and stirring for 10 minutes at a low temperature of 4 ℃ in an ice bath to obtain a reaction solution.
S103, slowly adding the precooled 1M sodium nitrite solution into the reaction liquid A, wherein the color is immediately changed into light yellow, and continuously reacting for 30 minutes at the temperature of 4 ℃ to obtain the benzoic acid hapten.
In an alternative embodiment, the present invention discloses a benzoic acid complete antigen having the formula:
in the structural formula, R is carrier protein, and n is a natural number of 5-20.
The invention also discloses a preparation method of the benzoic acid complete antigen, which comprises the steps S201-S204.
S201, preserving the benzoic acid hapten at 0-4 ℃ for later use to obtain a first reaction solution.
S202, preparing a phosphate buffer solution of carrier protein.
In particular, the molar concentration of the carrier protein is 1X 10-3mol/L, and the molar concentration of the phosphate is 0.01 mol/L.
S203, dropwise adding the first reaction solution into the phosphate buffer solution of the carrier protein, and stirring at 0-4 ℃ for reaction to obtain a mixed solution containing the benzoic acid complete antigen.
S204, dialyzing the mixed solution containing the benzoic acid complete antigen, and centrifuging to obtain a supernatant solution containing the benzoic acid complete antigen.
The structural formula of the benzoic acid complete antigen (MLT-BSA conjugate) is shown in the specification
R is carrier protein, and n is 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 also discloses a colloidal gold immunochromatographic reagent strip for detecting benzoic acid, which comprises a
The invention also provides a preparation method of the colloidal gold immunochromatographic reagent strip for detecting benzoic acid, which comprises the steps S301-S303.
S301, preparing a benzoic acid antibody, colloidal gold and a gold-labeled pad.
Specifically, the benzoic acid antibody is prepared by taking a benzoic acid complete antigen as an immunogen and adopting a conventional method. The colloidal gold is prepared by adopting a trisodium citrate reduction method.
The preparation method of the gold-labeled pad comprises the following steps of dipping the glass fiber by the treatment solution, drying, spraying the benzoic acid antibody gold-labeled compound on the pretreated glass fiber in an amount of 0.5-4 mu l/cm, drying at the ambient temperature of 25-30 ℃ to obtain the gold-labeled pad, and placing the gold-labeled pad in the environment of 2-8 ℃ for later use. When the benzoic acid antibody gold-labeled compound is prepared, the PH value of colloidal gold is adjusted to 7.0-9.0, under the stirring condition, the benzoic acid antibody is slowly added according to the dosage of adding 4-25 mu g of protein into each milliliter of colloidal gold solution, the mixture is kept stand for 10-30 min, bovine serum albumin BSA is added to the mixture until the final concentration is 0.5-5%, the mixture is kept stand for 10-30 min, the centrifugation is carried out, the supernatant is discarded, the precipitate is washed for 2-3 times by using the washing solution, the precipitate is resuspended by using the resuspension liquid with one tenth of the initial volume at the end, and the precipitate is kept at 4 ℃ for later use
And S302, setting a detection line and a quality control line.
Specifically, the detection line is set by taking a benzoic acid resistant complete antigen, diluting the benzoic acid resistant complete antigen with a coating buffer solution until the concentration of the benzoic acid resistant complete antigen is 1.0-1.5 mg/mL, taking a reaction pad, and scratching the benzoic acid resistant complete antigen on the reaction pad by using a film scratching instrument with the dosage of 1-10 mu l/cm to form the detection line.
The setting of the quality control line comprises diluting the goat anti-mouse antibody with a coating buffer solution until the concentration is 0.8-1.5 mg/mL, then scratching the goat anti-mouse antibody on the reaction pad with the dosage of 1-10 mul/cm to form the quality control line, and then placing the reaction pad at 37 ℃ for drying for later use.
S303, assembling a colloidal gold immunochromatographic reagent strip for detecting benzoic acid.
Specifically, get backplate, sample pad, absorb water the pad, the gold mark pad after handling and the reaction pad after handling and assemble, obtain the colloidal gold immunochromatographic assay reagent strip that detects benzoic acid, wherein sample pad, gold mark pad, reaction pad and absorb water the pad set gradually in on the backplate, sample pad and absorption water pad set up respectively in two tip of backplate, and the reaction pad sets up in the middle part of backplate, and the lower surface that is close to the tip of reaction pad of gold mark pad is laminated with the upper surface of reaction pad, and the upper surface that is close to the tip of sample pad on the gold mark pad is laminated with the lower surface of sample pad. The upper surface of one end of the reaction pad, which is far away from the gold mark pad, is attached to the upper surface of the water absorption pad, so that the liquid to be detected is fully overflowed onto the reaction pad through the structure and is finally absorbed by the water absorption pad. The reaction pad is provided with a detection line and a quality control line which are arranged in parallel at intervals, the detection line is coated with a benzoic acid complete antigen, the quality control line is coated with an antibody of an anti-benzoic acid antibody gold-labeled compound, and the gold-labeled pad is sprayed with the benzoic acid 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.
In an alternative embodiment, the benzoic acid antibody gold-labeled complex comprises benzoic acid antibody and colloidal gold nanoparticles, and the benzoic acid antibody is monoclonal antibody or polyclonal antibody. The benzoic acid antibody gold-labeled compound is formed by combining benzoic acid antibody and colloidal gold nanoparticles in an electrostatic manner. The antibody of the anti-benzoic acid antibody gold-labeled compound coated on the quality control line is a monoclonal antibody or a polyclonal antibody. 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 benzoic acid antibody is a monoclonal antibody of benzoic acid antibody, and the preparation method comprises steps S401-S404.
S401, animal immunization.
The conjugate MLT-BSA is used as an immunizing antigen to immunize Balb/c mice, and the weight is 100 mu g/kg. Mixing immunogen and equivalent Freund complete adjuvant to prepare emulsifier during first immunization, performing subcutaneous multipoint injection on neck and back, taking the same dose of immunogen and the equivalent Freund incomplete adjuvant at intervals of 2 weeks, mixing and emulsifying, continuing three-immunization, performing the same dose of method and two-immunization, taking blood from tail after one week of three-immunization to detect serum titer and inhibition, performing abdominal cavity impact immunization once when the requirement is met, and taking splenocytes after 3 days.
S401, 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.
S401, freezing and restoring cells.
Preparing hybridoma cells in logarithmic growth phase into 5 xl 06 cell suspension with freezing medium, subpackaging in freezing tube, 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.
S401, 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 measured by an ELISA method to be 1:128000, and the result shows that the purified antibody has higher specificity and sensitivity to the benzoic acid.
In an alternative embodiment, the benzoic acid antibody is a polyclonal benzoic acid antibody, and the preparation method comprises steps S501-S502.
S501, animal immunization.
The conjugate MLT-BSA is used as an immunizing antigen to immunize Balb/c mice of 8 weeks old with 1mg/kg body weight. Mixing the immunogen and an equal amount of Freund's complete adjuvant to prepare an emulsifier during first immunization, performing subcutaneous multipoint injection on the neck and the back, taking the same dose of immunogen and an equal amount of Freund's incomplete adjuvant at intervals of 2 weeks, mixing and emulsifying, and performing booster immunization once for 5 times in total.
S502, collection of antiserum and purification of 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 of the purified antibody is measured by an ELISA method at the ratio of 1:10000, and the purified antibody shows higher specificity and sensitivity to the benzoic acid.
In an alternative embodiment, the preparation of the colloidal gold specifically includes steps S601 to S602.
S601, dissolving 1g of chloroauric acid (HauCL4) in 100g of ultrapure water to prepare a 1% chloroauric acid solution 4C, and standing for later use;
s602, dissolving 1g of trisodium citrate dihydrate into 100g of ultrapure water to prepare a 1% trisodium citrate dihydrate solution 4C, and placing for later use;
s603, 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 to heat and stir for 15 minutes after the color is changed into wine red, and then stopping heating and stirring. Naturally cooling the prepared colloidal gold for later use; the prepared colloidal gold has the particle size of 25nm and the maximum absorption peak of 521 nm.
In an alternative embodiment, the specific preparation method of the benzoic acid antibody gold-labeled complex comprises steps S701-S702.
S701, 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 the benzoic acid antibody prepared in the fourth embodiment into the first hole, diluting the mixture in a multiple ratio to the last hole, 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).
S702, adjusting the pH value of colloidal gold to 8.0, slowly adding the benzoic acid antibody according to the dosage of adding 4-25 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, 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 a benzoic acid antibody gold-labeled compound, and standing for later use at 4 ℃.
The resuspension solution, namely the preservation solution, is borate, carbonate, phosphate, Tris-HCl or Tris-phosphate, acetate, barbital and the like containing 0.2-1% of macromolecular protein, the macromolecular protein can be bovine serum albumin, PEG20000, casein and the like, and the buffer solution is preferably a phosphate buffer solution, more preferably 0.5% BSA phosphate buffer solution with the pH value of 7.4.
In addition, the preparation method of the colloidal gold immunochromatographic reagent strip for detecting benzoic acid provided in this embodiment is only one of the ways of applying the benzoic acid complete antigen, the benzoic acid antibody gold-labeled complex, and the antibody against the benzoic acid antibody gold-labeled complex provided in the present invention. Other applications, particularly for immunoassay of benzoic acid, are considered to be within the scope of the embodiments of the present invention; in addition, the colloidal gold immunochromatographic reagent strip for detecting benzoic acid provided by the invention can be packaged, and comprises the following steps: and (3) assembling 1 part of cut test paper in a 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 in an outer bag together with a bag of drying agent, a specification and a sample injector, and storing the packaged powder at 4-25 ℃ in a dark place.
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.
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