WO2020248316A1 - 毒黄素半抗原、人工抗原、抗体及其合成方法和应用 - Google Patents

毒黄素半抗原、人工抗原、抗体及其合成方法和应用 Download PDF

Info

Publication number
WO2020248316A1
WO2020248316A1 PCT/CN2019/095182 CN2019095182W WO2020248316A1 WO 2020248316 A1 WO2020248316 A1 WO 2020248316A1 CN 2019095182 W CN2019095182 W CN 2019095182W WO 2020248316 A1 WO2020248316 A1 WO 2020248316A1
Authority
WO
WIPO (PCT)
Prior art keywords
toxoflavin
hapten
compound
antigen
toxaxanthin
Prior art date
Application number
PCT/CN2019/095182
Other languages
English (en)
French (fr)
Inventor
柯跃斌
李金峰
黎诚耀
丁晔
赵芳
刘博超
Original Assignee
深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） filed Critical 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
Publication of WO2020248316A1 publication Critical patent/WO2020248316A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present invention relates to the technical field of immunochemical analysis, in particular to toxoflavin haptens, artificial antigens, antibodies, and synthetic methods and applications thereof.
  • Fermented rice noodle poisoning is a bacterial food poisoning with a very high fatality rate in the vast rural areas of our country.
  • Pseudomonas cocotoxin fermented rice noodle subspecies is the cause of fermented rice noodle food poisoning and spoiled white fungus poisoning.
  • the pathogenic substances of this bacteria are two toxins produced during growth and reproduction: bongkrekic acid. acid, BA) and toxoflavin (TF). Both toxins are small molecules of fatty acids, which are highly toxic, heat-resistant, and highly toxic, and cannot be destroyed by general cooking methods. Therefore, food is easily poisoned after being contaminated. There is currently no specific antidote. The production of fermented acid is much higher than toxoflavin.
  • the methods used to detect rice yeast acid mainly include traditional detection methods, such as spectrophotometry, high performance liquid chromatography and their combined technology.
  • traditional detection methods such as spectrophotometry, high performance liquid chromatography and their combined technology.
  • the equipment is expensive and the detection time is long. , And require professional operation, so it is impossible to achieve real on-site detection.
  • the immunological detection and analysis technology has been widely used in the field of drug residue detection due to its high sensitivity, high specificity, rapidity, and easy operation. It has many advantages compared with inspection methods such as instruments. Therefore, the immunoassay provides a new analysis and detection method for the study of the residues of rice yeast acid.
  • the key technology is to be able to obtain antibodies with strong specificity and high sensitivity.
  • the prerequisite is to synthesize and prepare Appropriate hapten of rice yeast acid.
  • the rice yeast acid has a chain structure, and it is difficult for immunized animals to obtain high quality antibodies.
  • Toxaxanthin is a three-dimensional molecule composed of two parallel rings. Compared with orysonic acid, immunized animals are more likely to obtain highly sensitive and specific antibodies.
  • the immunodetection of toxoflavin can indirectly detect orycholic acid, which provides a simple and reliable detection scheme for the poisoning of orycholic acid.
  • the present invention provides toxoflavin haptens, artificial antigens, antibodies and their synthesis methods and applications.
  • Toxoflavin hapten which has the following structural formula:
  • n is the number of -CH 2 groups, and n is an integer of 2-8.
  • the present invention also provides a method for synthesizing the above-mentioned toxoflavin hapten, which includes the following steps:
  • compound 3 reacts with formaldehyde to obtain compound 4, which has the structural formula , Where n is the number of -CH 2 groups, and n is an integer of 2-8;
  • the compound 5 undergoes a hydrolysis reaction to obtain the toxin hapten.
  • the method for synthesizing the compound 1 is: (1) providing Intermediate 1, whose molecular formula is: , Where n is the number of -CH 2 groups, and n is an integer of 2-8; (2) The intermediate 1 is reacted with tert-butyl carbazate to obtain the compound 1.
  • the present invention also provides toxaxanthin artificial antigens, which include toxaxanthin immune antigens and toxaxanthin coating antigens, which are obtained by coupling the toxin haptens and carrier proteins.
  • the carrier protein is any one of bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HAS) or hemocyanin (KLH).
  • BSA bovine serum albumin
  • OVA ovalbumin
  • HAS human serum albumin
  • KLH hemocyanin
  • the structure of the toxin hapten when preparing the toxaxanthin immune antigen, the structure of the toxin hapten, 3 ⁇ n ⁇ 8; when preparing the toxoflavin coating antigen, the structure of the toxin hapten, 2 ⁇ n ⁇ 5.
  • n is 5 or 6 in the toxin hapten structure; when preparing the toxin hapten coating antigen, n is 3 in the toxin hapten structure.
  • the present invention also provides a method for synthesizing the toxaxanthin artificial antigen, which is prepared by coupling the toxin hapten with a carrier protein by a mixed acid anhydride method.
  • the present invention also provides a toxaxanthin antibody, which is obtained by immunizing animals with the above-mentioned toxlavin immune antigen.
  • sample to be tested is treated with methanol and hydroxylamine hydrochloride, wherein the concentration of the hydroxylamine hydrochloride is 0.2M-0.8M.
  • the immunological method is any one of immunological analysis methods including immunochromatography, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, and chemiluminescence immunoassay.
  • Et is ethyl
  • Ac is acetyl
  • Me is methyl
  • the linking arm of the fatty chain is derived from the nitrogen atom at position 1 of toxaxanthin, and the end of the linking arm of the derived fatty chain has a carboxyl group.
  • the toxin hapten of the present invention retains the characteristics of toxin to the greatest extent
  • the structure can better expose the antigenic determinants of the hapten, so that the immunogenicity of the toxoflavin hapten is significantly enhanced, and it has a carboxyl group that can be coupled to the carrier protein; the toxoflavin hapten is coupled to the carrier protein
  • the obtained toxaxanthin immune antigen to immunize the animal is more conducive to stimulating the animal’s immune response to produce more specific and sensitive antibodies.
  • the immunological method using this antibody can detect 0.02ng/ml toxin contamination , It provides a simple and quick solution for the rice yeast acidosis. The entire detection process only takes more than ten minutes, which can fully meet the actual detection needs.
  • the method for synthesizing the toxicoflavin hapten of the present invention has readily available raw materials, simple reaction operation, and easy control of reaction conditions.
  • the method for synthesizing the toxin hapten of the present invention has a simple synthesis route, the purity and yield of the toxin hapten is high, and the overall synthesis cost is more advantageous.
  • Figure 1 shows the synthetic route of the toxoflavin hapten of the present invention.
  • the present invention provides toxoflavin hapten, which has the following structural formula:
  • n is the number of -CH 2 groups, and n is an integer of 2-8.
  • Toxaxanthin is not immunogenic due to its small molecular weight (molecular weight is less than 1000) and cannot produce antibodies in animals. It must be coupled with a large molecular carrier protein to prepare artificial antigens to induce the production of specific antibodies. Since there is no active group on the toxaxanthin molecule that can be directly coupled to the carrier protein, it is necessary to introduce a suitable linking arm and an active group coupled to the carrier protein in the appropriate position on the toxoflavin molecule through the derivatization process. .
  • the linking arm of the fatty chain is derived from the nitrogen atom at position 1 of toxaxanthin, and the end of the linking arm of the derived fatty chain has a carboxyl group.
  • the toxin hapten of the present invention retains the characteristics of toxin to the greatest extent
  • the structure can better expose the antigenic determinants of the hapten, so that the immunogenicity of the toxoflavin hapten is significantly enhanced, and it has a carboxyl group that can be coupled to the carrier protein; the toxoflavin hapten is coupled to the carrier protein
  • the obtained toxaxanthin immune antigen to immunize the animal is more conducive to stimulating the animal’s immune response to produce more specific and sensitive antibodies.
  • the immunological method using this antibody can detect 0.02ng/ml toxin contamination , It provides a simple and quick solution for the rice yeast acidosis. The entire detection process only takes more than ten minutes, which can fully meet the actual detection needs.
  • the toxin hapten of the present invention is not only simple in synthesis method and high in purity, but also can be applied to synthesize an antigen system suitable for animal immunity, which makes up for the blank in the technical field of domestic toxin immunology methods, and is a toxin immunity The further development of detection methods laid the foundation.
  • the present invention provides a method for synthesizing the above-mentioned toxoflavin hapten.
  • the method for synthesizing the toxin hapten of the above structure includes the following steps:
  • the method for synthesizing the compound 1 is: (1) providing Intermediate 1, whose molecular formula is: , Where n is the number of -CH 2 groups and n is an integer of 2-8; (2) The intermediate 1 is reacted with tert-butyl carbazate to obtain the compound 1.
  • compound 2 reacts with trifluoroacetic acid to remove the Boc (tert-butoxycarbonyl) group.
  • the reaction principle of removing the Boc group can be learned by the skilled person through technical manuals or through conventional experimental methods.
  • the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can make routine adjustments according to actual needs.
  • compound 3 reacts with formaldehyde to obtain compound 4, which has the structural formula , Where n is the number of -CH 2 groups, and n is an integer of 2-8;
  • compound 3 and formaldehyde undergo a carbonyl amine condensation reaction under acidic conditions to form a carbon-nitrogen double bond.
  • the reaction principles of the carbonylamine condensation reaction are all known to the skilled person through technical manuals or through conventional experimental methods.
  • the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can make routine adjustments according to actual needs.
  • compound 4 undergoes a cyclization reaction.
  • the reaction principles are all known to the skilled person through technical manuals or through conventional experimental methods.
  • the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can follow the actual conditions. Regular adjustments are required.
  • the compound 5 undergoes a hydrolysis reaction to obtain the toxin hapten.
  • compound 5 undergoes a hydrolysis reaction.
  • the reaction principles are all known to the skilled person through technical manuals or through conventional experimental methods.
  • the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can according to actual needs. Make regular adjustments.
  • step S1 the n value of compound 1 is determined in step S1
  • the n value of each compound in the subsequent steps is also determined.
  • the present invention rationally designs the method for synthesizing toxaxanthin hapten. It uses 6-chloro-3-methylpyrimidine and compound 1 as starting materials to carry out substitution reaction, de-Boc, and carbonyl amine condensation successively.
  • the reaction, cyclization reaction and hydrolysis reaction have a simple synthesis route, readily available raw materials, simple reaction operation, and easy control of reaction conditions.
  • the purity and yield of the toxin hapten are high, and the overall synthesis cost is more advantageous.
  • the present invention provides toxaxanthin artificial antigens, which include toxaxanthin immune antigens and toxaxanthin coating antigens, which are obtained by coupling the toxin haptens described above with carrier proteins .
  • the carrier protein is any one of bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HAS) or hemocyanin (KLH), but is not limited to the materials listed above It may also be other materials that are not listed in this embodiment but are well known to those skilled in the art.
  • BSA bovine serum albumin
  • OVA ovalbumin
  • HAS human serum albumin
  • KLH hemocyanin
  • the structure of the toxin hapten when preparing toxaxanthin immune antigen, the structure of the toxin hapten, 3 ⁇ n ⁇ 8; when preparing the toxoflavin coating antigen, the structure of the toxin hapten, 2 ⁇ n ⁇ 5 .
  • n is 5 or 6 in the structure of toxin hapten; when preparing toxaxanthin coating antigen, n is 3 in the structure of toxin hapten.
  • the inventor found that the toxaxanthin-coated antigen was synthesized by the mixed acid anhydride method.
  • the toxoflavin-coated antigen n is 3, and the toxoflavin immune antigen is 5 or 6, the established immunological method has Higher sensitivity, compared with EDC method to synthesize toxoflavin-coated antigen and other combinations, the detection limit is increased by an order of magnitude, and unexpected technical effects have been achieved.
  • the present invention provides a method for synthesizing toxoflavin artificial antigen, which is prepared by coupling the toxoflavin hapten with a carrier protein by a mixed acid anhydride method, and the steps are as follows:
  • the present invention also provides a toxoflavin antibody, which is obtained by immunizing an animal with the above-mentioned toxoflavin immune antigen.
  • the present invention provides an immunological method for detecting toxoflavin, which uses the above-mentioned toxoflavin antibody and toxaxanthin to coat the antigen.
  • the immunological method is any one of immunological analysis methods including immunochromatography, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, and chemiluminescence immunoassay.
  • the treatment liquid is methanol and hydroxylamine hydrochloride, where the concentration of the hydroxylamine hydrochloride is 0.2M-0.8M, which can improve the sensitivity of detection.
  • the synthesis method of the compound 1 is as follows: (1) Intermediate 1 is provided, and the molecular formula of the intermediate 1 is: , Where n is the number of -CH 2 groups, and n is an integer of 2-8; (2) The intermediate 1 is reacted with tert-butyl carbazate to obtain the compound 1.
  • compound 3 reacts with formaldehyde to obtain compound 4, which has the structural formula , Where n is the number of -CH 2 groups, and n is an integer of 2-8;
  • the compound 5 undergoes a hydrolysis reaction to obtain the toxin hapten.
  • Example 2 Mix and emulsify the toxin immune antigen synthesized in Example 2 with an equal volume of Freund’s adjuvant (for the first time with Freund’s complete adjuvant, and for the second, third and fourth times with Freund’s incomplete adjuvant), to immunize Bal In B/C mice, ELISA was used to evaluate the immune effect of different toxoflavin immune antigens, and to prepare monoclonal antibodies to toxaxanthin. Table 1 shows the evaluation of toxaxanthin immune sera with different values of n.
  • Example 5 Immunological method for detecting toxoflavin
  • nanoparticle-labeled toxoflavin antibody prepare 40nm colloidal gold particles according to the Frens method (1973); adjust the pH according to the amount of 3 ⁇ L 0.2M potassium carbonate per ml of colloidal gold particles; add 3 ⁇ g toxin per ml of colloidal gold particles Flavin monoclonal antibody, react at room temperature for 15 minutes; add bovine serum albumin (BSA) to make the final concentration 1%, and let stand at room temperature for 15 minutes; centrifuge at 10000 rpm for 10 minutes; discard the supernatant and add 1/10 of the volume of colloidal gold resuspension (10mM Tris (PH8.0), 0.5% Tween20, 0.4% casein, 2% sucrose) resuspend the particles;
  • BSA bovine serum albumin
  • binding pad spray the nanoparticle-labeled toxiflavin antibody on the Osron 8964 glass cellulose membrane according to 5 ⁇ L/cm, dry it at 37°C, and cut it to a width of 0.4cm for use;
  • sample pad soak the polyester cellulose film with [100mM Tris (PH8.0), 0.5% Tween20], dry at 37°C, and cut it into 1.2cm width for use;
  • test strip Lap and paste the sample pad, bonding pad, chromatography membrane, and absorbent pad onto the PVC bottom plate in turn, cut them into 3.5mm wide test strips, dry and store them for later use;
  • test strips (1) Sample processing: Take 10g of sample (fungus, patient vomit, fermented rice noodle products, etc.), add 20ml sample processing solution, shake for 1min; centrifuge at 10000rpm for 1min, take the supernatant for testing; ( 2) Test sample: Insert one end of the sample pad of the test strip into the supernatant to be tested. Note that the binding pad should be on the top of the page and let it stand for 10 minutes to read the result. When the test line does not show color, the result is positive; when the test line goes out, the result is negative.
  • Example 6 The influence of using mixed acid anhydride method and EDC method to synthesize toxoflavin coated antigen
  • the immunological method was used to detect negative samples and evaluate the specificity of the method. A total of 98 samples (including 37 patient vomits, 13 fungus samples, and 48 fermented rice noodle products) were tested. No false positive results appeared, indicating that the method Has good specificity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

毒黄素半抗原、人工抗原、抗体及其合成方法和应用被公开，所述毒黄素1位的氮原子上衍生脂肪链连接臂，衍生的脂肪链连接臂末段带有羧基，所述毒黄素半抗原最大程度保留了毒黄素的特征结构，能更好地暴露半抗原的抗原决定簇，使得毒黄素半抗原的免疫原性明显增强，又具有可以与载体蛋白发生偶联的羧基；该毒黄素半抗原与载体蛋白偶联后得到的毒黄素免疫抗原去免疫动物，更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体，采用该抗体的免疫学方法可以检测出0.02ng/m1的毒黄素污染，为米酵菌酸中毒提供了简便快捷的解决方案，整个检测流程只需要十多分钟，完全能够满足实际检测需求。

Description

毒黄素半抗原、人工抗原、抗体及其合成方法和应用 技术领域
本发明涉及了免疫化学分析技术领域，特别是涉及了毒黄素半抗原、人工抗原、抗体及其合成方法和应用。
 背景技术
酵米面中毒是我国广大农村致死率极高的一种细菌性食物中毒。椰毒假单胞菌酵米面亚种是引起酵米面食物中毒和变质银耳中毒的原因，研究证明该菌的致病物质是其在生长繁殖过程中产生的两种毒素：米酵菌酸(bongkrekic acid,BA)和毒黄素(toxoflavin,TF)。两种毒素均为脂肪酸类的小分子物质，具有剧毒、耐热、毒性强，不能被一般的烹调方法所破坏，故食品被污染后极易引起中毒，目前尚无特效解毒药物，且米酵菌酸的产量远高于毒黄素。
目前用于检测米酵菌酸的方法主要有传统的检测方法，如分光光度法、高效液相色谱法及其联用技术等，以上方法虽然可以精确定量，但由于设备仪器昂贵，检测时间长，且需专业人员操作，因此无法实现真正意义上的现场检测。而免疫学检测分析技术以其高灵敏、特异性高、快速、操作简便等优点在药物残留检测领域已被广泛应用，比起仪器等检验方法有很多优势。所以免疫分析为米酵菌酸残留研究提供了一条新的分析检测方法。
在建立免疫学检测方法并应用该检测方法检测米酵菌酸残留量时，关键技术在于能够获取到特异性强、灵敏度高的抗体，而要实现这一目标，前提条件就是得合成、制备出合适的米酵菌酸半抗原。但是米酵菌酸为链状结构，免疫动物难以获得高质量的抗体。而毒黄素则是由两个并环组成的立体状的分子，相对于米酵菌酸，免疫动物更容易获得高灵敏、高特异的抗体。通过免疫检测毒黄素可以间接检测米酵菌酸，对米酵菌酸的中毒检测提供一种简便可靠的检测方案。但是，目前，国内还没有针对毒黄素半抗原的相关报道。
 技术问题
为了弥补已有技术的缺陷，本发明提供毒黄素半抗原、人工抗原、抗体及其合成方法和应用。
 技术解决方案
本发明所要解决的技术问题通过以下技术方案予以实现：
毒黄素半抗原，其具有如下结构式：
Figure 677429dest_path_image001
其中，n为-CH 2基团数目，n为2-8的整数。
本发明还提供上述毒黄素半抗原的合成方法，包括如下步骤：
S1.6-氯-3-甲基嘧啶与化合物1进行反应，得到化合物2，所述化合物1的分子式为：
Figure 767744dest_path_image002
，其中，n为-CH 2基团数目，n为2-8的整数；所述化合物2具有结构式
Figure 263841dest_path_image003
，其中，n为-CH 2基团数目，n为2-8的整数；
S2.所述化合物2与三氟乙酸进行反应，得到化合物3，所述化合物3具有结构式
Figure 515831dest_path_image004
，其中，n为-CH 2基团数目，n为2-8的整数；
S3.在酸性条件下，所述化合物3与甲醛进行反应，得到化合物4，所述化合物4具有结构式
Figure 415654dest_path_image005
，其中，n为-CH 2基团数目，n为2-8的整数；
S4.在亚硝酸钠和乙酸存在下，所述化合物4进行反应，得到化合物5，所述化合物5具有结构式
Figure 794814dest_path_image006
，其中，n为-CH 2基团数目，n为2-8的整数；
S5. 所述化合物5进行水解反应，得到所述毒黄素半抗原。
进一步地，所述化合物1的合成方法为：（1）提供中间体1，所述中间体1的分子式为：
Figure 158799dest_path_image007
，其中，n为-CH 2基团数目，n为2-8的整数；（2）所述中间体1与肼基甲酸叔丁酯进行反应，得到所述化合物1。
本发明还提供毒黄素人工抗原，所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原，其是由上述毒黄素半抗原与载体蛋白偶联得到的。
进一步地，所述载体蛋白为牛血清白蛋白(BSA)，卵清蛋白(OVA)，人血清白蛋白(HAS)或血蓝蛋白(KLH)中的任意一种。
进一步地，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，3≤n≤8；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，2≤n≤5。
进一步地，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，n为5或6；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，n为3。
本发明还提供毒黄素人工抗原的合成方法，其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到。
本发明还提供一种毒黄素抗体，它是由上述毒黄素免疫抗原经动物免疫得到。
一种检测毒黄素的免疫学方法，其采用上述毒黄素抗体。
进一步地，待测样品经甲醇和盐酸羟胺处理，其中，所述盐酸羟胺的浓度为0.2M-0.8M。
进一步地，所述免疫学方法为免疫层析法、酶联免疫吸附试验、时间分辨荧光免疫分析、化学发光免疫分析在内的任意一种免疫学分析方法。
 有益效果
本发明具有如下有益效果：
特别说明，本申请文件中化学结构式中Et为乙基；Ac为乙酰基；Me为甲基。
本发明中，在毒黄素1位上的氮原子上衍生脂肪链连接臂，衍生的脂肪链连接臂末段带有羧基，本发明的毒黄素半抗原最大程度保留了毒黄素的特征结构，能更好地暴露半抗原的抗原决定簇，使得毒黄素半抗原的免疫原性明显增强，又具有可以与载体蛋白发生偶联的羧基；该毒黄素半抗原与载体蛋白偶联后得到的毒黄素免疫抗原去免疫动物，更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体，采用该抗体的免疫学方法可以检测出0.02ng/ml的毒黄素污染，为米酵菌酸中毒提供了简便快捷的解决方案，整个检测流程只需要十多分钟，完全能够满足实际检测需求。
本发明中毒黄素半抗原的合成方法，使用的原料易得，反应操作较为简单，反应条件易于控制。本发明的毒黄素半抗原合成方法，合成路线简单，毒黄素半抗原的纯度和收率高，整体的合成成本更具优势。
 附图说明
图1为本发明毒黄素半抗原的合成路线。
 本发明的实施方式
第一方面，本发明提供毒黄素半抗原，其具有如下结构式：
其中，n为-CH 2基团数目，n为2-8的整数。
人工抗原的合成是免疫检测中抗体制备和建立免疫分析方法最关键的步骤，半抗原的合成是人工抗原合成的关键。毒黄素因分子量小而不具有免疫原性（分子量小于1000），不能在动物体内产生抗体，必须与大  分子载体蛋白偶联制备人工抗原，才能诱导特异性抗体的产生。由于毒黄素分子上没有可以直接与载体蛋白偶联的活性基团，因此需先通过衍生过程在毒黄素小分子上适当的位置引入合适的连接臂和与载体蛋白偶联的活性基团。不同结构的连接臂、不同的偶联方法或连接臂的引入位置，都影响后续建立的免疫学方法检测的灵敏度和特异性。目前，以毒黄素衍生物为半抗原制备合成抗体和人工抗原进行检测毒黄素的免疫学方法鲜有报道。
本发明中，在毒黄素1位上的氮原子上衍生脂肪链连接臂，衍生的脂肪链连接臂末段带有羧基，本发明的毒黄素半抗原最大程度保留了毒黄素的特征结构，能更好地暴露半抗原的抗原决定簇，使得毒黄素半抗原的免疫原性明显增强，又具有可以与载体蛋白发生偶联的羧基；该毒黄素半抗原与载体蛋白偶联后得到的毒黄素免疫抗原去免疫动物，更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体，采用该抗体的免疫学方法可以检测出0.02ng/ml的毒黄素污染，为米酵菌酸中毒提供了简便快捷的解决方案，整个检测流程只需要十多分钟，完全能够满足实际检测需求。
本发明的毒黄素半抗原，不仅合成方法简便、纯度较高，而且能应用于合成适于动物免疫的抗原体系，弥补了国内毒黄素免疫学方法技术领域的空白，为毒黄素免疫检测方法的进一步发展奠定了基础。
第二方面，本发明提供上述毒黄素半抗原的合成方法。
具体地，上述结构的毒黄素半抗原的合成方法包括如下步骤：
S1.6-氯-3-甲基嘧啶与化合物1进行反应，得到化合物2，所述化合物1的分子式为：
Figure 830958dest_path_image008
，其中，n为-CH 2基团数目，n为2-8的整数；所述化合物2具有结构式
Figure 483656dest_path_image009
，其中，n为-CH 2基团数目，n为2-8的整数；
此步骤中，6-氯-3-甲基嘧啶与化合物1进行取代反应，其原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知，本发明对反应的具体操作不作具体限定， 本领域技术人员可以根据实际需要进行常规调整。
所述化合物1的合成方法为：（1）提供中间体1，所述中间体1的分子式为：
Figure 666507dest_path_image010
，其中，n为-CH 2基团数目，n为2-8的整数；（2）所述中间体1与肼基甲酸叔丁酯进行反应，得到所述化合物1。
 
S2.所述化合物2与三氟乙酸进行反应，得到化合物3，所述化合物3具有结构式
Figure 619419dest_path_image011
，其中，n为-CH 2基团数目，n为2-8的整数；
此步骤中，化合物2与三氟乙酸反应，脱去Boc（叔丁氧羰基）基团，脱Boc基团的反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知，本发明对反应的具体操作不作具体限定， 本领域技术人员可以根据实际需要进行常规调整。
 
S3.在酸性条件下，所述化合物3与甲醛进行反应，得到化合物4，所述化合物4具有结构式
Figure 416474dest_path_image012
，其中，n为-CH 2基团数目，n为2-8的整数；
此步骤中，化合物3和甲醛在酸性条件下进行羰胺缩合反应，形成碳氮双键。羰胺缩合反应的反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知，本发明对反应的具体操作不作具体限定， 本领域技术人员可以根据实际需要进行常规调整。
S4.在亚硝酸钠和乙酸存在下，所述化合物4进行反应，得到化合物5，所述化合物5具有结构式
Figure 351543dest_path_image013
，其中，n为-CH 2基团数目，n为2-8的整数；
此步骤中，化合物4进行成环反应，反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知，本发明对反应的具体操作不作具体限定， 本领域技术人员可以根据实际需要进行常规调整。
S5. 所述化合物5进行水解反应，得到所述毒黄素半抗原。
此步骤中，化合物5进行水解反应，反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知，本发明对反应的具体操作不作具体限定， 本领域技术人员可以根据实际需要进行常规调整。
可以理解，步骤S1中化合物1的n值确定后，后续步骤中各化合物的n值也确定了。
本发明根据毒黄素的结构特点，合理设计毒黄素半抗原的合成方法，以6-氯-3-甲基嘧啶与化合物1为起始原料，先后进行取代反应、脱Boc、羰胺缩合反应、成环反应和水解反应，合成路线简单，使用的原料易得，反应操作较为简单，反应条件易于控制。本发明的毒黄素半抗原合成方法，毒黄素半抗原的纯度和收率高，整体的合成成本更具优势。
第三方面，本发明提供毒黄素人工抗原，所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原，其是由上述毒黄素半抗原与载体蛋白偶联得到的。
所述载体蛋白为牛血清白蛋白(BSA)，卵清蛋白(OVA)，人血清白蛋白(HAS)或血蓝蛋白(KLH)中的任意一种，但不限于前面所列举的几种材料，也可以是其他未列举在本实施例中的但被本领域技术人员所熟知的其他材料。
其中，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，3≤n≤8；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，2≤n≤5。
更优选地，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，n为5或6；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，n为3。发明人发现，采用混合酸酐法合成毒黄素包被抗原，当毒黄素包被抗原中，n为3，而毒黄素免疫抗原中n为5或6时，所建立的免疫学方法具有更高的灵敏度，较之EDC法合成毒黄素包被抗原和其它组合，检测下限提高了一个数量级，取得了预料不到的技术效果。
第四方面，本发明提供毒黄素人工抗原的合成方法，其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到，步骤如下：
称取5mg毒黄素半抗原，溶于1ml DMF中，置于4℃下10min，加入8.5μl 三乙胺，再加入6.3ul 氯甲酸异丁酯，4℃搅拌反应30min，得甲液；
取20mg OVA溶于1.8ml 50%DMF中，预冷至4℃，置于冰浴不断搅拌，用1M NaOH调节PH至8.0，得毒黄素人工抗原。
发明人发现，采用混合酸酐法合成毒黄素包被抗原，相较于EDC法合成毒黄素包被抗原，所建立的免疫学方法具有更高的灵敏度。
第五方面，本发明还提供一种毒黄素抗体，它是由上述毒黄素免疫抗原经动物免疫得到。
第六方面，本发明提供一种检测毒黄素的免疫学方法，其采用上述毒黄素抗体和毒黄素包被抗原。
所述免疫学方法为免疫层析法、酶联免疫吸附试验、时间分辨荧光免疫分析、化学发光免疫分析在内的任意一种免疫学分析方法。
检测毒黄素的免疫学方法中，需要采用处理液对待测样品进行处理。发明人在实践中发现，处理液为甲醇和盐酸羟胺，其中，所述盐酸羟胺的浓度为0.2M-0.8M时，可以提高检测的灵敏性。
下面结合实施例对本发明进行详细的说明，实施例仅是本发明的优选实施方式，不是对本发明的限定。
实施例1：毒黄素半抗原的合成
S1.6-氯-3-甲基嘧啶与化合物1进行反应，得到化合物2，所述化合物1的分子式为：
Figure 587352dest_path_image014
，其中，n为-CH 2基团数目，n为2-8的整数；所述化合物2具有结构式
Figure 676662dest_path_image015
，其中，n为-CH 2基团数目，n为2-8的整数；
所述化合物1的合成方法为：（1）提供中间体1，所述中间体1的分子式为：
Figure 441355dest_path_image010
，其中，n为-CH 2基团数目，n为2-8的整数；（2）所述中间体1与肼基甲酸叔丁酯进行反应，得到所述化合物1。
 
S2.所述化合物2与三氟乙酸进行反应，得到化合物3，所述化合物3具有结构式
Figure 334225dest_path_image016
，其中，n为-CH 2基团数目，n为2-8的整数；
S3.在酸性条件下，所述化合物3与甲醛进行反应，得到化合物4，所述化合物4具有结构式
Figure 357414dest_path_image017
，其中，n为-CH 2基团数目，n为2-8的整数；
S4.在亚硝酸钠和乙酸存在下，所述化合物4进行反应，得到化合物5，所述化合物5具有结构式
Figure 19339dest_path_image018
，其中，n为-CH 2基团数目，n为2-8的整数；
S5. 所述化合物5进行水解反应，得到所述毒黄素半抗原。
实施例2：毒黄素免疫抗原的合成
取载体蛋白（牛血清白蛋白，BSA）20mg溶于4ml 磷酸盐缓冲液（pH7.4）中，加入5mg毒黄素半抗原，混匀，加入10mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)，室温（25℃）反应2小时，透析除去未结合到载体蛋白上的小分子，收集透析后的蛋白质即为毒黄素免疫抗原。
实施例3：毒黄素包被抗原的合成（混合酸酐法）
称取5mg毒黄素半抗原，溶于1ml DMF中，置于4℃下10min，加入8.5μl 三乙胺，再加入6.3ul 氯甲酸异丁酯，4℃搅拌反应30min，得甲液；
取20mg OVA溶于1.8ml 50%DMF中，预冷至4℃，置于冰浴不断搅拌，用1M NaOH调节PH至8.0，得毒黄素包被抗原。
实施例4：毒黄素抗体
将实施例2合成的毒黄素免疫抗原与等体积弗氏佐剂（首次用弗氏完全佐剂，第二、三、四次用弗氏不完全佐剂）混合、乳化，免疫Bal B/C鼠，采用ELISA评价不同毒黄素免疫抗原的免疫效果，并制备毒黄素单克隆抗体。表1为n值不同的毒黄素免疫抗原免疫血清评价情况。
其中，毒黄素单克隆抗体的制备流程为：
（1）     融合前3天，用50μg抗原经腹腔加强免疫一次；
（2）     融合前一天，制备饲养细胞，按照100μL/孔铺到96孔板中；
（3）     摘眼球取血，脱颈处死，于75%酒精中浸泡5min；
（4）     取出脾脏，于纱网上碾磨，显微镜下计数；
（5）     1200rpm，5min，弃掉上清；
（6）     按照1个骨髓瘤细胞对应5个脾细胞的比例加入骨髓瘤细胞，补加1640培养基至30mL，充分混匀；
（7）     1200rpm，10min，弃上清；
（8）     轻弹管底，充分重悬细胞；
（9）     于37℃下， 1min内沿管壁加入50% PEG1450 1mL；
（10）   缓慢加入25mL 1640培养基；
（11）   900rpm，7min，弃上清；
（12）   120mL HAT培养液重悬细胞，按照100μL/孔铺到准备好的饲养细胞板中；于37℃ 5%二氧化碳培养箱中培养；
（13）   第8天用HT培养基换液；
（14）   第10天ELISA检测；
（15）   用完全培养基稀释ELISA检测呈强阳性的孔中的细胞，使细胞浓度为5cells/mL；
（16）   将细胞悬液按照200μL/孔分装到96孔板中，置于37℃ 5%二氧化碳培养箱中培养；
（17）   待微孔中的细胞长到约100cells/孔时，进行ELISA检测；
（18）   再次进行克隆筛选，直至所有有细胞的孔均呈阳性反应；
（19）   随后转入24孔细胞培养板进行培养，随后用细胞培养瓶进行培养；
（20）   按照100万个细胞/只小鼠的剂量腹腔注射BalB/C小鼠；
（21）   收集腹水，纯化其中的抗体。
Figure 440087dest_path_image019
实施例5：检测毒黄素的免疫学方法，
1.纳米颗粒标记毒黄素抗体的制备：按照Frens法（1973）制备40nm胶体金颗粒；按照每毫升胶体金颗粒加入3μL 0.2M碳酸钾的量调节PH；按照每毫升胶体金颗粒加入3μg毒黄素单克隆抗体，室温反应15min；加入牛血清白蛋白（BSA），使其终浓度为1%，室温静置15min；10000rpm离心10min；弃掉上清，加入1/10胶体金体积的重悬液(10mM Tris（PH8.0）,0.5%Tween20、0.4%酪蛋白、2%蔗糖)重悬颗粒；
2.结合垫的制备：按照5μL/cm喷纳米颗粒标记毒黄素抗体于奥斯龙8964玻璃纤维素膜上，置于37℃烘干，裁成0.4cm宽，备用；
3.层析膜的制备：按照1μL/cm喷涂浓度为1mg/mL的毒黄素包被抗原于硝酸纤维素膜（Millipore 135）上，作为检测线；按照1μL/cm喷涂浓度为0.2mg/mL的羊抗鼠IgG作为质控线，置于室温晾干，备用；
4.样品垫的制备：用[100mM Tris（PH8.0），0.5%Tween20]浸泡聚酯纤维素膜，置于37℃烘干，裁成1.2cm宽，备用；
5.试纸条的组装：将样品垫、结合垫、层析膜、吸水垫依次搭接粘贴到PVC底板上，裁成3.5mm宽的试纸条，干燥保存，备用；
6.试纸条的使用：（1）样品处理：取样品（木耳、病人呕吐物、发酵米面制品等）10g，加入20ml样品处理液，振摇1min；10000rpm离心1min，取上清检测；（2)检测样品：将试纸条样品垫一端插入待检上清中，注意结合垫应在页面上方，静置10min，判读结果。当检测线不显色时，结果为阳性；当检测线出线时，结果为阴性。
表2为不同处理液处理样品时，试纸条的检测下限比对结果。从结果来看，联合应用甲醇和盐酸羟胺可以提高检测的灵敏性，其中盐酸羟胺的浓度在0.2M-0.8M之间时，检测下限最佳。而单独应用盐酸羟胺（0.4M），则不能改善检测的灵敏性。
Figure 85832dest_path_image020
实施例6：采用混合酸酐法和EDC法合成毒黄素包被抗原的影响
采用实施例3中的混合酸酐法制备不同的毒黄素包被抗原，按照实施例5的方法建立检测毒黄素的免疫学方法，采用混合酸酐法制备的不同的毒黄素包被抗原的免疫学方法的检测下限参见表4。
采用EDC法制备不同的毒黄素包被抗原：取载体蛋白（卵清蛋白，OVA）20mg溶于4ml 磷酸盐缓冲液（pH7.4）中，加入5mg毒黄素半抗原，混匀，加入10mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)，室温（25℃）反应2小时，透析除去未结合到载体蛋白上的小分子，收集透析后的蛋白质即为毒黄素包被抗原；按照实施例5的方法建立检测毒黄素的免疫学方法，采用EDC法制备的不同的毒黄素包被抗原的免疫学方法的检测下限参见表3。
我们发现，采用混合酸酐法制备的包被抗原与某些抗体组合可以建立更为灵敏的免疫层析法，与表3结果相比，当包被抗原（n=3）、免疫抗原（n=5或6）时，所建立的方法具有很高的灵敏性，检测下限整整较EDC法偶联抗原提高了一个数量级。
Figure 335548dest_path_image021
Figure 635336dest_path_image022
实施例7：特异性评价
采用实施例3中的混合酸酐法制备毒黄素包被抗原（n为3），采用实施例4的方法合成毒黄素抗体（n为5），按照实施例5的方法建立检测毒黄素的免疫学方法检测阴样本，评价该方法的特异性，一共检测了98份样本（其中病人呕吐物37份、木耳样品13份、发酵米面制品48份），没有假阳性结果出现，说明该方法具有良好的特异性。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本发明的保护范围之内。

Claims (10)

  1. 毒黄素半抗原，其特征在于，其具有如下结构式：
    Figure 952280dest_path_image001
    其中，n为-CH 2基团数目，n为2-8的整数。
  2. 如权利要求1所述的毒黄素半抗原的合成方法，其特征在于，其包括如下步骤：
    S1.6-氯-3-甲基嘧啶与化合物1进行反应，得到化合物2，所述化合物1的分子式为：
    Figure 237899dest_path_image002
    ，其中，n为-CH 2基团数目，n为2-8的整数；所述化合物2具有结构式
    Figure 413665dest_path_image003
    ；
    S2.所述化合物2与三氟乙酸进行反应，得到化合物3，所述化合物3具有结构式
    Figure 650480dest_path_image004
    ；
    S3.在酸性条件下，所述化合物3与甲醛进行反应，得到化合物4，所述化合物4具有结构式
    Figure 937105dest_path_image005
    ；
    S4.在亚硝酸钠和乙酸存在下，所述化合物4进行反应，得到化合物5，所述化合物5具有结构式
    Figure 546072dest_path_image006
    ；
    S5. 所述化合物5进行水解反应，得到所述毒黄素半抗原。
  3. 如权利要求2所述的毒黄素半抗原的合成方法，其特征在于，所述化合物1的合成方法为：（1）提供中间体1，所述中间体1的分子式为：
    Figure 892740dest_path_image007
    ，其中，n为-CH 2基团数目，n为2-8的整数；（2）所述中间体1与肼基甲酸叔丁酯进行反应，得到所述化合物1。
  4. 毒黄素人工抗原，其特征在于，所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原，其是由权利要求1所述的毒黄素半抗原与载体蛋白偶联得到的。
  5. 如权利要求4所述的毒黄素人工抗原，其特征在于，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，3≤n≤8；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，2≤n≤5。
  6. 如权利要求4所述的毒黄素人工抗原，其特征在于，制备毒黄素免疫抗原时，所述毒黄素半抗原结构中，n为5或6；制备毒黄素包被抗原时，所述毒黄素半抗原结构中，n为3。
  7. 如权利要求4所述的毒黄素人工抗原的合成方法，其特征在于，其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到。
  8. 一种毒黄素抗体，其特征在于，其是由权利要求4所述的毒黄素免疫抗原经动物免疫得到。
  9. 一种检测毒黄素的免疫学方法，其特征在于，其采用权利要求8所述的毒黄素抗体。
  10. 如权利要求9所述的免疫学方法，其特征在于，待测样品经甲醇和盐酸羟胺处理，其中，所述盐酸羟胺的浓度为0.2M-0.8M。
PCT/CN2019/095182 2019-06-14 2019-07-09 毒黄素半抗原、人工抗原、抗体及其合成方法和应用 WO2020248316A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910558883.0 2019-06-14
CN201910558883.0A CN110305139B (zh) 2019-06-14 2019-06-14 用于间接检测米酵菌酸的毒黄素半抗原及其合成方法

Publications (1)

Publication Number Publication Date
WO2020248316A1 true WO2020248316A1 (zh) 2020-12-17

Family

ID=68076356

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/095182 WO2020248316A1 (zh) 2019-06-14 2019-07-09 毒黄素半抗原、人工抗原、抗体及其合成方法和应用

Country Status (2)

Country Link
CN (1) CN110305139B (zh)
WO (1) WO2020248316A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111175499A (zh) * 2020-02-28 2020-05-19 江南大学 一种用于检测睾酮素的elisa试剂盒的制备方法
CN111978271B (zh) * 2020-08-05 2022-08-30 重庆市农业科学院 噻螨酮半抗原及其制备方法、噻螨酮抗原、抗体及其应用
CN113801220B (zh) * 2021-09-18 2023-07-07 青岛普瑞邦生物工程有限公司 一种米酵菌酸复合抗原、米酵菌酸抗体及其制备方法和酶联免疫试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105319313A (zh) * 2015-12-09 2016-02-10 山东出入境检验检疫局检验检疫技术中心 一种毒黄素的液相色谱-串联质谱检测方法
CN105974018A (zh) * 2016-05-07 2016-09-28 浙江省质量检测科学研究院 基于多功能净化柱-高效液相色谱法检测食品中毒黄素的方法
CN106018659A (zh) * 2016-05-07 2016-10-12 浙江省质量检测科学研究院 一种食品中毒黄素的快速检测方法
CN109824604A (zh) * 2019-02-25 2019-05-31 中国农业大学 磺胺类药物半抗原和人工抗原及其制备方法与应用
CN110294762A (zh) * 2019-06-14 2019-10-01 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 毒黄素半抗原、人工抗原、抗体及其合成方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57146796A (en) * 1981-03-09 1982-09-10 Haruo Ogura Glycoside derivative
WO2010072807A2 (en) * 2008-12-23 2010-07-01 Fondation Jerome Lejeune Inhibitors of cystathionine beta synthase to reduce the neurotoxic overproduction of endogenous hydrogen sulfide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105319313A (zh) * 2015-12-09 2016-02-10 山东出入境检验检疫局检验检疫技术中心 一种毒黄素的液相色谱-串联质谱检测方法
CN105974018A (zh) * 2016-05-07 2016-09-28 浙江省质量检测科学研究院 基于多功能净化柱-高效液相色谱法检测食品中毒黄素的方法
CN106018659A (zh) * 2016-05-07 2016-10-12 浙江省质量检测科学研究院 一种食品中毒黄素的快速检测方法
CN109824604A (zh) * 2019-02-25 2019-05-31 中国农业大学 磺胺类药物半抗原和人工抗原及其制备方法与应用
CN110294762A (zh) * 2019-06-14 2019-10-01 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 毒黄素半抗原、人工抗原、抗体及其合成方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, RONGQING ET AL.: "Progress in the Synthesis of Artificial Antigen", CHINA BIOTECHNOLOGY, vol. 38, no. 12, 31 December 2018 (2018-12-31), DOI: 20200215110422 *
WENG, CHENHUI ET AL.: "Advances in the Synthesis, Separation and Detection Technology of Toxoflavin", CHINESE BULLETIN OF LIFE SCIENCES, vol. 29, no. 4, 30 April 2017 (2017-04-30), DOI: 20200215110247 *

Also Published As

Publication number Publication date
CN110305139A (zh) 2019-10-08
CN110305139B (zh) 2021-01-19

Similar Documents

Publication Publication Date Title
WO2020248316A1 (zh) 毒黄素半抗原、人工抗原、抗体及其合成方法和应用
CN110845444B (zh) 烯酰吗啉半抗原、人工抗原和抗体及其制备方法和应用
WO2022183762A1 (zh) 一种抗非那西丁单克隆抗体杂交瘤细胞株ad及其制备方法与应用
WO2018133039A1 (zh) 制备抗体对的方法、试剂盒及试剂盒的应用和制备抗体对的系统
JP5276924B2 (ja) オクラトキシンに対する抗体、その抗体を用いた親和性カラム、およびオクラトキシンの免疫学的検出用キット
WO2020248317A1 (zh) 毒黄素半抗原、人工抗原、抗体及其合成方法和应用
CN110879293A (zh) 一种筛选分泌配对单克隆抗体的杂交瘤细胞株的方法与应用
CN110713986B (zh) 一株维生素b1单克隆抗体杂交瘤细胞株cbdd及其应用
CN111943882A (zh) 腐霉利半抗原、人工抗原和抗体及其制备方法和应用
EP0311383B1 (en) Monoclonal antibody to methamphetamine, preparation of the same, assay method and assay kit of methamphetamine
CN110872344B (zh) 一种氯霉素完全抗原及其制备方法与应用
US5283066A (en) Method of stimulating an immune response by using a hapten
CN111763658A (zh) 一株分泌抗二硝托胺单克隆抗体的杂交瘤细胞株及其应用
Eisenstein et al. Isolation and characterization of a monoclonal antibody directed against type 1 fimbriae organelles from Escherichia coli
KR20120132227A (ko) 다중 타입 구제역 바이러스 검출용 단일클론 항체 및 이를 이용한 구제역 바이러스 검출방법
CN111377888B (zh) 一种闹羊花毒素iii半抗原及其制备方法与应用
CN106929477B (zh) 一株抗前列腺素F2α的特异性单克隆抗体杂交瘤细胞株WXX-2及其应用
CN107226795B (zh) 利奈唑胺半抗原和完全抗原及其制备方法和应用
CN111157735B (zh) 凝集性卡他莫拉菌单克隆抗体及其制备方法和应用
CN117069580B (zh) 一种三氯生半抗原及其制备方法和应用
CN114317448B (zh) 一株分泌抗乙螨唑单克隆抗体杂交瘤细胞株及其应用
CN114181102B (zh) 己雷琐辛半抗原、人工抗原、抗体及其合成方法和应用
CN113087776B (zh) 一种丝状噬菌体多克隆抗体及其制备方法与应用
CN116640076A (zh) 一种甲砜霉素半抗原、人工抗原、抗体及其制备和应用
JPH0568584A (ja) モノクローナル抗体、これを用いた測定法、試薬キツト及び検索法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19932746

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19932746

Country of ref document: EP

Kind code of ref document: A1