CN114989098A - Ethirimol hapten, full antigen, antibody, preparation method and application - Google Patents
Ethirimol hapten, full antigen, antibody, preparation method and application Download PDFInfo
- Publication number
- CN114989098A CN114989098A CN202210866370.8A CN202210866370A CN114989098A CN 114989098 A CN114989098 A CN 114989098A CN 202210866370 A CN202210866370 A CN 202210866370A CN 114989098 A CN114989098 A CN 114989098A
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- Prior art keywords
- ethirimol
- formula
- hapten
- antibody
- preparation
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- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- 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
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- C07K14/765—Serum albumin, e.g. HSA
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- 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
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention belongs to the technical field of immunoassay, and particularly relates to an ethirimol hapten, a full antigen, an antibody, a preparation method and application thereof. The ethirimol hapten provided by the invention has a structure shown in a formula I, wherein R in the formula I 1 Is an active group capable of direct coupling to a carrier protein. The ethirimol hapten provided by the invention keeps basic structure of ethirimolBased on the structure, the original-CH of ethirimol 2 ‑CH 2 ‑CH 2 ‑CH 2 -the structure is used as a linker arm, and an active group R capable of directly coupling to a carrier protein is introduced at a site on the linker arm remote from the ethirimol feature 1 The nonspecific binding caused by an external connecting arm is effectively avoided, the introduced active group can fully expose the characteristic structure of the ethirimol main body molecule after being coupled with the carrier protein, the prepared ethirimol complete antigen has immunogenicity, the prepared ethirimol-resistant antibody has good specificity and high sensitivity, can be used for establishing an ethirimol immunological detection method or detection reagent, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of immunoassay, and particularly relates to an ethirimol hapten, a full antigen, an antibody, a preparation method and application thereof.
Background
Ethirimol is named prometryn in English and has a chemical name: 5-butyl-2-ethylamino-4-hydroxy-6-methylpyrimidine with CAS accession number: 23947-60-6, the molecular formula is: c 11 H 19 N 3 O, molecular weight: 209.3, the Chinese category name: ethirimol, pyrimethanil, ethimidone, aminopyrimidine, 5-butyl-2-ethylamino-4-hydroxy-6-methylpyrimidine, ethirimol or ethirimol.
The ethirimol has actual control effect on powdery mildew of a plurality of crops such as strawberries, watermelons, cucumbers, grapes and the like, and has higher safety on the crops than other imported or domestic like products. However, unreasonable ethirimol application mode and dosage can cause environmental pollution and pesticide residue, and residual ethirimol can be continuously transferred and migrated in a food chain through enrichment, so that adverse effects on quality, safety and ecological environment of agricultural products are caused, and human health is further harmed. Therefore, the residual level of ethirimol needs to be monitored.
Many methods have been established to detect residual ethirimol in environmental samples, food and agricultural products. Instrumental methods of analysis are sufficiently accurate and highly sensitive, and Liquid Chromatography (LC), LC-tandem mass spectrometry (LC-MS/MS), Gas Chromatography (GC) and GC-tandem mass spectrometry (GC-MS/MS) are all commonly used detection methods. However, these instrumental methods typically require trained personnel, expensive instrumentation, complex sample pre-treatment and time-consuming analytical procedures, and are not suitable for on-site residual detection of ethirimol.
The immunoassay method has the advantages of rapidness, simplicity, convenience, real-time property, easy field detection, simple sample pretreatment, high sensitivity, strong selectivity, suitability for high-throughput analysis and the like due to the specific recognition effect of the immunoassay method on the antigen and the antibody, and can greatly reduce the detection cost.
Disclosure of Invention
In view of the above, the purpose of the present invention is to provide an ethirimol hapten, a complete antigen, an antibody preparation method and applications, the ethirimol hapten provided by the present invention can fully highlight ethirimol antigenic determinants after coupling, and the prepared ethirimol complete antigen has immunogenicity and can specifically recognize ethirimol, so that the obtained anti-ethirimol antibody has good specificity and high sensitivity, and can be further used for research and development of an ethirimol immunoassay method.
The invention provides an ethirimol hapten which has a structure shown in a formula I:
r in the formula I 1 Is an active group capable of direct coupling to a carrier protein.
Preferably, said R is 1 Comprises- (CH) 2 ) n -COOH、-(CH 2 ) n -NH 2 、-(CH 2 ) n -OH、-(CH 2 ) n -COOR 2 、
n is an integer of 0 to 3, m is an integer of 0 to 4, and the formula- (CH) 2 ) n -COOR 2 
R in (1) 2 Included
Preferably, the ethirimol hapten has a structure represented by formula I-1 or formula I-2:
the invention provides a preparation method of ethirimol hapten, which comprises the following steps:
r in the structure of the compound with the structure shown in formula II 3 Replacement of a group by R by chemical reaction 1 Obtaining group to obtain the ethirimol hapten with the structure shown in the formula I;
r in the formula II 3 is-CH 3 or-COO-CH 2 -CH 3 。
Preferably, the method for preparing the ethirimol hapten with the structure shown in the formula I-1 comprises the following steps:
dissolving a compound with a structure shown in a formula 5 and inorganic strong base in a mixed solution of an organic solvent and water for hydrolysis reaction to obtain an ethirimol hapten with a structure shown in a formula I-1;
the invention provides an ethirimol complete antigen which is obtained by coupling the ethirimol hapten prepared by the technical scheme or the ethirimol hapten prepared by the preparation method of the technical scheme with carrier protein.
The invention provides an ethirimol antibody, which is obtained by emulsifying the ethirimol complete antigen in the technical scheme and then immunizing a host animal.
The invention provides a preparation method of the ethirimol antibody in the technical scheme, which comprises the following steps:
emulsifying the ethirimol complete antigen in the technical scheme and then immunizing a host animal;
the preparation method of the ethirimol polyclonal antibody comprises the following steps: after the immune host animal is boosted, collecting blood of the immune animal and separating to obtain the ethirimol polyclonal antibody;
the preparation method of the ethirimol monoclonal antibody comprises the following steps: extracting splenocytes of an immunized host animal, fusing the splenocytes with SP2/0 tumor cells, screening monoclonal hybridoma cell strains capable of secreting ethirimol antibodies, and preparing ethirimol monoclonal antibodies based on the monoclonal hybridoma cell strains;
the preparation method of the ethirimol nano antibody comprises the following steps: extracting mRNA transcribed by B lymphocytes in the blood of the immunized host animal, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by taking the cDNA as a substrate to obtain diversified nano antibody gene segments, constructing a phage or yeast antibody expression library by using the diversified nano antibody gene segments, and screening to obtain the ethirimol nano antibody.
The invention provides a reagent or a kit for detecting ethirimol, which contains the ethirimol antibody in the technical scheme or the ethirimol antibody prepared by the preparation method in the technical scheme.
The invention provides an ethirimol antibody in the technical scheme, or an ethirimol antibody prepared by the preparation method in the technical scheme, or an application of a reagent or a kit in the technical scheme in detection of ethirimol.
The invention provides an ethirimol hapten which has a structure shown in a formula I:
r in the formula I 1 Is an active group capable of direct coupling to a carrier protein.
The ethirimol hapten provided by the invention is prepared from the original-CH of ethirimol on the basis of keeping the basic structure of ethirimol 2 -CH 2 -CH 2 -CH 2 -structure as a full or partial linking structure of a linker arm, in-CH 2 -CH 2 -CH 2 -CH 2 Introduction of an active group R capable of direct coupling to a carrier protein, at a site structurally distant from the characteristic structure of ethirimol 1 The ethirimol complete antigen has immunogenicity, and after the host animal is immunized with the ethirimol complete antigen, the prepared ethirimol-resistant serum or ethirimol-resistant antibody can be used for establishing an ethirimol immunological detection method or detection reagent, so that the method has a wide application prospect.
The invention provides an ethirimol complete antigen which is obtained by coupling the ethirimol hapten prepared by the technical scheme or the ethirimol hapten prepared by the preparation method of the technical scheme with carrier protein. The ethirimol-resisting serum or the ethirimol-resisting antibody obtained by immunizing the ethirimol complete antigen provided by the invention can efficiently recognize and specifically bind ethirimol, and can provide a technical basis for the immune application of ethirimol.
The invention provides an ethirimol antibody, which is obtained by emulsifying the ethirimol complete antigen in the technical scheme and then immunizing a host animal. The ethirimol-resistant serum or the ethirimol-resistant antibody obtained by immunizing the ethirimol complete antigen provided by the invention can efficiently recognize and specifically bind ethirimol.
In conclusion, the ethirimol hapten is designed and synthesized for the first time, and the ethirimol complete antigen prepared by coupling the ethirimol hapten with the carrier protein has immunogenicity. According to the invention, the ethirimol complete antigen is used for immunizing a host animal to prepare the ethirimol-resistant antibody with high sensitivity and specificity, so that a technical basis can be provided for establishing an immunoassay method of the ethirimol-resistant antibody. Moreover, the immunoassay method developed based on the ethirimol-resistant antibody prepared by the invention can meet the requirement of rapidly detecting ethirimol on site, and has wide application prospect.
Drawings
FIG. 1 is a scheme for synthesizing an ethirimol hapten of the formula I-1 in example 1;
FIG. 2 is a scheme for synthesizing ethirimol hapten of formula I-2 in example 2;
FIG. 3 is a scheme of the synthesis of the ethirimol complete antigen of formula III according to the invention;
FIG. 4 is a liquid chromatogram of ethirimol hapten shown in formula I-1 in example 1;
FIG. 5 is a mass spectrum of LCMS of ethirimol hapten shown in formula I-1 in example 1;
FIG. 6 is a drawing showing the ethirimol hapten of formula I-1 in example 1 1 H NMR spectrum;
FIG. 7 is a MALDI-TOF-MS spectrum of OVA standard in example 3;
FIG. 8 is a MADLI-TOF-MS spectrum of the ethirimol complete antigen of formula III-1 in example 3;
FIG. 9 is a MADLI-TOF-MS spectrum of the BSA standard in example 4;
FIG. 10 is a MALDI-TOF-MS spectrum of the ethirimol complete antigen shown in formula III-2 in example 4.
Detailed Description
The invention provides an ethirimol hapten which has a structure shown in a formula I:
r in the formula I 1 Is an active group capable of direct coupling to a carrier protein.
In the present invention, said R 1 Preferably comprises- (CH) 2 ) n -COOH、-(CH 2 ) n -NH 2 、-(CH 2 ) n -OH、-(CH 2 ) n -COOR 2 、
n is an integer of 0 to 3, m is an integer of 0 to 4, and the formula- (CH) 2 ) n -COOR 2 
R in (1) 2 Included
In the present invention, n is preferably an integer of 0 to 3, and more preferably 0.
In the present invention, said R 1 Particularly preferred are-OH and-NH 2 -COOH or-COOR 2 。
In the present invention, the- (CH) 2 ) n -COOR 2 R in (1) 2 It is particularly preferable that
In particular embodiments of the invention, the ethirimol hapten is particularly preferably of the formula I-1 or formula I-2:
the invention provides a preparation method of ethirimol hapten, which comprises the following steps:
r in the structure of the compound with the structure shown in formula II 3 Replacement of a group by R by chemical reaction 1 Obtaining group to obtain the ethirimol hapten shown in the formula I;
r in the formula II 3 is-CH 3 or-COO-CH 2 -CH 3 。
In the present invention, the method for preparing the ethirimol hapten with the structure shown in formula I-1 preferably comprises the following steps:
dissolving a compound with a structure shown in a formula 5 and inorganic strong base in a mixed solution of an organic solvent (hereinafter referred to as a first organic solvent) and water for hydrolysis reaction to obtain an ethirimol hapten with a structure shown in a formula I-1;
in the present invention, the method for preparing the compound having the structure represented by formula 5 preferably comprises the steps of:
dissolving a compound having a structure shown in formula 1, a compound having a structure shown in formula 2 and sodium hydride (NaH) in an organic solvent (hereinafter referred to as a second organic solvent) in a protective gas atmosphere to perform an alkylation reaction, thereby obtaining a compound having a structure shown in formula 3;
the compound with the structure shown in the formula 3, the compound with the structure shown in the formula 4 and sodium methoxide (CH) 3 ONa) dissolved in an organic solvent (hereinafter referred to as a third organic solvent) to carry out a condensation reaction, thereby obtaining a compound having a structure represented by formula 5;
in a specific embodiment of the present invention, the compound having a structure represented by formula 4 is specifically guanidine ethylsulfate.
The compound with the structure shown in the formula 1, the compound with the structure shown in the formula 2 and sodium hydride (NaH) are dissolved in Tetrahydrofuran (THF) in a protective gas atmosphere to carry out alkylation reaction, so that the compound shown in the formula 3 is obtained.
In the present invention, the molar ratio of the compound having the structure represented by formula 1 to the compound having the structure represented by formula 2 is preferably 3: 2.
In the present invention, the molar ratio of the compound having the structure represented by formula 1 to NaH is preferably 1: 1.
In the present invention, the second polar organic solvent is particularly preferably Tetrahydrofuran (THF).
The invention has no special requirement on the dosage of the THF, and the compound with the structure shown in the formula 1, the compound with the structure shown in the formula 2 and NaH are completely dissolved.
In the present invention, the alkylation reaction is preferably carried out under heating under reflux.
In the present invention, the time for the alkylation reaction is preferably 12 hours.
In the present invention, the protective gas atmosphere is preferably a nitrogen atmosphere.
In the invention, alkylation reaction liquid is obtained after the alkylation reaction, and the alkylation reaction liquid is preferably subjected to post-treatment to obtain the compound with the structure shown in the formula 3. In the present invention, the post-treatment preferably includes: adding saturated ammonium chloride aqueous solution, adding water, extracting with ethyl acetate, mixing organic phases, washing the organic phase with saturated NaCl aqueous solution, drying, concentrating, and separating and purifying by column chromatography. In the present invention, the volume ratio of the saturated ammonium chloride aqueous solution to the THF is preferably 1: 3; the volume ratio of the water to the saturated aqueous ammonium chloride solution is preferably 5: 1; the number of times of ethyl acetate extraction is preferably 2 washes, and the ratio of the volume of ethyl acetate used in each extraction to the volume of water is preferably 1: 1; the drying is preferably carried out by using anhydrous sodium sulfate; the invention has no special requirements on the specific implementation process of the concentration; in the invention, the column chromatography is preferably silica gel column chromatography, the eluent used for the column chromatography is preferably petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 10: 1.
In the invention, the compound with the structure shown in the formula 3 is light yellow oily matter.
After obtaining the compound shown in the formula 3, the compound shown in the formula 4 (guanidine ethyl sulfate) and sodium methoxide are dissolved in a third organic solvent for condensation reaction, and the compound shown in the formula 5 is obtained.
In the present invention, the molar ratio of the compound represented by formula 3 to the compound represented by formula 4 is preferably 2: 1.
In the present invention, the molar ratio of the compound represented by the formula 3 to sodium methoxide is preferably 1: 1.
In the present invention, the third organic solvent is particularly preferably methanol.
The invention has no special requirement on the using amount of the methanol, and the compound shown in the formula 3, the compound shown in the formula 4 and sodium methoxide are completely dissolved.
In the present invention, the temperature of the condensation reaction is preferably 70 ℃.
In the present invention, the time of the condensation reaction is preferably 1 hour.
In the present invention, a condensation reaction solution is obtained after the condensation reaction, and the present invention preferably performs a post-treatment on the condensation reaction solution to obtain a compound having a structure represented by formula 5. In the present invention, the post-treatment preferably comprises: removing the ethanol solvent, adding saturated ammonium chloride aqueous solution, extracting by using ethyl acetate, washing an organic phase by using water, washing by using saturated NaCl solution, drying the washed organic phase, concentrating, and separating and purifying by column chromatography. In the present invention, the volume ratio of the saturated aqueous ammonium chloride solution to ethanol is preferably 4: 3; the volume ratio of the ethyl acetate to the saturated ammonium chloride aqueous solution is preferably 1: 2; the invention preferably adopts anhydrous sodium sulfate to dry the washed organic phase; the invention has no special requirements on the specific implementation process of the concentration; in the present invention, the column chromatography is preferably silica gel column chromatography, the eluent used for the column chromatography is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 50: 1.
In the present invention, the strong inorganic base is preferably NaOH.
In the present invention, the first organic solvent is particularly preferably ethanol.
In the present invention, the mass ratio of the compound having the structure represented by formula 5 to the inorganic strong base is preferably 11.56: 8.
In the present invention, the volume ratio of ethanol to water is preferably 1: 1.
In the present invention, the temperature of the hydrolysis reaction is preferably room temperature.
In the present invention, the time for the hydrolysis reaction is preferably 16 hours.
In the invention, hydrolysis reaction liquid is obtained after the hydrolysis reaction, and the invention preferably carries out post-treatment on the hydrolysis reaction liquid to obtain the ethirimol hapten with the structure shown in the formula I-1. In the present invention, the post-treatment preferably comprises: sequentially carrying out: concentrating, adjusting the pH value of the concentrated solution to 4 with acetic acid to precipitate solid, carrying out solid-liquid separation, collecting the solid product, washing with water, pulping with methanol, and drying. The invention has no special requirements on the specific implementation process of the concentration; the ratio of the volume of the water for washing to the volume of the methanol for beating is preferably 1: 2; the invention has no special requirements for the specific implementation process of the drying.
In the present invention, the method for preparing the ethirimol hapten with the structure shown in formula I-2 preferably comprises the following steps:
dissolving the ethirimol hapten with the structure shown in the formula I-1, N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) in N, N-Dimethylformamide (DMF) for substitution reaction to obtain the ethirimol hapten with the structure shown in the formula I-2.
In the present invention, the molar ratio of the ethirimol hapten to the NHS having the structure represented by the formula I-1 is preferably 0.021: 0.042.
In the present invention, the molar ratio of the ethirimol hapten to the EDC having the structure represented by the formula I-1 is preferably 0.021: 0.042.
In the invention, the ethirimol hapten with the structure shown in the formula I-1, NHS and EDC are dissolved in a reaction solution formed by DMF, and the molar concentration of the ethirimol hapten with the structure shown in the formula I-1 is preferably 0.042 mol/L.
In the present invention, the temperature of the substitution reaction is preferably 4 ℃.
In the present invention, the incubation time for the substitution reaction is preferably 10 hours.
In the present invention, the substitution reaction is preferably carried out under stirring, and the stirring is preferably magnetic stirring.
The invention provides an ethirimol complete antigen which is obtained by coupling the ethirimol hapten prepared by the technical scheme or the ethirimol hapten prepared by the preparation method of the technical scheme with carrier protein.
In the present invention, the carrier protein preferably comprises Bovine Serum Albumin (BSA), Ovalbumin (OVA) or Keyhole Limpet Hemocyanin (KLH).
In the present invention, the structural formula of the ethirimol complete antigen is preferably as shown in formula III:
in a specific embodiment of the present invention, the structural formula of the ethirimol complete antigen is particularly preferably as shown in formula III-1 or formula III-2:
the invention provides a preparation method of the ethirimol complete antigen in the technical scheme, which preferably comprises the following steps:
and mixing the ethirimol hapten with a buffer solution of the carrier protein for coupling reaction to obtain the ethirimol complete antigen.
In the invention, when the ethirimol hapten is preferably an ethirimol hapten with a structure shown in a formula I-1, the ethirimol hapten with the structure shown in the formula I-1 is preferably prepared according to the preparation method of the ethirimol hapten shown in the formula I-2 to obtain the ethirimol hapten shown in the formula I-2, and then the ethirimol hapten and a carrier protein are subjected to coupling reaction.
In the present invention, the ethirimol hapten is preferably coupled to the carrier protein in the form of an organic solution of the ethirimol hapten.
In a specific embodiment of the present invention, the ethirimol hapten is preferably a substitution reaction supernatant obtained by a method for preparing the ethirimol hapten shown in formula I-2 according to the above technical scheme, and the substitution reaction supernatant is used as a raw material to perform a coupling reaction with the carrier protein.
In the invention, in the reaction system of the coupling reaction, the molar ratio of the ethirimol hapten to the carrier protein is preferably (10-60): 1, and more preferably 60: 1.
In the invention, the reaction temperature of the coupling reaction is preferably 0-50 ℃, and more preferably 4 ℃; the coupling reaction time is preferably 8-36 h, and more preferably 12 h; the pH value of the coupling reaction is preferably 5-9, and more preferably 7.4.
In the present invention, the buffer solution of the carrier protein is preferably at least one of a Carbonate Buffer Solution (CBS), a Phosphate Buffer Solution (PBS), a borate buffer solution, and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution of the carrier protein. In the present invention, the pH of the buffer solution of the carrier protein is preferably 5 to 9, and more preferably 7.4.
In the present invention, after the coupling reaction, the present invention preferably further comprises dialyzing the reaction system of the coupling reaction, wherein the dialysate used for dialysis is preferably a PBS solution; the pH value of the PBS solution is preferably 7-10, and more preferably 7.4; the concentration of the PBS solution is preferably 0.01-0.2 mol/L, and more preferably 0.01 mol/L.
The ethirimol hapten has good stability, few synthesis steps, low synthesis cost, simple reaction conditions, high purity of the synthesized hapten, and solubility and stability which can meet the requirements of coupling carrier protein.
The invention also provides application of the hapten or the ethirimol complete antigen in the scheme in preparation of an ethirimol antibody.
In the present invention, the ethirimol antibody is preferably an anti-ethirimol serum.
The invention provides an ethirimol antibody, which is obtained by emulsifying the ethirimol complete antigen in the technical scheme and then immunizing a host animal.
The invention provides a preparation method of the ethirimol antibody in the technical scheme, which comprises the following steps:
emulsifying the ethirimol complete antigen in the technical scheme and then immunizing a host animal;
the preparation method of the ethirimol polyclonal antibody comprises the following steps: after the immune host animal is strengthened, collecting blood of the immune animal and separating to obtain the ethirimol polyclonal antibody;
the preparation method of the ethirimol monoclonal antibody comprises the following steps: extracting splenocytes of an immunized host animal, fusing the splenocytes with SP2/0 tumor cells, screening monoclonal hybridoma cell strains capable of secreting ethirimol antibodies, and preparing ethirimol monoclonal antibodies based on the monoclonal hybridoma cell strains;
the preparation method of the ethirimol nano antibody comprises the following steps: extracting mRNA transcribed by B lymphocytes in the blood of the immunized host animal, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by taking the cDNA as a substrate to obtain diversified nano antibody gene segments, constructing a phage or yeast antibody expression library by using the diversified nano antibody gene segments, and screening to obtain the ethirimol nano antibody.
In the present invention, in the preparation of the ethirimol nanobody, the host animal is preferably camelidae or cartilaginous fish.
In the present invention, the monoclonal hybridoma cell line is easily prepared by those skilled in the art, for example, spleen cells obtained by immunizing a host animal with the complete antigen are taken, fused with SP2/0 tumor cells in vitro, and monoclonal hybridoma cells capable of secreting ethirimol are selected by a selective medium to obtain a monoclonal hybridoma cell line; the ethirimol nano antibody can also be obtained by means of genetic engineering technology and the like, and the technologies are easy to realize for the technicians in the field; based on this, it is within the scope of the present invention to use the hapten or the complete antigen provided by the present invention no matter what method is used to prepare the ethirimol monoclonal antibody, the ethirimol polyclonal antibody or the ethirimol nano antibody.
On the basis of the hapten or complete antigen disclosed in the invention, the person skilled in the art can easily think of preparing anti-ethirimol antibody by adopting a proper antibody preparation method, no matter what kind of animal is used for immunization, no matter how the immunization condition or parameter is set or changed, as long as the hapten or complete antigen provided by the invention is used, and the invention belongs to the protection scope.
The antibody or antiserum prepared by immunizing a host animal with the ethirimol complete antigen provided by the invention can be specifically combined with ethirimol, and has a good immune effect.
The invention provides a reagent or a kit for detecting ethirimol, which contains an ethirimol antibody in the technical scheme or an anti-ethirimol antibody prepared by the preparation method in the technical scheme.
The invention provides an ethirimol antibody in the technical scheme, an anti-ethirimol antibody prepared by the preparation method in the technical scheme, or an application of a reagent or a kit in the technical scheme in ethirimol detection.
The ethirimol hapten provided by the invention has the advantages of simple synthetic route, low synthetic cost, good solubility and stability; the antiserum obtained by immunizing a host animal with the complete antigen has high titer and good characteristics, and splenocytes obtained by immunizing a mouse with the complete antigen can be effectively used for fusing hybridoma cells and preparing the anti-ethirimol monoclonal antibody. The alpaca is immunized by the complete antigen, the peripheral blood of the alpaca is collected, B lymphocytes of the alpaca are collected, a phage display nano antibody expression library is constructed by a phage display technology, and the nano antibody for specifically recognizing the ethirimol can be obtained by four rounds of solid phase competition panning. The ethirimol monoclonal antibody, the ethirimol polyclonal antibody or the ethirimol nano antibody prepared by the ethirimol complete antigen provided by the invention has good specificity and low minimum detection limit, can realize specific recognition of ethirimol, can be used for establishing an ethirimol immunological detection method or detection reagent, and has wide application prospect.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Examples some sources of materials:
hydrochloric acid, sodium dihydrogen phosphate dodecahydrate, sodium chloride, gelatin, citric acid monohydrate and Tween-20 were all purchased from national drug group chemical agents, Inc.; anhydrous N, N-Dimethylformamide (DMF) was purchased from alatin; 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (EDC), N-hydroxysuccinimide (NHS), Freund's complete adjuvant, Freund's incomplete adjuvant, Bovine Serum Albumin (BSA) and Ovalbumin (OVA) were purchased from Sigma; goat anti-mouse IgG-HRP was purchased from Jackson.
Example 1
Synthesizing an ethirimol hapten with a structure shown as a formula I-1: the structure is as follows:
the synthetic route is shown in figure 1.
NaH (60%, 1.20g,30mmol) was added to dry THF (30mL) under nitrogen to give a mixture which was then cooled to 0 ℃; to the mixture system was added ethyl acetoacetate (1, 3.90g, 30mmol in FIG. 1) with stirring, followed by stirring at room temperature for 30min, and to the system was added ethyl 5-bromo-pentanoate (2, 4.18g, 20mmol in FIG. 1); the reaction mixture was heated to reflux for 12 h. Cooled to room temperature, saturated ammonium chloride solution (10mL) was added followed by water (50 mL); the resulting organic phase was combined with ethyl acetate (extraction (50mL × 2)), washed with saturated brine (50mL), dried over sodium sulfate, and concentrated to give an oil, which was subjected to silica gel column chromatography (petroleum ether: ethyl acetate: 10:1, v: v, elution) to give a compound having a structure represented by formula 3 as a pale yellow oil (3.28g, yield 60%) (3 in fig. 1).
A compound having a structure represented by formula 4 (guanidine ethylsulfate, 8.5g, 31mmol) was dissolved in methanol (150mL), cooled to 0 ℃ in an ice bath, and a 30 wt% methanol solution of sodium methoxide (11.2g, 62mmol) was added thereto and stirred at the same temperature for 30 min; then, the compound of formula 3 (16.0g, 62mmol) was added to the system and the mixture was heated to 70 ℃ and stirred for 1 h. Concentrating the obtained reaction liquid to remove ethanol; to the residue was added a saturated ammonium chloride solution (200 mL); extraction with ethyl acetate (100 mL); the organic phase was washed with water (200mL), saturated brine (100mL) and dried over sodium sulfate; the concentrated residue was subjected to silica gel column chromatography (dichloromethane: methanol 50:1, v: v, elution gave a compound having a structure represented by formula 5 (12.4g, yield 71%).
The compound having a structure represented by formula 5 (11.56g, 40mmol) was dissolved in ethanol (100mL), and a sodium hydroxide solution (NaOH 8.0g, 100mL water) was added thereto, and the resulting reaction mixture was stirred at room temperature for reaction for 16 hours. Concentrating the reaction solution, adjusting the pH value to 4 with acetic acid, stirring for 10min, separating out a solid, filtering and collecting a solid product, washing the solid product with water (50mL), pulping with methanol (100mL), and drying to obtain a white solid, namely the ethirimol hapten with the structure shown in the formula I-1.
FIG. 4 is a liquid chromatogram of ethirimol hapten, shown in formula I-1 in example 1; the liquid chromatogram (FIG. 4) shows that only one sample peak is present, and the ethirimol hapten has the molecular formula C 12 H 19 N 3 O 3 254.15, exact theoretical molecular weight value is 253.30.
The product prepared in example 1 was purified by liquid chromatography, LC-MS (FIG. 5), 1 H NMR (FIG. 6) confirmed the ethirimol hapten shown in formula I-1: a 5- (2- (ethylamino) -4-hydroxy-6-methylpyrimidin-5-yl) pentanoic acid compound, having the english name: 5- (2- (ethyllamino) -4-hydroxy-6-methylpyrimidin-5-yl) pentanic acid.
1 H NMR(400MHz,DMSO-d6)δ1.07(t,J＝6.4Hz,3H),1.29-1.37(m,2H),1.44-1.52(m,2H),2.05(s,3H),2.19-2.28(m,4H),3.18-3.25(m,2H),6.12(s,1H),10.66(s,1H),11.91(s,1H)。
Example 2
Synthesizing an ethirimol hapten shown as a formula I-2, wherein the structure is as follows:
the synthetic route is shown in figure 2.
5.37mg (0.021mmol) of ethirimol immunogen represented by formula I-2 prepared in example 1, 4.88mg (0.042mmol) of NHS and 8.13mg (0.042mmol) of EDC were weighed out and sufficiently dissolved in 0.5mL of anhydrous DMF, and the reaction was magnetically stirred at 4 ℃ for 10 hours. After the reaction is finished, reaction liquid containing the ethirimol hapten shown as the formula I-2 is obtained, and the reaction liquid containing the ethirimol hapten shown as the formula I-2 prepared in the example 2 can be directly used for coupling with a buffer solution of carrier protein.
Example 3
Synthesizing an ethirimol complete antigen of the structure shown in formula III-1:
the synthetic route is shown in figure 3.
The synthesis method comprises the following steps:
the supernatant of the reaction solution containing the ethirimol hapten shown in the formula I-2 obtained in the example 2 is slowly dripped into PBS buffer solution of OVA, wherein the PBS buffer solution of OVA is obtained by dissolving 10mg of OVA in 1mL of PBS buffer solution with the pH value of 7.4 and uniformly mixing, the feeding molar ratio of the ethirimol hapten shown in the formula I-2 to the OVA is 40:1, the reaction is stirred at 25 ℃ for 4 hours, the obtained reaction solution is dialyzed for six times in PBS buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L, and the completely dialyzed reaction product solution is diluted into 1mg/mL solution. The dialysis is used for removing ethirimol hapten shown in formula I-2 or other small molecules in the unreacted liquid to obtain ethirimol complete antigen shown in formula III-1, namely a conjugate of ethirimol hapten shown in formula I-1 and OVA.
The OVA (figure 7) and the ethirimol complete antigen with the structure shown in the formula III-1 (figure 8) obtained by MALDI-TOF-MS detection have 44721.036 and 47354.014 single charge ion peaks. The coupling ratio of the carrier protein OVA to the ethirimol hapten as described in formula I-1 is calculated as: coupling ratio (complete antigen molecular weight-carrier protein molecular weight)/hapten molecular weight. The coupling ratio of the ethirimol complete antigen of the structure shown in the formula III-1 is calculated to be 1:10 by a coupling ratio formula.
Example 4
Synthesizing an ethirimol complete antigen having the structure shown in formula III-2:
slowly dripping the supernatant of the reaction liquid containing the ethirimol hapten shown in the formula I-2 obtained in the example 2 into a PBS buffer solution of BSA, wherein the PBS buffer solution of BSA is obtained by dissolving 20mg of BSA in 5 mLphosphate buffer solution (PBS) with the pH value of 7.4 and uniformly mixing, the feeding molar ratio of the ethirimol hapten shown in the formula I-2 to the BSA is 40:1, stirring and reacting for 4 hours at 25 ℃, dialyzing the obtained reaction liquid for six times in the PBS buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L, diluting the completely dialyzed reaction product solution into a solution with the concentration of 1mg/mL, quickly freezing the solution by liquid nitrogen, and freezing and storing at-20 ℃ for later use. Obtaining the ethirimol complete antigen with the structure shown in the formula III-2, namely the conjugate of the ethirimol hapten and BSA shown in the formula I-1.
MALDI-TOF-MS detects the obtained BSA standard (figure 9) and the ethirimol complete antigen with the structure shown in formula III-2 (figure 10) with single charge ion peaks 67510.359 and 75544.340. The coupling ratio of the ethirimol complete antigen of the structure shown in the formula III-2 is calculated to be 1:31 by a coupling ratio formula.
Example 5
Application of ethirimol complete antigen
First, ethirimol antibody was prepared using the ethirimol complete antigen of the structure represented by formula III-2 prepared in example 4
(1) A6-8 week-old Balb/c mouse was used as an experimental animal (the weight of an 8 week-old Balb/c mouse was about 23-25 g).
(2) Primary immunization: the diluted ethirimol complete antigen solution (with the concentration of 1mg/mL) with the structure shown in the formula III-2 obtained in the example 4 is filtered by a sterile filter, added with equal volume of Freund's complete adjuvant, and fully stirred and emulsified until the ethirimol complete antigen solution does not diffuse when dropped into water. The ethirimol complete antigen with the structure shown in the formula III-2 which is well emulsified is injected into abdominal cavities and subcutaneous backs of mice, and the total injection dose is 0.1mg of emulsified antigen per mouse.
(3) And (3) boosting immunity: after 2 weeks of primary immunization, 1mL of the diluted ethirimol complete antigen solution with the structure shown in formula III-2 is added with 1mL of Freund's incomplete adjuvant, and the mixture is fully stirred and emulsified until the ethirimol complete antigen solution is not dispersed when being dropped into water. The emulsified ethirimol complete antigen with the structure shown in the formula III-2 is used for carrying out intraperitoneal injection and back subcutaneous multi-point injection on mice, and the total injection dose is 0.1mg of emulsified antigen per mouse. The boosting immunization is carried out once every 14 days, blood is collected from the eye orbit of the mice 3 days after the third boosting immunization, and the titer of ethirimol antibody in serum is determined by the test method: ic-ELISA. Coating 1mg/mL ethirimol complete antigen with a structure shown in a formula III-1, diluting serum to obtain required gradient, removing eyeballs and collecting blood after the titer is more than or equal to 1:64000 (the titer is defined as the dilution multiple of the serum when the color value of a zero hole is about 1.0), standing the blood in a constant-temperature incubator at 37 ℃ for 30min, standing the blood in a refrigerator at 4 ℃ for 2h, centrifuging the blood in a centrifuge at 4 ℃ and 10000r/min for 5min, and separating the serum to obtain the ethirimol antiserum. Was used in the following experiments.
Second, potency detection of antibody in anti-ethirimol serum
The various buffers used in the following experiments were as follows:
(1) coating buffer (CBS, pH 9.6, 0.05M carbonate buffer) Na was weighed 2 CO 3 1.5g、NaHCO 3 2.94 g, and keeping the volume of ultrapure water to 1000 mL;
(2) phosphate buffer (0.01M PBS, pH 7.4): weighing KH 2 PO 4 0.2 g、NaCl 8g、NaH 2 PO 4 ·12H 2 O2.92 g and ultrapure water with constant volume of 1L;
(3) wash buffer (PBST): adding Tween-20 with the volume ratio of 0.1% into the prepared phosphoric acid buffer solution;
(4) sample dilution (PBSTG): adding 1% Tween-20 and 1g gelatin (heated and melted in a microwave oven) into the prepared phosphoric acid buffer solution;
(5) substrate buffer (pH 5.5): weighing Na 2 HPO 3 ·12H 2 9.22g of O, 2.55g of citric acid monohydrate, 0.5mL of Tween-20, and constant volume of 1L of ultrapure water;
(6) stop solution (1M HCl): 440mL of distilled water was measured out, and 40mL of 98% (v/v) concentrated hydrochloric acid was added dropwise with stirring.
(one) determination of the potency and inhibition of the antibody in ethirimol-resistant serum
1. Preparation of ethirimol-coated antigen solution with structure shown as formula III-1
The ethirimol complete antigen of the structure shown in the formula III-1 prepared in the example 3 is subjected to gradient dilution by CBS according to the ratio of 1:1000, 1:2000, 1:4000 and 1:8000 to obtain coating antigen solutions of ethirimol complete antigens of the structure shown in the formula III-1 with different concentrations.
2. Preparation of ethirimol standard solution
(1) Weighing 10mg of ethirimol standard sample, and fully dissolving the ethirimol standard sample in 10mL of methanol to obtain 1mg/mL ethirimol standard solution.
(2) The 1mg/mL ethirimol standard solution of (1) above was prepared using PBSTG to a final concentration of 1000ng/mL ethirimol standard solution.
3. Preparation of ethirimol antiserum diluent
And (3) carrying out gradient dilution on the ethirimol antiserum prepared in the step (a) by using PBSTG according to needs to obtain an ethirimol antiserum diluent.
4. Determination of ethirimol antiserum titer and inhibition rate
Coating: and (3) adding 100 mu L of the ethirimol complete antigen coating antigen solution with the structure shown in the formula III-1, which is prepared in the step (1), into each hole of a 96-hole enzyme label plate, incubating for 3h at the constant temperature of 37 ℃, washing for 3 times by using PBST, and drying.
Secondly, competition: in zero wells, 50 μ LPBSTG per well; and (3) adding 50 mu L of ethirimol standard solution prepared in the step (2) into each inhibition hole.
③ adding ethirimol antiserum diluent into an enzyme label plate (50 mu L/hole), incubating for 30min at 37 ℃, washing the plate for 3 times by PBST, and drying.
Adding an enzyme labeled secondary antibody: goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) was diluted to a working concentration with PBS (0.1M, pH 9.6), added at 100. mu.L per well, incubated at 37 ℃ for 30min, washed 3 times with PBST, and spun-dried.
Color development: the chromogenic substrate is prepared at present, 100 mu LTMB single-component chromogenic solution is added into each hole, and the mixture is shaded and developed for 15min at room temperature.
Sixthly, termination and detection: the reaction was stopped by adding 50. mu.L of 1M HCl stop solution to each well, and the absorbance (OD value) of each well was measured at 450nm using a microplate reader.
Determining the potency: when the OD value is about 1.0, the maximum dilution multiple of the ethirimol antiserum is the ethirimol antiserum titer.
Determining specificity, namely judging the specificity of the serum according to the inhibition rate.
The calculation formula of the inhibition rate is as follows: the inhibition ratio ═ [ (control well OD value-inhibition well OD value)/control well OD value ] × 100%, the ethirimol antiserum titer results after the third and fourth immunizations are shown in table 1.
TABLE 1 ethirimol-resistant serum titers and inhibition rates (TMB color development 15min, 1000ng/mL standard inhibition)
Note: i represents the inhibition hole in the ELISA plate, C represents the control hole in the ELISA plate, IR represents the inhibition rate, and K represents 1000 times.
TABLE 2 serum titers and inhibition rates of ethirimol fusion mice (TMB color 15min, 1000ng/mL standard inhibition)
Note: i represents an inhibition well in the microplate, C represents a control well in the microplate, IR represents an inhibition rate, and K represents 1000 times.
The results in Table 1 show that after the fourth immunization, when the coating antigen (ethirimol hapten-OVA) is diluted 8000 times, the anti-ethirimol serum titer is 256000, and the inhibition rate is 76.2%. The ethirimol complete antigen with the structure shown in the formula III-2 prepared in the example 4 has immunogenicity and good specificity, and can be used for preparing anti-ethirimol antibodies.
The results in Table 2 show that the fused mice have high serum titer and the inhibition rate is more than 80%. It is demonstrated that spleen cells of ethirimol fusion mice having the structure of formula III-2 prepared in example 5 above can be used in fusion experiments to prepare anti-ethirimol antibodies.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. An ethirimol hapten having the structure of formula I:
r in the formula I 1 Is an active group capable of direct coupling to a carrier protein.
2. The ethirimol hapten according to claim 1, wherein said R is 1 Comprises- (CH) 2 ) n -COOH、-(CH 2 ) n -NH 2 、-(CH 2 ) n -OH、-(CH 2 ) n -COOR 2 、
n is an integer of 0 to 3, m is an integer of 0 to 4, and the formula- (CH) 2 ) n -COOR 2 Or
R in (1) 2 Included
3. The ethirimol hapten according to claim 1 or claim 2, wherein said ethirimol hapten has the structure of formula I-1 or formula I-2:
4. a process for the preparation of an ethirimol hapten as claimed in any one of claims 1 to 3 comprising the steps of:
combining the structure shown in the formula IIR in the structure of a substance 3 Replacement of a group by R by chemical reaction 1 The group is obtained to obtain the ethirimol hapten with the structure shown in the formula I;
r in the formula II 3 is-CH 3 or-COO-CH 2 -CH 3 。
5. The method according to claim 4, wherein the ethirimol hapten of the formula I-1 is prepared by a method comprising the steps of:
dissolving a compound with a structure shown in a formula 5 and inorganic strong base in a mixed solution of an organic solvent and water to perform hydrolysis reaction to obtain an ethirimol hapten with a structure shown in a formula I-1;
6. an ethirimol complete antigen obtained by coupling the ethirimol hapten prepared by any one of claims 1 to 3 or the ethirimol hapten prepared by the preparation method of claim 4 or 5 with a carrier protein.
7. An ethirimol antibody produced by immunizing a host animal with an emulsified ethirimol complete antigen of claim 6.
8. A method for producing an ethirimol antibody as claimed in claim 7, comprising the steps of:
immunizing a host animal after emulsifying the ethirimol complete antigen of claim 6;
the preparation method of the ethirimol polyclonal antibody comprises the following steps: after the immune host animal is boosted, collecting blood of the immune animal and separating to obtain the ethirimol polyclonal antibody;
the preparation method of the ethirimol monoclonal antibody comprises the following steps: extracting splenocytes of immune host animals, fusing the splenocytes with SP2/0 tumor cells, screening monoclonal hybridoma cell strains capable of secreting ethirimol antibodies, and preparing ethirimol monoclonal antibodies based on the monoclonal hybridoma cell strains;
the preparation method of the ethirimol nano antibody comprises the following steps: extracting mRNA transcribed by B lymphocytes in the blood of an immune host animal, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by taking the cDNA as a substrate to obtain diversified nano antibody gene segments, constructing a phage or yeast antibody expression library by using the diversified nano antibody gene segments, and screening to obtain the ethirimol nano antibody.
9. A reagent or kit for detecting ethirimol, comprising the ethirimol antibody of claim 7 or the ethirimol antibody prepared by the preparation method of claim 8.
10. Use of the ethirimol antibody of claim 7, or the ethirimol antibody produced by the production method of claim 8, or the reagent or kit of claim 9, for the detection of ethirimol.
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