CN108164472B - 4H-triazole ring structure-based limiting N- (2-guanidino-ethylimino) -morpholine antigen, antibody and application - Google Patents
4H-triazole ring structure-based limiting N- (2-guanidino-ethylimino) -morpholine antigen, antibody and application Download PDFInfo
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- CN108164472B CN108164472B CN201711308739.9A CN201711308739A CN108164472B CN 108164472 B CN108164472 B CN 108164472B CN 201711308739 A CN201711308739 A CN 201711308739A CN 108164472 B CN108164472 B CN 108164472B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
- C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D249/14—Nitrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention discloses a 4H-triazole ring structure-based limiting N- (2-guanidino-ethylimino) -morpholine hapten, artificial antigen, antibody and application. An N- (2-guanidino-ethylimino) -morpholine hapten, which has the structure shown in formula I:
wherein R is carboxyl and p-carboxyphenyl. The N- (2-guanidino-ethylimino) -morpholine artificial antigen and antibody are prepared, the detection efficiency of the N- (2-guanidino-ethylimino) -morpholine is high, the operation is simple and convenient, and the method can be used for the enzyme-linked immunosorbent assay rapid detection of the N- (2-guanidino-ethylimino) -morpholine; the antigen-antibody synthesis process is simple, the cost is low, and the application prospect is good.
Description
Technical Field
The invention belongs to the technical field of drug residue analysis and detection. More particularly, relates to a 4H-triazole ring structure-based limiting N- (2-guanidino-ethylimino) -morpholine hapten, artificial antigen and antibody, and a preparation method and application thereof.
Background
N- (2-guanidino-ethylimino) -morpholine Hydrochloride (Moroxydine Hydrochloride, commonly called Moroxydine Hydrochloride) with core effective component N- (2-guanidino-ethylimino) -morpholine and molecular formula of C6H13N5O.HCl with molecular weight of 207.66. Because N- (2-guanidyl-ethylimino) -morpholine can effectively reduce or inhibit the activity of DNA polymerase and RNA polymerase of viruses and the synthesis of proteins, and the like, the N- (2-guanidyl-ethylimino) -morpholine serving as a non-nucleoside broad-spectrum antiviral drug is not only widely applied to the treatment of diseases caused by viruses such as influenza, herpes, varicella and the like, but also widely applied to the prevention and treatment of viral diseases in the breeding industry and the planting industry.
In 2014, the drug has attracted extensive attention by people due to the fact that the drug has been exposed to more than one 'viron' event in the fields of Xian, Jilin and the like. The event is mainly caused by that children take N- (2-guanidino-ethylimino) -morpholine hydrochloride tablets for a long time, so that a plurality of students have the conditions of abdominal pain, palpitation, skin itch, vomiting, dizziness and the like, and serious students also have the reactions of palpitation, hypoglycemia, shortness of breath, shiver and the like. Therefore, the N- (2-guanidino-ethylimino) -morpholine hydrochloride as an antiviral drug still has the problems of uncertain curative effect, reasonable prescription and the like. At present, the residue of N- (2-guanidino-ethylimino) -morpholine can be detected from tobacco, brown rice, tomatoes, honey, animal tissues and the like, and in addition, the residue condition in environmental soil is not too optimistic, so that the drug-resistant virus can be increased, and potential safety hazards are buried for the health of people. Therefore, the method has important significance for detecting the N- (2-guanidino-ethylimino) -morpholine residue in animal and plant derived food and environment.
At present, conventional methods for detecting N- (2-guanidino-ethylimino) -morpholine in medicines and foods mostly depend on conventional instrumental analysis methods, such as high performance liquid chromatography, liquid chromatography-electrospray tandem mass spectrometry and the like. The methods have the defects of long period, strong specialization, high cost and the like, are difficult to meet the requirement of on-site rapid detection, and cannot be widely popularized in application. The enzyme-linked immunoassay (ELISA) based on antigen-antibody specific recognition has the advantages of rapidness, sensitivity, accuracy and the like, and is particularly suitable for field high-throughput detection. In addition, ELISA has played an important role in food safety testing because it can compensate for the above-mentioned shortcomings of the instrumental methods.
The ELISA method has high requirements for selection of antigens and immunogens, small molecular compounds (MW is less than or equal to 1000Da) generally have no immunogenicity, cannot directly immunize animals to generate specific antibodies, but can react with the antibodies, namely have reactogenicity. If small molecules are linked to immunogenic macromolecules (usually proteins) to make artificial antigens, it is possible to mount an immune response in the body, generating antibodies to the small molecule structure. The design of small molecule hapten structures is important because no small molecule attached to a macromolecular carrier can produce specific antibodies. The key to establishing the enzyme-linked immunoassay method is the design and synthesis of hapten and artificial antigen.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the problems, defects and shortcomings of the existing N- (2-guanidino-ethylimino) -morpholine detection technology, such as long period, strong professional property, high cost, unsuitability for field large-scale rapid detection, incapability of realizing direct rapid detection of N- (2-guanidino-ethylimino) -morpholine and the like, and provide a hapten with a limited N- (2-guanidino-ethylimino) -morpholine structure, an artificial antigen prepared by coupling the hapten with a carrier protein, and an organism generating a specific antibody aiming at N- (2-guanidino-ethylimino) -morpholine after immunizing an experimental animal; can be used for detecting N- (2-guanidino-ethylimino) -morpholine, and has high detection efficiency and simple operation.
The invention aims to provide an N- (2-guanidino-ethylimino) -morpholine hapten.
The invention also aims to provide the N- (2-guanidino-ethylimino) -morpholine artificial antigen.
It is another object of the present invention to provide an N- (2-guanidino-ethylimino) -morpholine antibody.
The above purpose of the invention is realized by the following technical scheme:
an N- (2-guanidino-ethylimino) -morpholine hapten, which has the structure shown in formula I:
wherein R is carboxyl and p-carboxyphenyl.
When R is carboxyl, the structure of the N- (2-guanidino-ethylimino) -morpholine hapten is shown as formula Ia:
when R is p-carboxyl phenyl, the structure of the N- (2-guanidino-ethylimino) -morpholine hapten is shown as formula Ib:
the preparation method of the N- (2-guanidino-ethylimino) -morpholine hapten (Ia and Ib) comprises the following steps: at room temperature, firstly, enabling N-cyano dithioimine dimethyl carbonate to react with morpholine to prepare an intermediate b, then reacting with 80% hydrazine hydrate to prepare an intermediate c, then adding glyoxylic acid or p-aldehyde benzoic acid to perform Mannich reaction with the intermediate c to prepare N- (2-guanidino-ethylimino) -morpholine haptens Ia and Ib, wherein the reaction formula is as follows:
key factors in the process for the preparation of N- (2-guanidino-ethylimino) -morpholine hapten Ia:
(1) the mol ratio of dimethyl N-cyanodithioiminocarbonate to morpholine is 1:1, the solvent is methanol, the mixture is stirred for 3 hours at room temperature, a large amount of white precipitate is generated when the reaction is finished, ethanol is removed by suction filtration, and the mixture is washed with diethyl ether for three times to obtain an intermediate b;
(2) the molar ratio of the intermediate b to 80% hydrazine hydrate is 1: 1.7-1.8, condensing and refluxing are carried out, TLC monitoring is carried out during condensing and refluxing, and the product needs to be separated and purified by column chromatography to obtain an intermediate c;
(3) the molar ratio of the intermediate c to glyoxylic acid is 1:2, a solvent methanol is added with a proper amount of glacial acetic acid, the mixture is stirred at room temperature, a crude product of the N- (2-guanidino-ethylimino) -morpholine hydrochloride hapten III is prepared through a Mannich reaction, the crude product is subjected to suction filtration to remove ethanol, filter residue is washed with ethanol for three times, and the hapten N- (2-guanidino-ethylimino) -morpholine hapten Ia is obtained after drying.
Key factors in the process for the preparation of N- (2-guanidino-ethylimino) -morpholine hapten Ib:
and (3) stirring the intermediate c and p-aldehyde benzoic acid at a molar ratio of 1:1 by using a solvent methanol at room temperature for reaction until no yellow precipitate is generated, performing suction filtration to obtain yellow filter residue, washing with methanol for three times, and drying to obtain the hapten N- (2-guanidino-ethylimino) -morpholine hapten Ib.
In addition, the application of the N- (2-guanidino-ethylimino) -morpholine hapten in preparing the N- (2-guanidino-ethylimino) -morpholine artificial antigen is also within the protection scope of the invention.
An artificial N- (2-guanidino-ethylimino) -morpholine antigen is prepared through coupling the hapten of N- (2-guanidino-ethylimino) -morpholine with carrier protein.
The structural formula of the N- (2-guanidino-ethylimino) -morpholine artificial antigen is shown as the structures of II a and II b:
preferably, the carrier protein is Bovine Serum Albumin (BSA) or Ovalbumin (OVA), wherein BSA is used to prepare the immunogen and OVA is used to prepare the coating antigen.
More preferably, the N- (2-guanidino-ethylimino) -morpholine artificial antigen is prepared by an active ester method, and the specific steps comprise: dissolving N- (2-guanidino-ethylimino) -morpholine hapten, DCC and NHS in a proper amount of DMF, stirring and reacting overnight at 4 ℃, centrifuging and taking supernatant to obtain the activated N- (2-guanidino-ethylimino) -morpholine derivative. Dropwise adding the activated hapten derivative into PBS buffer solution containing 5mg/mL carrier protein, stirring at 4 ℃ for reaction for 12h, dialyzing with PBS at 4 ℃ for 3 days, changing dialysate for 3 times per day to obtain N- (2-guanidino-ethylimino) -morpholine artificial antigen, subpackaging, and storing at low temperature for later use; the molar ratio of the N- (2-guanidino-ethylimino) -morpholine hapten to the carrier protein is 40-100: 1.
More preferably, the preparation method of the N- (2-guanidino-ethylimino) -morpholine artificial antigen comprises the following steps: dissolving N- (2-guanidino-ethylimino) -morpholine hapten, DCC and NHS in a molar ratio of 1:2.4:2.4 in a proper amount of DMF, stirring and reacting at 4 ℃ overnight, and centrifuging to obtain a supernatant so as to obtain the activated N- (2-guanidino-ethylimino) -morpholine derivative. Dropwise adding the activated hapten derivative into PBS buffer solution (0.01M, pH 7.4) containing 5mg/mL carrier protein, stirring at 4 ℃ for reaction for 12h, dialyzing with PBS at 4 ℃ for 3 days, changing dialyzate 3 times per day to obtain N- (2-guanidino-ethylimino) -morpholine artificial antigen, subpackaging, and storing at low temperature for later use; the molar ratio of the N- (2-guanidino-ethylimino) -morpholine hapten to the carrier protein is 60: 1.
In addition, the application of the N- (2-guanidino-ethylimino) -morpholine artificial antigen in preparing anti-N- (2-guanidino-ethylimino) -morpholine polyclonal antibodies, monoclonal antibodies or genetic engineering antibodies is also within the protection scope of the invention.
An N- (2-guanidino-ethylimino) -morpholine antibody is prepared by immunizing animals with the N- (2-guanidino-ethylimino) -morpholine artificial antigen. The N- (2-guanidino-ethylimino) -morpholine antibody is a polyclonal antibody, a monoclonal antibody or a genetic engineering antibody.
The application of the N- (2-guanidino-ethylimino) -morpholine antibody in the immunoassay detection of N- (2-guanidino-ethylimino) -morpholine is also within the protection scope of the invention.
A rapid immunoassay method for directly detecting N- (2-guanidino-ethylimino) -morpholine comprises the following steps:
(1) preparing an anti-N- (2-guanidino-ethylimino) -morpholine polyclonal antibody by immunizing animals with the N- (2-guanidino-ethylimino) -morpholine artificial antigen serving as an immunogen;
(2) coating the N- (2-guanidino-ethylimino) -morpholine artificial antigen as a coating antigen on a microporous plate, and adding the anti-N- (2-guanidino-ethylimino) -morpholine polyclonal antibody prepared in the previous step into the microporous plate;
(3) antiserum titer was detected by indirect competitive ELISA.
The invention has the following beneficial effects:
the invention provides an N- (2-guanidino-ethylimino) -morpholine hapten and an artificial antigen prepared based on the hapten, and explains preparation methods of the N- (2-guanidino-ethylimino) -morpholine hapten and the artificial antigen and application of the N- (2-guanidino-ethylimino) -morpholine hapten in antibody preparation.
The antigen-antibody synthesis process of the invention is simple, the cost is lower, and the invention lays a foundation for developing an enzyme-linked immunosorbent assay rapid detection tool with low cost, high detection efficiency and simple operation, and has good application prospect.
Drawings
FIG. 1 shows the synthesis of N- (2-guanidino-ethylimino) -morpholine haptens Ia (A) and N- (2-guanidino-ethylimino) -morpholine haptens Ib (B).
In FIG. 2, A is the mass spectrum of N- (2-guanidino-ethylimino) -morpholine hapten Ia; b is an antibody performance identification chart of the N- (2-guanidino-ethylimino) -morpholine hapten Ia.
In FIG. 3, A is the mass spectrum of the hapten Ib of N- (2-guanidino-ethylimino) -morpholine prepared by the invention; b is an antibody performance identification chart of the N- (2-guanidino-ethylimino) -morpholine hapten Ib.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Synthesis of N- (2-guanidino-ethylimino) -morpholine hapten Ia
As shown in FIG. 1, 1.46g (10mM) of dimethyl N-cyanodithioiminocarbonate, 0.87g (10mM) of morpholine and 15mL of ethanol were sequentially added to a 50mL single-neck round-bottom flask, and the mixture was magnetically stirred at room temperature for 3 hours. At the end of the reaction, a large amount of white precipitate was produced, and ethanol was removed by suction filtration and washed three times with diethyl ether to give 1.5g of a white solid powder, which was designated as intermediate b. 930mg (5.5mM) of intermediate b and 20mL of ethanol were placed in a 50mL single-neck round-bottom flask, heated to dissolve, 600g (9.6m M) of 80% hydrazine hydrate was added, the mixture was refluxed for 0.5h at 80 ℃, monitored by TLC during the reaction, after completion of the reaction, the ethanol was evaporated to dryness, the obtained solid was dissolved in a solution of chloroform-n-hexane 1:1, and filtered to obtain a white powder, which was dried to a mass of 750mg to obtain intermediate c. Then, 338mg (2mM) of intermediate c and 148mg (2mM) of glyoxylic acid were added in this order to a 50mL single-neck round-bottom flask in 20mL of ethanol as a solvent, and 16 drops of glacial acetic acid were added dropwise under stirring at room temperature. After 4h of reaction a white precipitate was produced, during which time TLC was used for monitoring. And after the reaction is finished, filtering to remove ethanol, washing filter residue with ethanol for three times, and drying to obtain the hapten Ia with the mass of 386 mg.
EXAMPLE 2 Synthesis of N- (2-guanidino-ethylimino) -morpholine hapten Ib
As shown in FIG. 1, 169mg (1mM) of intermediate c of example 3 and 20mL of methanol were placed in a 50mL single-neck round-bottom flask, and the solid was dissolved by stirring, then 10mL of a methanol solution containing 150mg (1mM) of p-aldehyde benzoic acid was slowly added dropwise while causing yellow precipitate, and the reaction was stirred at room temperature for 30 min. And carrying out suction filtration to obtain yellow filter residue, washing with methanol for three times, and drying to obtain hapten Ib with the mass of 242 mg.
Example 3 Synthesis of Artificial antigen
The preparation method of the artificial antigen mainly comprises the synthesis of immunogen and coating antigen. The preparation of the immunogen and the preparation of the coating antigen are different in the type of carrier protein, wherein the carrier protein adopted by the immunogen is Bovine Serum Albumin (BSA); the carrier protein adopted by the coating antigen is Ovalbumin (OVA). A method for preparing immunogen. The method for synthesizing immunogen/coating antigen adopted by the invention is an active ester method.
(1) Synthesis of immunogens
11.2mg (0.05mmol) of the hapten Ia of N- (2-guanidino-ethylimino) -morpholine described in example 1 are dissolved in 1.5mL of DMF, 24.8mg of DCC and 13.8mg of NHS are added with stirring, the reaction is carried out overnight at 4 ℃ with magnetic stirring, and the supernatant is taken as solution A after centrifugation. 20mg of carrier protein was weighed and dissolved in 4mL of PBS buffer solution (0.01M, pH 7.4), and the solution B was prepared by stirring and dissolving. And (3) sucking the solution A and dropwise adding the solution A into the solution B under magnetic stirring, and reacting for 12 hours under magnetic stirring at 4 ℃. After centrifugation, the supernatant was dialyzed against PBS at 4 ℃ for 3 days, and the dialysate was changed 3 times a day. Immunogen Ia-BSA was obtained, adjusted to a concentration of 1mg/mL with PBS, and 500. mu.L per tube was dispensed into 0.5mL centrifuge tubes. Freezing in-20 deg.C refrigerator for use.
The hapten in the previous step was replaced with 15.1mg (0.05mmol) of N- (2-guanidino-ethylimino) -morpholine hapten Ib described in example 2, and the same synthetic procedure was followed to give N- (2-guanidino-ethylimino) -morpholine immunogen Ib-BSA, which was adjusted to 1mg/mL with PBS, and 500. mu.L per tube was dispensed into 0.5mL centrifuge tubes. Freezing in-20 deg.C refrigerator for use.
(2) Synthesis of coatingen
For the above method for synthesizing coatingen corresponding to hapten, the synthesis steps are identical to those of immunogen, and only difference is that carrier protein is changed into OVA, so that coatingen Ia-OVA and Ib-OVA can be obtained. The concentration was adjusted to 1mg/mL with PBS, and 500. mu.L of each tube was dispensed into a 0.5mL centrifuge tube. Freezing in-20 deg.C refrigerator for use.
EXAMPLE 4 preparation of N- (2-guanidino-ethylimino) -morpholine monoclonal antibodies, engineered antibodies and polyclonal antibodies
1. Preparation of monoclonal antibodies
(1) Animal immunization: mixing and emulsifying the immunogen Ia-BSA and the immunogen Ib-BSA prepared in the example 3 with equal amount of adjuvant (complete Freund adjuvant is used for the first time, and incomplete Freund adjuvant is used for the later time), carrying out interval immunization on Balb/c mice, detecting by indirect immunoassay, and obtaining the spleen of the mice containing N- (2-guanidino-ethylimino) -morpholine specific antibodies in blood.
(2) Cell fusion and cloning: and (3) fusing Balb/c mouse spleen cells generating specific antibodies with myeloma cells SP20, measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method or a micro-cloning method to obtain and establish a hybridoma cell strain producing the monoclonal antibody.
(3) Freezing and recovering cells: and (3) preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging the cell suspension into freezing tubes, and storing the cells 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.
(4) Preparation and purification of monoclonal antibody: by in vivo induction method, Balb/c mice (8 weeks old) are injected with sterilized paraffin oil in the abdominal cavity, hybridoma cells are injected in the abdominal cavity 7-14 days later, and ascites is collected 7-10 days later. Purifying ascites by caprylic acid-saturated ammonium sulfate method or affinity chromatography, identifying purity by SDS-PAGE electrophoresis, subpackaging small bottles, and storing at-20 ℃.
2. Preparation of genetically engineered antibodies
Extracting RNA of N- (2-guanidino-ethylimino) -morpholine monoclonal cells or spleen cells of mice immunized by N- (2-guanidino-ethylimino) -morpholine immunogen, reverse transcribing into cDNA, designing antibody light and heavy chain amplification primer, amplifying light and heavy chain gene of antibody by using PCR technology, inserting expression plasmid TGI strain, expressing in colibacillus, purifying by using immunoaffinity method to obtain gene engineering antibody, identifying purity by SDS-PAGE electrophoresis, subpackaging in small bottles, and preserving at-20 ℃.
3. Preparation of polyclonal antibodies
Mixing and emulsifying the immunogens Ia-BSA and Ib-BSA prepared in the example 3 and the same amount of adjuvant (complete Freund's adjuvant is used for the first time, and then incomplete Freund's adjuvant is used for the second time), and immunizing healthy New Zealand white rabbits with the weight of 2.5-3 kg by adopting a plurality of injection modes of back subcutaneous injection, leg muscle injection and ear marginal vein injection, wherein each immunogen is injected two correspondingly. The first immunization was boosted every three weeks after four weeks. Blood was taken from the marginal vein 1 week after the third booster immunization, and the serum titer was measured by indirect ELISA, and when the titer did not rise, the marginal vein was used for booster immunization. Collecting blood from heart after two days, standing at room temperature for 0.5-1 hr, centrifuging at 4 deg.C and 12000r/min for 10min, collecting supernatant, packaging in centrifuge tube, and storing at-20 deg.C for use.
4. Antibody testing
In FIG. 2, A is the mass spectrum of N- (2-guanidino-ethylimino) -morpholine hapten Ia; b is an antibody performance identification chart of the N- (2-guanidino-ethylimino) -morpholine hapten Ia.
In FIG. 3, A is the mass spectrum of the hapten Ib of N- (2-guanidino-ethylimino) -morpholine prepared by the invention; b is an antibody performance identification chart of the N- (2-guanidino-ethylimino) -morpholine hapten Ib.
The indirect competition ELISA is used for measuring the antibody titer based on the light absorption value of 1.0-1.5, and the result shows that the titer of the polyclonal antiserum corresponding to the N- (2-guanidino-ethylimino) -morpholine hapten Ia corresponding to the formula Ia is 1:128000, and the inhibition rate is 90%; the potency of the polyclonal antiserum against the N- (2-guanidino-ethylimino) -morpholine hapten Ib, which corresponds to the formula Ib, is 1:32000, and the inhibition rate is 86%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An N- (2-guanidino-ethylimino) -morpholine hapten which is characterized by having a structure shown in a formula I:
wherein R is carboxyl and p-carboxyphenyl.
2. A process for the preparation of a hapten of N- (2-guanidino-ethylimino) -morpholine according to claim 1, comprising the steps of: at room temperature, firstly, enabling N-cyano dithioimine dimethyl carbonate to react with morpholine to prepare an intermediate b, then reacting with 80% hydrazine hydrate to prepare an intermediate c, then adding glyoxylic acid or p-aldehyde benzoic acid to perform Mannich reaction with the intermediate c to prepare N- (2-guanidino-ethylimino) -morpholine haptens Ia and Ib, wherein the reaction formula is as follows:
3. use of the hapten of N- (2-guanidino-ethylimino) -morpholine according to claim 1 for the production of artificial antigens of N- (2-guanidino-ethylimino) -morpholine.
4. An artificial antigen of N- (2-guanidino-ethylimino) -morpholine derived from the hapten of N- (2-guanidino-ethylimino) -morpholine of claim 1 coupled to a carrier protein.
5. The N- (2-guanidino-ethylimino) -morpholine artificial antigen as claimed in claim 4 wherein the carrier protein is bovine serum albumin or ovalbumin.
6. The N- (2-guanidino-ethylimino) -morpholine artificial antigen as claimed in claim 4, which is prepared by an active ester method, and comprises the following steps: dissolving N- (2-guanidino-ethylimino) -morpholine hapten, DCC and NHS in a proper amount of DMF, stirring at 4 ℃ for reacting overnight, centrifuging and taking supernatant to obtain an activated N- (2-guanidino-ethylimino) -morpholine derivative, dropwise adding the activated hapten derivative into PBS buffer solution containing 5mg/mL carrier protein, stirring at 4 ℃ for reacting for 12h, dialyzing at 4 ℃ for 3 days by PBS, replacing dialysate for 3 times every day to obtain the N- (2-guanidino-ethylimino) -morpholine artificial antigen, subpackaging and preserving at low temperature for later use; the molar ratio of the N- (2-guanidino-ethylimino) -morpholine hapten to the carrier protein is 40-100: 1.
7. Use of the artificial antigen of N- (2-guanidino-ethylimino) -morpholine as claimed in claim 4 for the production of polyclonal, monoclonal or genetically engineered antibodies against N- (2-guanidino-ethylimino) -morpholine.
8. A polyclonal N- (2-guanidino-ethylimino) -morpholine antibody prepared from the artificial antigen of N- (2-guanidino-ethylimino) -morpholine of claim 4.
9. Use of the polyclonal N- (2-guanidino-ethylimino) -morpholine antibody of claim 8 for the immunological detection of N- (2-guanidino-ethylimino) -morpholine.
10. A rapid immunoassay method for directly detecting N- (2-guanidino-ethylimino) -morpholine is characterized by comprising the following steps: (1) immunizing an animal with the artificial N- (2-guanidino-ethylimino) -morpholine antigen of claim 4 as an immunogen to produce polyclonal antibodies against N- (2-guanidino-ethylimino) -morpholine;
(2) coating the N- (2-guanidino-ethylimino) -morpholine artificial antigen as a coating on a microplate, and adding the anti-N- (2-guanidino-ethylimino) -morpholine polyclonal antibody prepared in the previous step into the microplate;
(3) antiserum titer was detected by indirect competitive ELISA.
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| CN101407501A (en) * | 2008-11-21 | 2009-04-15 | 华南农业大学 | Preparation of 5-methyl morpholine-3-amino-2-oxazolidinone derivative hapten, antigen and antibody |
| WO2013082338A1 (en) * | 2011-11-30 | 2013-06-06 | The Johns Hopkins University | Homomultivalent and heteromultivalent inhibitors of prostate specific membrane antigen (pmsa) and uses thereof |
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| CN1680406A (en) * | 2005-01-21 | 2005-10-12 | 浙江大学 | Triazoline artificial semi-antigen, antigen, specific antibody and use thereof |
| CN101407501A (en) * | 2008-11-21 | 2009-04-15 | 华南农业大学 | Preparation of 5-methyl morpholine-3-amino-2-oxazolidinone derivative hapten, antigen and antibody |
| WO2013082338A1 (en) * | 2011-11-30 | 2013-06-06 | The Johns Hopkins University | Homomultivalent and heteromultivalent inhibitors of prostate specific membrane antigen (pmsa) and uses thereof |
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