CN109575123B - Preparation method and application of fluoroacetamide hapten and monoclonal antibody - Google Patents
Preparation method and application of fluoroacetamide hapten and monoclonal antibody Download PDFInfo
- Publication number
- CN109575123B CN109575123B CN201811324738.8A CN201811324738A CN109575123B CN 109575123 B CN109575123 B CN 109575123B CN 201811324738 A CN201811324738 A CN 201811324738A CN 109575123 B CN109575123 B CN 109575123B
- Authority
- CN
- China
- Prior art keywords
- fluoroacetamide
- hapten
- solution
- antibody
- carrier protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
- C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
- C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
- C07C233/54—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of a saturated carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/58—Preparation of carboxylic acid halides
- C07C51/60—Preparation of carboxylic acid halides by conversion of carboxylic acids or their anhydrides or esters, lactones, salts into halides with the same carboxylic acid part
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
-
- 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
Abstract
The invention relates to the technical field of biochemical engineering, and particularly discloses a preparation method and application of fluoroacetamide hapten and monoclonal antibody. The invention takes ethyl fluoroacetate and p-aminophenylacetic acid as main raw materials to synthesize fluoroacetamide hapten; the conjugate is prepared by coupling with carrier protein Bovine Serum Albumin (BSA) or hemocyanin (KLH) by an active lipid method. Experiments prove that the conjugate prepared by the invention can enable an organism to generate an antibody aiming at fluoroacetamide, namely the prepared conjugate is a fluoroacetamide artificial antigen. The fluoroacetamide artificial antigen prepared by the invention is suitable for enzyme-linked immunoassay of fluoroacetamide.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a preparation method and application of fluoroacetamide hapten and monoclonal antibody.
Background
Fluoroacetamide (also known as 2-fluoroacetamide, aphimide, and Hippodamia Hipponica King et al. The pure product is odorless white needle crystal or white powder, is easily soluble in water (1:5), and can be hydrolyzed into fluoroacetic acid (fluoroacetic acid) in acidic and neutral aqueous solution and into sodium fluoroacetate (sodium fluoroacetate) in alkaline aqueous solution. The preparation process is simple and the product is obtained by fluorinating chloroacetamide. Fluoroacetamide can enter a body through skin, respiratory tract and digestion to cause a poisoning event, if rescue is not timely initiated to cause death, the generation of adenosine triphosphate and the metabolic process of an intermediate product are influenced, the generated fluoroacetamide and the fluorocitric acid can cause damage to multiple organs such as a heart, a brain, a lung, a liver and the like, the fluoroacetamide can prevent the cycle process of tricarboxylic acid to cause the sharp increase of high-activity oxygen free radicals, the generation of ATP is reduced, the structure of a cell membrane is loose, and the electronic structure is unstable, so that electrons are easily taken off by the oxygen free radicals, and the cell membrane is easily attacked by the free radicals. Because of strong neurotoxicity and secondary toxic phenomenon, the compound has 5.3mg/kg of fluoroacetamide and 0.22mg/kg of sodium fluoroacetate to acute oral lethal dose (LD50) of white rats, is easy to be absorbed by skin, has no alertness and is easy to cause secondary poisoning. Rats killed by cats eating fluoroacetamide or sodium fluoroacetate will be poisoned and killed, pests killed by birds eating fluoroacetamide will be poisoned and killed, and birds killed by cats and dogs will also be poisoned and killed.
The fluoroacetamide has the characteristics of high toxicity, quick acting, simple synthesis, low price, difficult generation of drug resistance and the like, and is used for the toxic event, so the damage of the fluoroacetamide to the human health can be reduced to the greatest extent by perfecting a quick detection mechanism and adopting a symptomatic treatment method. At present, the detection method of fluoroacetamide mainly comprises analysis methods based on physicochemical and immunological methods, wherein the former methods comprise thin layer chromatography, chemical color development, gas chromatography, high performance liquid chromatography and the like. The chemochromic method is a commonly used rapid detection method at present, the fluoroacetamide is hydrolyzed to generate ammonia when meeting with a strongly alkaline Neisseria reagent, and the ammonia reacts with the Neisseria reagent to generate a yellowish-brown precipitate, the minimum detected amount is 0.5 mu g, and the positive rate is as follows: 51.57%, although the operation is simple and convenient, the cost is low, and the detection is rapid, the requirements of the detection limit are difficult to meet due to the chemical semi-quantitative detection method, low sensitivity, low positive rate and the like; while other instrument analysis methods have high sensitivity and good reproducibility, but have the characteristics of time consumption, expensive detection instruments, complex operation and the like, and cannot meet the requirement of on-site rapid detection; immunological detection methods such as enzyme chain immunoadsorption reaction, immune chip and the like are gradually paid more attention by relevant departments of quality inspection in China due to the fact that the immunological detection methods are time-saving, low in cost, simple and convenient to operate and suitable for field detection.
Fluoroacetamide is used as hapten, and can be coupled with carrier protein to stimulate animal to produce antibody immunogenicity. However, fluoroacetamide is a small molecule substance, and has a relative molecular weight of only 76, and therefore, it is not immunogenic, and fluoroacetamide has a naked amino group, but it is difficult to cause a chemical reaction due to the formation of an amide bond. This brings great difficulty to the research of the immunoassay method of fluoroacetamide, and also hinders the application and popularization of the immunoassay method to a certain extent.
For the above reasons, it is very necessary to study the preparation method of fluoroacetamide conjugates.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method and application of a fluoroacetamide hapten and a monoclonal antibody.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, a fluoroacetamide hapten has the molecular structural formula shown in formula I:
wherein A is selected from benzene ring or (CH)2)n；n＝1～20。
In a specific embodiment of the invention, A is a phenyl ring, and A is (CH)2)4By way of illustration, both have the chemical structures shown in formula II and formula III:
further, based on the development of the above hapten, the present invention provides a fluoroacetamide antigen obtained by coupling a carrier protein to the carboxyl carbon of the above fluoroacetamide hapten (FLU) by an active ester method, wherein the carrier protein is hemocyanin (KLH) or Bovine Serum Albumin (BSA), and the obtained fluoroacetamide antigen is FLU-KLH or FLU-BSA.
In the subsequent experimental procedures and preparation of the kit, FLU-KLH was used as the immunogen and FLU-BSA was used as the coating antigen.
The preparation method of the fluoroacetamide antigen specifically comprises the following steps:
(1) dissolving the fluoroacetamide hapten in Dimethylformamide (DMF) to obtain a hapten solution;
(2) adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) into the hapten solution, and stirring for reaction;
(3) dissolving carrier protein in PBS buffer solution to obtain protein solution;
(4) dropwise adding the liquid phase obtained in the step (2) into the protein solution prepared in the step (3) while stirring;
preferably, the method specifically comprises the following steps:
(1) weighing 50mg of the fluoroacetamide hapten and dissolving the fluoroacetamide hapten in 1mL of Dimethylformamide (DMF) to obtain a hapten solution;
(2) adding 80mg of Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the hapten solution, and stirring at room temperature for reacting for 2 hours;
(3) weighing 160mg of carrier protein, and dissolving the carrier protein in 20mL of PBS buffer solution to obtain a protein solution;
(4) dropwise adding the liquid phase obtained in the step (2) into the protein solution prepared in the step (3) while stirring;
the mass and volume involved in the above steps may be scaled up or down.
In a second aspect, the invention provides a fluoroacetamide antibody prepared by immunizing an animal with a fluoroacetamide antigen of the invention.
The fluoroacetamide antibody can be a monoclonal antibody or a polyclonal antibody prepared by adopting an antibody preparation method which is conventional in the field.
When the fluoroacetamide antibody is used for detection of a fluoroacetamide compound, it is preferable that the antibody is a fluoroacetamide monoclonal antibody.
The invention further provides application of the fluoroacetamide antibody in detection of fluoroacetamide compounds. The cross reaction rate of the fluoroacetamide monoclonal antibody with 4-aminophenylacetic acid, bromoacetic acid, chloroacetamide, 1-chloro-3-fluoroisopropanol, difluoroacetic acid, 1, 3-difluoro-2-propanol, ethyl (4-aminophenyl) acetate, iodoacetic acid, iodoacetamide, sodium chloroacetate, thiosemicarbazide and 2,2, 2-trifluoroacetamide samples is less than 0.1 percent, and no cross reaction exists, so that the specificity of the fluoroacetamide monoclonal antibody is very good.
Preferably, the method for preparing the fluoroacetamide monoclonal antibody comprises the following steps by taking the fluoroacetamide antigen FLU-KLH as an immunogen:
(1) emulsifying the immunogen with an equal volume of Freund complete adjuvant, immunizing a Balb/c mouse for the first time, halving the dose of the immunogen used in the first immunization, emulsifying with an equal volume of Freund incomplete adjuvant, and performing boosting immunization on the Balb/c mouse immunized for the first time;
(2) the dosage of the immunogen for the first immunization is 0.1 mg/mouse, the immunization dosage of each Balb/c mouse after emulsification is 0.2 ml/mouse, and the immunization mode is 4-8 points of neck and back immunization;
(3) the boosting immunization frequency is 3 times, blood is collected from eyeballs one week after each immunization, antiserum is collected, and the titer and inhibition condition of the antiserum of the mouse are determined by enzyme linked immunosorbent assay (ELISA);
(4) the boosting immunization is specifically carried out once on each 21 days after the first immunization;
(5) two weeks after the last booster immunization, the eyeballs were picked up for blood collection, and the mouse antiserum was collected.
As a preferred embodiment of the present invention, the preparation of the monoclonal antibody specifically comprises the steps of:
(1) animal immunization
Immunizing Balb/c mice with immunogen FLU-KLH with a single immunization dose of 100 mug for 4 times at an interval of 2 weeks, wherein the first three times of immunization is subcutaneous multipoint injection on the back of the neck, and the last time of immunization is intraperitoneal injection;
(2) cell fusion and cloning
3 days after the fourth immunization, splenocytes are taken and fused with SP2/0 myeloma cells according to the ratio of 5:1 (number ratio), cell supernatants are measured by adopting indirect competitive ELISA, and positive holes are screened;
cloning the positive hole by using a limiting dilution method to obtain a hybridoma cell capable of secreting a fluoroacetamide monoclonal antibody;
(3) preparation and purification of monoclonal antibodies
An incremental culture method:
the preparation method of the cell culture medium comprises the following steps: adding calf serum and sodium bicarbonate into a PRIM-1640 culture medium, wherein the final concentration of the calf serum is 20% (mass percentage content) and the final concentration of the sodium bicarbonate is 0.2% (mass percentage content);
placing the hybridoma cells in a culture medium, culturing at 37 ℃ for 2 days, and purifying the obtained culture solution by using an octanoic acid-saturated ammonium sulfate method to obtain a monoclonal antibody solution, wherein the monoclonal antibody solution is stored at (-20 ℃);
protein concentration (mg/mL) ═ 1.45 OD280-0.74 OD260 in the monoclonal antibody
The protein concentration in the monoclonal antibody is calculated by adopting the formula, and the growth condition of the hybridoma cells can be monitored.
Preparing ascites:
balb/c mice were injected intraperitoneally with sterile paraffin oil (0.5 mL/mouse). After 7 days, the hybridoma cells (5 × 105 cells/cell) were intraperitoneally injected, and after 7 days, ascites was collected, and the obtained culture solution was purified by the octanoic acid-saturated ammonium sulfate method to obtain a monoclonal antibody solution (stored at-20 ℃).
Further, the freezing and thawing of the hybridoma cells can be performed by the following method:
making hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. During recovery, the freezing tube is taken out and immediately placed into a water bath kettle at 37 ℃ for instant dissolution, and after centrifugation, the freezing solution is removed and then the tube is transferred into a culture bottle for culture.
Further, the invention provides an enzyme-linked immunoassay kit, which contains the fluoroacetamide antibody, preferably the fluoroacetamide monoclonal antibody.
The kit may further comprise reagents/ingredients conventionally used in the art, such as a coating source, a coating solution, a blocking solution, a second antibody labeled with a bio-enzyme, a washing solution, a color-developing agent, and a stop solution.
Preferably, hemocyanin (KLH) is used as an antigen of a carrier protein as an immunogen, an immune animal is used for preparing an antibody, and Bovine Serum Albumin (BSA) is used as an antigen of the carrier protein as a coating antigen.
Preferably, in the kit:
the coating is originally FLU-BSA;
the coating solution is 0.05M carbonate buffer solution, and the pH value is 9.6;
the sealing liquid is 2% of skimmed milk;
the second antibody marked by the biological enzyme is goat anti-mouse IgG, and the biological enzyme is Horse Radish Peroxidase (HRP);
the washing solution is PBST; namely adding Twen-20 into 0.01M PBS to prepare the product;
the color developing agent is 2% of tetramethyl benzidine (TMB) and 30% of hydrogen peroxide.
The above stop solution is 2M H2SO4。
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the invention firstly provides the fluoroacetamide hapten, prepares the fluoroacetamide antibody and fills the blank at home and abroad. There has been no information on the preparation of fluoroacetamide antibodies and the related immunoassay methods. The conjugate of the hapten and the carrier protein provided by the invention is used for preparing the fluoroacetamide antibody, and the preparation process is simple, economic, high in sensitivity and high in practical value. The kit prepared by the polyclonal antibody prepared by the invention has good application prospect.
Drawings
FIG. 1 is a scheme showing the synthesis of fluoroacetamide hapten-fluoroacetylphenylacetic acid (fluoroacetamide phe) in example 1.
FIG. 2 is a scheme showing the synthesis of fluoroacetamide hapten fluoroacetylhexanoic acid (fluoroacetamide liner) in example 1.
FIG. 3 shows the fluoroacetamide hapten p-fluoroacetophenone acetate (fluoroacetamide phe) in example 11H NMR spectrum.
FIG. 4 shows fluoroacetamide hapten fluoroacetylhexanoic acid (fluoro) in example 1Acetamide liner) of1H NMR spectrum.
FIG. 5 is a flyweight spectrum of BSA in example 3.
FIG. 6 is a flight mass spectrum of the coupling product of BSA and fluoroacetamide hapten p-fluoroacetylphenylacetic acid (fluoroacetamide phe) in example 3.
FIG. 7 is a flight mass spectrum of the coupling product of BSA and fluoroacetamide hapten fluoroacetylhexanoic acid in example 3.
FIG. 8 is a flight mass spectrum of the coupling product of BSA and fluoroacetic acid in example 3.
FIG. 9 shows the sensitivity of detection of monoclonal antibodies prepared from the fluoroacetamide artificial antigen in example 4.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged. The hemocyanin is called KLH for short, and the bovine serum albumin is called BSA for short.
Example 1 Fluoroacetamide hapten (fluoroacetamide phe) and Synthesis and identification
1. Synthesis of fluoroacetamide phe (shown in FIG. 1)
1) Synthesis of fluoroacetyl chloride
An aqueous solution of KOH (19g,0.339mol) (34mL) was slowly added dropwise to ethyl fluoroacetate (30g, 0.283mol) in ethanol (300mL) at room temperature to form a white precipitate. The mixture was stirred at room temperature (30-35 ℃ C.) overnight. And (3) spin-drying the solvent, re-dissolving the obtained sodium salt in hydrochloric acid (3M, 200mL), adding NaCl solid to saturate the solution, then extracting with diethyl ether for 4-5 times, drying the combined organic phases with anhydrous sodium sulfate, filtering, and spin-drying the filtrate to obtain the fluoroacetic acid (15 g).
Adding PCl into a three-mouth bottle with a thermometer and a condenser inserted therein5(44g, 0.211mol, 1.1eq), cooling to 0-5 ℃ in an ice bath, slowly dropwise adding fluoroacetic acid, stirring the reaction solution for 0.5h at room temperature, and then heating to 80 ℃ for reaction for 1 h. The reaction solution was transferred to a single-neck flask and distilled under atmospheric pressure, and a 70-72 ℃ fraction (12g, 44% yield) was collected as a colorless transparent liquid.
2) Synthesis of ethyl p-aminophenylacetate (1-010-A2)
SOCl2(17.3g,0.146mol) was slowly added dropwise to ethanol (90mL) of p-aminophenylacetic acid (11g, 0.0728mol), and the mixture was cooled in an ice bath. The temperature is increased to reflux for 1 h. Cooling to room temperature, removing the solvent by spinning off, and adding Na to the residue2CO3Adjusting the pH value of the solution to 8-9, extracting the EtOAc for 2-3 times, washing the combined organic phase with water and saturated salt water, and drying the organic phase with anhydrous sodium sulfate. Filtration and spin-drying of the filtrate gave a yellow liquid (13.0g, 100%) as a clean product without further purification.
3) Synthesis of 1-010-A3
A THF solution (100mL) of the compound 1-010-A2(13g,0.075mol) is cooled to 0-5 ℃, fluoroacetyl chloride (9.45g,0.098mol) is slowly added dropwise, and the reaction is carried out for 2-3 h at room temperature (30-35 ℃) after the dropwise addition is finished. Appropriate amount of water was added, EtOAc extraction was performed 2-3 times, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the filtrate was spun dry, and column chromatography (DCM/MeOH ═ 80: 1) gave compound 1-010-a2(12.5g, 72%) as a pale yellow powder.
4) Synthesis of ENO-1-010
An aqueous solution (25mL) of KOH (2.53g, 0.045mol) was slowly added dropwise to ethanol (70mL) of 1-010-A3(7.2g, 0.03mol), and the mixture was stirred at room temperature (32-35 ℃ C.) for 1 h. Adjusting pH to about 5 with 15% HOAc, removing ethanol by rotary evaporation to obtain yellow powder, precipitating, and stirring for 0.5 hr. Filtration and drying of the filter cake gave pale yellow crystals (3.7g, 59%).
2. Identification of fluoroacetamide phe
The structure is determined by hydrogen nuclear magnetic resonance spectroscopy:
1H NMR(DMSO-d6,400MHz)，3.52(s,2H)，4.92(s,1H),5.03(s,1H),7.21(d,J＝4.2Hz,2H),7.57(d,J＝4.2Hz,2H),10.07(br,1H)。
the structural formula of the fluoroacetamide phe is shown as the formula (II)1H NMR spectrum 3.
Example 2 fluoroacetamide hapten (fluoroacetamide liner) and Synthesis and characterization
1. Synthesis of fluoroacetamide liner (shown in FIG. 2)
Adding 10g (76mmol) of 6-aminocaproic acid into 100ml of anhydrous methanol, enabling the solution to be turbid, adding 23g (227mml) of triethylamine, stirring for 10min at room temperature, dropwise adding 12g (113mmol) of ethyl fluoroacetate, carrying out reflux reaction overnight, enabling the solution to become clear, cooling to room temperature, adding acetic acid to adjust the pH value to 5-6, concentrating the reaction solution, carrying out oil pump drying to obtain an oily liquid, adding EtOAc (ethyl acetate) for dissolution, washing EA twice with saturated saline, drying, spin drying, and carrying out oil pump drying to obtain 7g of a product, wherein the yield is about 48%.
2. Identification of fluoroacetamide liner
The structure is determined by hydrogen nuclear magnetic resonance spectroscopy:
1H NMR(400MHz,CDCl3):6.48(bs,1H,NH)；4.87(s,1H,CH2)；4.75(s,1H,CH2)3.35(q,2H,CH2,J＝6.8Hz)；2.36(t,2H,CH2,J＝8.4Hz)；1.67(m,2H,CH2),1.59(m,2H,CH2),1.40(m,2H,CH2)。
the structural formula of the fluoroacetamide liner is shown as a formula (III), and the fluoroacetamide liner has1The H NMR spectrum is shown in FIG. 4.
EXAMPLE 3 preparation of fluoroacetamide Artificial antigen
1. Preparation and identification of fluoroacetamide coatingen
1.1 preparation of Fluoroacetamide Encapsulates
Dissolving 50mg of fluoroacetamide hapten (shown in formula II or formula III) in 1ml of N, N-Dimethylformamide (DMF), adding 80mg of N, N' -Dicyclohexylcarbodiimide (DCC) and 60mg of N-hydroxysuccinimide (NHS) into the fluoroacetamide hapten solution, and stirring at room temperature for reacting for 2 hours; BSA 160mg dissolved in 20ml of PBS buffer (0.01mol/L pH 7.4), followed by dropwise addition of the activating drug to the protein solution under stirring, and reaction at 4 ℃ overnight; then putting the reaction solution into a dialysis bag, dialyzing with a 4 ℃ physiological saline solution for 48h, and changing water for 4 times; the dialysate was dispensed into centrifuge tubes and stored at-20 ℃.
1.2 identification of Fluoroacetamide Encapsulates
Fluoroacetamide artificial antigen was dissolved in PBS for flight mass spectrometry analysis, and BSA was used as a control (as shown in FIG. 5). The flight mass spectrum result shows that: the fluoroacetamide hapten and BSA have a coupling reaction (the coupling number of fluoroacetamide phe, fluoroacetamide liner and fluoroacetic acid are 36, 34 and 78 respectively), so that a fluoroacetamide hapten BSA conjugate is generated, and the flight mass spectrum of the fluoroacetamide phe, fluoroacetamide liner, fluoroacetic acid and BSA conjugate is shown in FIGS. 6-8. 2. Preparation and characterization of fluoroacetamide immunogens
2.1 preparation of fluoroacetamide immunogens
And (3) replacing Bovine Serum Albumin (BSA) with hemocyanin (KLH), and performing the other steps 1.1 in the step (1) to obtain a fluoroacetamide hapten KLH conjugate, namely the fluoroacetamide immunogen.
Fluoroacetamide immunogen is called FLU-KLH for short, and fluoroacetamide coating original solution is called FLU-BSA solution for short. The concentration of FLU-KLH in the FLU-KLH solution was 1 mg/mL.
EXAMPLE 4 preparation of monoclonal antibody against fluoroacetamide Compound
Balb/c mice: purchased from Beijing Wittiulihua laboratory animal technology, Inc.;
SP2/0 myeloma cells: available from sigma-aldrich, catalog No. 08060101.
First, animal immunization
A fluoroacetamide immunogen was prepared according to example 3 using haptens of formula II and III, respectively, and Balb/c mice were immunized with a solution of FLU-KLH, each mouse was immunized with 100. mu.g of FLU-KLH in a single dose 4 times, each time at 2 weeks intervals, the former three immunizations were performed by subcutaneous multiple injections into the back of the neck, and the latter immunization was performed intraperitoneally.
Second, cell fusion and cloning
1. 3 days after the fourth immunization, splenocytes were taken and fused with SP2/0 myeloma cells at a ratio of 5:1 (quantitative ratio), cell supernatants were assayed by indirect competitive ELISA, and positive wells were screened.
2. Cloning the positive hole by using a limiting dilution method to obtain hybridoma cells capable of secreting fluoroacetamide monoclonal antibodies, and respectively obtaining two hapten structure anti-fluoroacetamide monoclonal antibody hybridoma cells which are respectively named as FLU-B6 and FLU-C11 according to different immunogens.
Thirdly, freezing and recovering cells
Making hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per ml, preserved for long period in liquid nitrogen. During recovery, the freezing tube is taken out and immediately placed into a water bath kettle at 37 ℃ for instant dissolution, and after centrifugation, the freezing solution is removed and then the tube is transferred into a culture bottle for culture.
Preparation and purification of monoclonal antibody
1. Method of incremental culture
The preparation method of the cell culture medium comprises the following steps: calf serum and sodium bicarbonate are added into the PRIM-1640 culture medium, wherein the final concentration of the calf serum is 20% (mass percentage content) and the final concentration of the sodium bicarbonate is 0.2% (mass percentage content).
The hybridoma cells were cultured in a medium at 37 ℃ for 2 days, and the resulting culture medium was purified by the octanoic acid-saturated ammonium sulfate method to obtain a monoclonal antibody solution (stored at-20 ℃).
Protein concentration (mg/mL) ═ 1.45 OD280-0.74 OD260 in the monoclonal antibody
The protein concentration in the monoclonal antibody is calculated by adopting the formula, and the growth condition of the hybridoma cells can be monitored.
2. Preparation of ascites
Balb/c mice were injected intraperitoneally with sterile paraffin oil (0.5 mL/mouse). 7 days later, hybridoma cells (5X 10) were injected intraperitoneally5One/one), ascites was collected after 7 days, and the obtained culture solution was purified by the octanoic acid-saturated ammonium sulfate method to obtain a monoclonal antibody solution (preservation at (-20 ℃).
Fifth, identification of monoclonal antibody
The monoclonal antibody solutions prepared by the incremental culture method in the fourth step are respectively identified as follows:
1. the subtype of the monoclonal antibody was detected using an ELISA monoclonal antibody subtype detection kit (Sigma, catalog No. 19285), and the immunoglobulin subclass of the monoclonal antibody was kappa class and IgG1 subtype.
2. Determination of antibody titer
(1) The solution of FLU-BSA prepared in example 3 (adjusted in concentration with carbonate buffer) was used for coating at 100. mu.L/well and the concentration of the solution of FLU-BSA was 1.0. mu.g/mL.
(2) Incubate at 4 ℃ for 16 hours.
(3) The plate was closed and washed.
(4) Adding 100 μ L of monoclonal antibody solution or its diluent (PBS buffer for gradient dilution) prepared by step four middle increment culture method into each well
(5) Incubate for 2h at room temperature and wash the plate.
(6) mu.L of horseradish peroxidase-labeled IgG was added to each well and incubated at room temperature for 2 hours.
(7) And (5) washing the plate.
(8) Adding TMB color development solution, and developing in dark for 15 min.
(9) The reaction was stopped by adding 50. mu.l of 2mol/L sulfuric acid to each well and the OD450 was read.
Positive wells were obtained when the OD reached about 1.0. The antibody titer was 1: 27000.
3. calculation of monoclonal antibody sensitivity
Wherein the immunogen is KLH-FLU-phe coated by BSA-FLU-liner, KLH-FLU-acid (fluoroacetic acid) is coated by BSA-FLU-acid, and KLH-FLU-liner is coated by BSA-FLU-phe, so that the sensitivity is high.
(1) - (3) the same as (1) - (3) of step 2.
(4) Add 50. mu.l TETS standard solution to each well (consisting of TETS and PBS buffer, wells with only PBS buffer added as control wells); each hole is provided with 3 multiple holes
(5) 50 μ L of the monoclonal antibody solution prepared by the step four increment culture method or a dilution thereof (PBS buffer for gradient dilution) was added to each well, incubated at room temperature for 2 hours, and the plate was washed.
(6) mu.L of horseradish peroxidase-labeled IgG was added to each well and incubated at room temperature for 2 hours.
(7) Washing plate
(8) Adding TMB color development solution, and developing in dark for 15 min.
(9) Add 50. mu.L of 2mol/L sulfuric acid per well to stop the reaction, read the OD450The value is obtained.
The absorbance values (average values of three multiple wells) obtained by using the standard solutions of the respective concentrations were divided by the absorbance values of the control wells, and then multiplied by 100 as ordinate, and a graph was drawn with the logarithmic value of the natural number of the fluoroacetamide concentration in each standard solution as abscissa. The detection result of the sensitivity of the monoclonal antibody shows that the monoclonal antibodies with immunogen KLH-FLU-phe and KLH-FLU-liner have higher sensitivity. Wherein, the immunogen is monoclonal antibody prepared by KLH-FLU-phe, the sensitivity is the highest, and the sensitivity detection curve is shown in figure 9.
Referring to FIG. 9, the concentration of fluoroacetamide corresponding to a value on the ordinate equal to 50% is obtained, i.e., IC50The value is obtained. Sensitivity of monoclonal antibodies to fluoroacetamide (IC)50) The value was 1.5 mg/mL.
6. Specific assay
Respectively dissolving 4-aminophenylacetic acid, bromoacetic acid, chloroacetamide, 1-chloro-3-fluoroisopropanol, difluoroacetic acid, 1, 3-difluoro-2-propanol, ethyl (4-aminophenyl) acetate, iodoacetic acid, iodoacetamide, sodium chloroacetate, thiosemicarbazide and 2,2, 2-trifluoroacetamide by using a coating buffer solution to prepare solutions with the following concentration gradients: 0. 37, 111, 333, 1000, 3000 and 9000ng/mL, and the half inhibitory amounts (IC) of 4-aminophenylacetic acid, bromoacetic acid, chloroacetamide, 1-chloro-3-fluoroisopropanol, difluoroacetic acid, 1, 3-difluoro-2-propanol, ethyl (4-aminophenyl) acetate, iodoacetic acid, iodoacetamide, sodium chloroacetate, thiosemicarbazide and 2,2, 2-trifluoroacetamide samples against fluoroacetamide monoclonal antibody (monoclonal antibody prepared by KLH-FLU-phe immunization was selected, BSA-FLU-liner was used as the coating antigen) and fluoroacetamide coating antigen were calculated according to the procedures in step one50) And the cross-reaction rate of the reaction,the formula for the cross-reactivity is: the cross-reactivity ratio (%) × 100% (concentration of fluoroacetamide causing 50% inhibition of reaction of fluoroacetamide monoclonal antibody with fluoroacetamide peridium antigen/concentration of other substance causing 50% inhibition of reaction of fluoroacetamide monoclonal antibody with fluoroacetamide peridium antigen). The experiment was repeated 3 times.
The results showed that the half inhibition amounts (IC50) of the fluoroacetamide monoclonal antibody to the fluoroacetamide coating antigen were each greater than 1000. mu.g/mL for 4-aminophenylacetic acid, bromoacetic acid, chloroacetamide, 1-chloro-3-fluoroisopropanol, difluoroacetic acid, 1, 3-difluoro-2-propanol, ethyl (4-aminophenyl) acetate, iodoacetic acid, iodoacetamide, sodium chloroacetamide, thiosemicarbazide and 2,2, 2-trifluoroacetamide samples, fluoroacetamide monoclonal antibody to the fluoroacetamide coating antigen, fluoroacetamide to 4-aminophenylacetic acid, bromoacetic acid, chloroacetamide, 1-chloro-3-fluoroisopropanol, difluoroacetic acid, 1, 3-difluoro-2-propanol, ethyl (4-aminophenyl) acetate, iodoacetic acid, iodoacetamide, sodium chloroacetate, thiosemicarbazide and 2, the cross reaction rate of the 2, 2-trifluoroacetamide sample is less than 0.1%, and no cross reaction exists, which indicates that the specificity of the fluoroacetamide monoclonal antibody is very good.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. It should be understood that the technical solutions of the above embodiments, in which the amounts of reagents or raw materials used are proportionally increased or decreased, are substantially the same as those of the above embodiments. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. A fluoroacetamide hapten, characterized by the molecular structural formula as shown in formula (II):
2. a fluoroacetamide antigen obtainable by conjugating a fluoroacetamide hapten according to claim 1 to a carrier protein, wherein the carrier protein is hemocyanin or bovine serum albumin.
3. A process for the preparation of a fluoroacetamide antigen as claimed in claim 2, wherein a carrier protein is coupled to the carboxyl carbon of the fluoroacetamide hapten as claimed in claim 1 by the active ester method.
4. The preparation method according to claim 3, characterized by comprising the following steps:
(1) dissolving the fluoroacetamide hapten of claim 1 in dimethylformamide to provide a hapten solution;
(2) adding dicyclohexylcarbodiimide and N-hydroxysuccinimide into the hapten solution, and stirring for reaction;
(3) dissolving carrier protein in PBS buffer solution to obtain protein solution;
(4) and (3) dropwise adding the liquid phase obtained in the step (2) into the protein solution prepared in the step (3) while stirring.
5. The preparation method according to claim 3 or 4, characterized by comprising the following steps:
(1) weighing 50mg of the fluoroacetamide hapten as defined in claim 1 and dissolving the fluoroacetamide hapten in 1mL of dimethylformamide to obtain a hapten solution;
(2) adding 80mg of dicyclohexylcarbodiimide and 60mg of N-hydroxysuccinimide into the hapten solution, and stirring at room temperature for reacting for 2 hours;
(3) weighing 160mg of carrier protein, and dissolving the carrier protein in 20mL of PBS buffer solution to obtain a protein solution;
(4) dropwise adding the liquid phase obtained in the step (2) into the protein solution prepared in the step (3) while stirring;
the mass and volume involved in the above steps may be scaled up or down.
6. A fluoroacetamide antibody produced by immunizing an animal with the fluoroacetamide antigen of claim 2.
7. The fluoroacetamide antibody according to claim 6, wherein the antibody is a fluoroacetamide monoclonal antibody or a polyclonal antibody.
8. A hybridoma cell secreting a fluoroacetamide antibody of claim 6 or 7.
9. Use of the antibody of claim 6 or 7 for the detection of fluoroacetamides for non-disease diagnostic purposes.
10. An enzyme-linked immunoassay kit comprising the antibody of claim 6 or 7.
11. The kit according to claim 10, wherein hemocyanin is used as an antigen of a carrier protein as an immunogen, an immune animal produces an antibody, and bovine serum albumin is used as an antigen of a carrier protein as a coating antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811324738.8A CN109575123B (en) | 2018-11-08 | 2018-11-08 | Preparation method and application of fluoroacetamide hapten and monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811324738.8A CN109575123B (en) | 2018-11-08 | 2018-11-08 | Preparation method and application of fluoroacetamide hapten and monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109575123A CN109575123A (en) | 2019-04-05 |
| CN109575123B true CN109575123B (en) | 2020-12-15 |
Family
ID=65921817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811324738.8A Active CN109575123B (en) | 2018-11-08 | 2018-11-08 | Preparation method and application of fluoroacetamide hapten and monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109575123B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551220A (en) * | 2019-08-29 | 2019-12-10 | 北京勤邦生物技术有限公司 | Preparation and application of DDT monoclonal antibody |
| CN112094810B (en) * | 2020-08-26 | 2022-07-05 | 中国农业大学 | Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403755A (en) * | 2008-10-16 | 2009-04-08 | 江南大学 | Immunity chromatography detection test paper for fussol poison and preparation method thereof |
| CN104262250A (en) * | 2014-09-01 | 2015-01-07 | 深圳市美凯特科技有限公司 | Triiodothyroxin hapten luminous marker and synthetic method thereof |
| CN104402817A (en) * | 2014-10-22 | 2015-03-11 | 深圳市美凯特科技有限公司 | Novel thyroxine hapten luminescent marker and synthetic method thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3267108D1 (en) * | 1982-04-20 | 1985-12-05 | Teijin Ltd | Novel vitamin d3 derivatives, processes for preparation thereof, and antigens comprising said derivatives to be used for preparation of antibodies for immunochemical assay and antibodies prepared therefrom |
| JPS61112042A (en) * | 1985-07-31 | 1986-05-30 | Wakunaga Seiyaku Kk | Compound suitable as crosslinking reagent |
| US9274105B2 (en) * | 2011-07-13 | 2016-03-01 | Optrotrace (SuZhou) Technologies, Inc. | Analyzing chemical and biological substances using nano-structure based spectral sensing |
| CN106928079B (en) * | 2017-03-21 | 2019-02-22 | 广东第二师范学院 | Trefanocide haptens and its preparation method and application |
| CN108047071B (en) * | 2017-12-07 | 2020-07-28 | 杭州同舟生物技术有限公司 | Carcinone artificial hapten, artificial antigen, preparation method and application thereof |
| CN108517317A (en) * | 2018-04-04 | 2018-09-11 | 江南大学 | A kind of anti-Clorprenaline monoclonal antibody hybridoma cell strain and its application |
| CN108640850B (en) * | 2018-05-14 | 2021-04-13 | 广东达元绿洲食品安全科技股份有限公司 | Malachite green hapten, artificial antigen and application of malachite green hapten and artificial antigen in fluorescence quantitative immunochromatography |
-
2018
- 2018-11-08 CN CN201811324738.8A patent/CN109575123B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101403755A (en) * | 2008-10-16 | 2009-04-08 | 江南大学 | Immunity chromatography detection test paper for fussol poison and preparation method thereof |
| CN104262250A (en) * | 2014-09-01 | 2015-01-07 | 深圳市美凯特科技有限公司 | Triiodothyroxin hapten luminous marker and synthetic method thereof |
| CN104402817A (en) * | 2014-10-22 | 2015-03-11 | 深圳市美凯特科技有限公司 | Novel thyroxine hapten luminescent marker and synthetic method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Synthesis of Substituted Thioureas and Their Sulfur Heterocyclic Systems of p-Amino Salicylic Acid as Antimycobacterial Agents;Mohammed Saleh I. T. Makki等;《Journal of Chemistry》;20130131;摘要 * |
| 半抗原间隔臂尺度对免疫原性的影响研究;白宇琛等;《农药学学报》;20080430;Article ID 862463 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109575123A (en) | 2019-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111057064B (en) | 14-hydroxyl gelsemine hapten and artificial antigen as well as preparation method and application thereof | |
| CN110845444B (en) | Dimethomorph hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN109265401B (en) | Preparation method and application of iprodione hapten and antigen | |
| CN109575123B (en) | Preparation method and application of fluoroacetamide hapten and monoclonal antibody | |
| CN108680755B (en) | Sulfonamide synergist hapten and holoantigen as well as preparation method and application thereof | |
| CN110627726B (en) | Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN109824645B (en) | Warfarin hapten and artificial antigen as well as preparation method and application thereof | |
| CN111333503A (en) | Dicofol hapten, artificial antigen and antibody as well as preparation methods and applications thereof | |
| CN109212200A (en) | A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof | |
| CN102336831A (en) | Anti-sildenafil specific antibody | |
| CN104744294B (en) | (α ethyl hexyls) ester hapten, artificial antigen and its preparation method for antibody of phthalic acid two and application | |
| CN109970550B (en) | Sorbic acid hapten, artificial antigen, antibody, synthetic method and application thereof | |
| CN110938007B (en) | Dicofol hapten, artificial antigen, antibody, synthetic method and application thereof | |
| CN109608479A (en) | The preparation method and application of a kind of Tetramine haptens and monoclonal antibody | |
| CN112480167B (en) | Isocarbophos hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN109305963A (en) | A kind of ketoconazole haptens, artificial antigen and the preparation method and application thereof | |
| CN114315722B (en) | Preparation and application of tolfenpyrad artificial hapten and antibody thereof | |
| JP2005035893A (en) | Alachlor hapten and antibody against alachlor and immunoassay using the same | |
| CN114480295B (en) | Hybridoma cell strain secreting anti-butralin monoclonal antibody and application thereof | |
| CN116041211B (en) | Rapid detection device for malachite green in aquatic product, and preparation and application thereof | |
| CN112939832B (en) | Tiamulin hapten TMLO, artificial antigen, antibody and preparation method and application thereof | |
| CN111060690B (en) | Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof | |
| CN114644572B (en) | Bromhexine hapten, artificial antigen, antibody, and preparation method and application thereof | |
| CN116120242B (en) | Quick detection device for fenazaquin in tea, and preparation and application thereof | |
| CN111943881B (en) | Chlorfenapyr hapten, artificial antigen and antibody as well as preparation methods and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |