WO2018054394A1 - Hybridoma cell line nan-1 which secretes sulfonamide antibiotic-resistant monoclonal antibody, and application of hybridoma cell line - Google Patents
Hybridoma cell line nan-1 which secretes sulfonamide antibiotic-resistant monoclonal antibody, and application of hybridoma cell line Download PDFInfo
- Publication number
- WO2018054394A1 WO2018054394A1 PCT/CN2017/110882 CN2017110882W WO2018054394A1 WO 2018054394 A1 WO2018054394 A1 WO 2018054394A1 CN 2017110882 W CN2017110882 W CN 2017110882W WO 2018054394 A1 WO2018054394 A1 WO 2018054394A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- monoclonal antibody
- cell line
- hybridoma cell
- sulfa
- nan
- Prior art date
Links
Images
Classifications
-
- 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
- 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 a hybridoma cell line NaN-1 secreting a monoclonal antibody against sulfa antibiotics and the use thereof, and relates to a hybridoma cell line NaN-1 and a monoclonal antibody against sulfa antibiotics thereof, belonging to food safety immunology Testing area.
- SAs Sulfonamide antibiotics
- SAs Sulfonamide antibiotics
- Such antibiotics are also a class of antibacterial drugs widely used in aquaculture, aquaculture and other aquaculture production, which are often used for the prevention and treatment of livestock diseases, and as feed additives to promote animal growth.
- residue in food due to its potential harm to human body and the emergence of resistance to pathogenic bacteria, its residue in food has caused widespread concern.
- various countries have strengthened the monitoring of SAs and formulated the maximum residue of SAs in animal-derived foods.
- the Codex Alimentarius Commission, the European Union and some countries in Europe and the United States clearly stipulate that the total content of SAs in food should not exceed 0.1 mg/kg.
- the detection methods of SAs mainly include instrumental detection methods (such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry), enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) and colloidal gold immunochromatography.
- instrumental detection methods such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry
- enzyme-linked immunosorbent assay ELISA, Enzyme-Linked Immunosorbent Assay
- colloidal gold immunochromatography mainly include instrumental detection methods (such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry), enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) and colloidal gold immunochromatography.
- ELISA enzyme-linked immunosorbent assay
- colloidal gold immunochromatography colloidal gold immunochromatography
- the object of the present invention is to provide a group-selective monoclonal antibody hybridoma cell line NaN-1 resistant to sulfa antibiotics, and the accession number is CGMCC No.12028, and the antibody prepared from the cell line can recognize twenty-five kinds of SAs, and It has good detection sensitivity and can be used to establish immunological detection methods for SAs.
- the basic steps for preparing the NaN-1 cell strain provided by the present invention are as follows:
- the hapten is a product having a carboxyl group and is coupled by a carbodiimide method.
- the specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8. Mg N-hydroxysuccinimide was activated at room temperature for 4 h; 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution and allowed to react at room temperature overnight. The unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen.
- mice After complete emulsification of complete antigen and an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. The last time was directly immunized with complete antigen (without adjuvant), and serum titer and inhibition were detected by indirect ELISA;
- the present invention also provides a monoclonal antibody against sulfa antibiotics which is secreted by the anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 of the accession number CGMCC No. 12028.
- the anti-SAs monoclonal antibody has good detection sensitivity to twenty-five sulfa antibiotics, among which, sulfadiazine (SD), Sulfamerazine (SMR), sulfamethoxine (Sulfamerazine, SMR)
- SD sulfadiazine
- SMR Sulfamerazine
- SMR sulfamethoxine
- SMR sulfamerazine
- the IC 50 of Sulfadimidine, SDM) and Sulfathizzole (STZ) were 4.51, 1.80, 1.98 and 0.53 ⁇ g/L, respectively.
- the anti-sulfonamide antibiotic monoclonal antibody can be used in the field of food safety to detect the content of sulfa antibiotics in food.
- the invention provides an anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1, and the monoclonal antibody secreted by the cell line can recognize twenty-five sulfa antibiotics, and has good detection sensitivity, wherein
- the IC 50 of sulfadiazine, sulfamethazine, sulfadimethoxine and sulfathiazole are: 4.51, 1.80, 1.98 and 0.53 ⁇ g/L, respectively, which can be used for multi-residue detection of sulfonamides in food safety.
- the monoclonal antibody hybridoma cell line NaN-1 has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, referred to as CGMCC. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China. The Institute of Microbiology, Chinese Academy of Sciences, is classified as a monoclonal cell strain with the accession number CGMCC No.12028.
- Figure 1 Standard curve for inhibition of sulfadiazine (SD) by monoclonal antibodies secreted by NaN-1.
- the invention screens cell supernatant by cell fusion, HAT selective medium culture, indirect ELISA and indirect competition ELISA by immunizing mice with complete antigen of SAs, and finally obtains wide recognition and good sensitivity to SAs.
- Carbonate buffer (CBS): Weigh 1.59g of Na 2 CO 3 and 2.93g of NaHCO 3 , dissolve in a small amount of double distilled water, mix with double distilled water to about 800mL, adjust the pH to 9.6, add Double distilled water to a volume of 1000mL, stored at 4 ° C for use;
- PBST PBS containing 0.05% Tween 20;
- TMB color developing solution liquid A: Na 2 HPO 4 ⁇ 12H 2 O 18.43 g, citric acid 9.33 g, pure water to 100 mL; B liquid: 60 mg TMB dissolved in 100 mL of ethylene glycol. A and B liquids are mixed with a volume ratio of 1..5 to be a TMB coloring liquid, which is currently used.
- the hapten is a product having a carboxyl group and is coupled by a carbodiimide method.
- the specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8.
- Mg N-hydroxysuccinimide was activated at room temperature for 4 h, and 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution, and reacted at room temperature overnight.
- the unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen and characterized by UV.
- the original synthesis of the coating is similar to the synthesis of the immunogen, and the carrier protein is simply replaced with chicken egg albumin.
- mice Healthy 6-8 week old BALB/c mice were selected for immunization. After the complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. Blood was collected 7 days after the third immunization, and the serum titer and inhibition of the mice were determined by indirect competitive ELISA. The mice with high titer and more SAs were selected, and the immunization was performed 18 days after the four immunizations without adjuvant. injection.
- cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows: (1) The mouse spleen was aseptically taken, ground and passed through a 200 mesh cell sieve to obtain spleen cell suspension.
- PEG polyethylene glycol, molecular weight 4000
- the RPMI-1640 screening medium was semi-exchanged on the third day of cell fusion, and the whole day was changed with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100 ⁇ HT on the 5th day.
- the cell supernatant was taken for screening on day 7.
- the screening is divided into two steps: the first step is to select positive cell wells by indirect ELISA, and the second step is to select SD, SMR, SDM and STZ as standard products, and the inhibition effect of positive cells is determined by indirect competition ELISA. Cell wells with good inhibition of these four standards were selected, subcloned by limiting dilution method, and detected by the same method. This was repeated three times to obtain a cell strain NaN-1.
- mice 8-8 weeks old BALB/c mice were injected with 1 mL of paraffin oil per mouse. After 7 days, each mouse was intraperitoneally injected with 1 ⁇ 10 6 hybridoma cell NaN-1, and ascites was collected from the 7th day. Ascites was purified by caprylic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20 °C.
- Drug name IC 50 ( ⁇ g/L) Cross-reaction rate (%) Sulfadiazine (SD) 4.51 100.00 Sulfamethazine (SMR) 1.8 250.56 Sulfamethoxine 1.09 413.76 Sulfadimethoxypyrimidine (SDM) 1.98 227.78 Sulfamethoxazole 0.15 3006.67 Sulfamethoxazole 0.21 2147.62
- the monoclonal antibody prepared by the hybridoma cell line NaN-1 by in vivo ascites was applied to the ELISA addition recovery test of several sulfa antibiotics (SD, SMR, SDM, STZ). The specific steps are as follows:
- Termination and measurement 50 ⁇ L of the stop solution was added to each well to terminate the reaction, and then the OD 450 value of each well was measured with a microplate reader.
- the ELISA titer of serum is the highest dilution factor of serum corresponding to 2.1 times of OD 450 value (ie P/N ⁇ 2.1).
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A hybridoma cell line NaN-1 which secretes a sulfonamide antibiotic-resistant monoclonal antibody, and an application of the hybridoma cell line, belonging to the field of food safety immunoassays. The antibody secreted by the hybridoma cell line NaN-1 is capable of identifying twenty-five sulfonamide antibiotics. The hybridoma cell line has been stored in the China General Microbiological Culture Collection Center (CGMCC), the accession number being CGMCC No. 12028. The monoclonal antibody secreted by the cell line NaN-1 is capable of identifying twenty-five sulfonamide antibiotics and has good detection sensitivity. The IC50 for sulfadiazine (SD), sulfamerazine (SMR), sulfadimethoxine (SDM) and sulfathiazole (STZ) is respectively: 4.51, 1.80, 1.98 and 0.53 μg/L. The present invention can be used for sulfonamide antibiotic multi-residue detection within the field of food safety.
Description
本发明涉及一株分泌抗磺胺类抗生素单克隆抗体的杂交瘤细胞株NaN-1及其应用,涉及杂交瘤细胞株NaN-1及其产生的抗磺胺类抗生素单克隆抗体,属于食品安全免疫学检测领域。The present invention relates to a hybridoma cell line NaN-1 secreting a monoclonal antibody against sulfa antibiotics and the use thereof, and relates to a hybridoma cell line NaN-1 and a monoclonal antibody against sulfa antibiotics thereof, belonging to food safety immunology Testing area.
磺胺类抗生素(Sulfonamide antibiotics,SAs)是人工合成的抗菌药,用于临床已近50年,它具有抗菌谱较广、性质稳定、使用简便等优点。这类抗生素也是畜牧、水产等养殖生产中广泛应用的一类抗菌药物,其常常用于预防和治疗畜禽疾病,以及作为饲料添加剂促进动物生长。但由于其对人体潜在的危害性以及致病菌耐药性的产生,其在食品中的残留问题引起了广泛关注。近年来,各个国家加强了对SAs的监控,分别制定了动物源性食品中SAs的最大残留量。国际食品法典委员会、欧盟及欧美一些国家明确规定食品中SAs总含量不得超过0.1mg/kg。Sulfonamide antibiotics (SAs) are synthetic antibacterial drugs that have been used in clinical practice for nearly 50 years. They have the advantages of wide antibacterial spectrum, stable nature and easy to use. Such antibiotics are also a class of antibacterial drugs widely used in aquaculture, aquaculture and other aquaculture production, which are often used for the prevention and treatment of livestock diseases, and as feed additives to promote animal growth. However, due to its potential harm to human body and the emergence of resistance to pathogenic bacteria, its residue in food has caused widespread concern. In recent years, various countries have strengthened the monitoring of SAs and formulated the maximum residue of SAs in animal-derived foods. The Codex Alimentarius Commission, the European Union and some countries in Europe and the United States clearly stipulate that the total content of SAs in food should not exceed 0.1 mg/kg.
目前SAs的检测方法主要有仪器检测法(如高效液相色谱法,高效液相色谱串联质谱分析法),酶联免疫检测方法(ELISA,Enzyme-Linked ImmunosorbentAssay)以及胶体金免疫层析技术。由于仪器分析方法的样品前处理繁琐、耗时、仪器贵重、需要专业人员操作,不能实现大量样品的同时检测。ELISA是一种极为高效、灵敏的检测方法,检测时对样本的纯度要求不高、操作简便,适用于大量样本的现场快速检测。但要实现对磺胺类抗生素的同时检测,制备群选性的单克隆抗体是前提。At present, the detection methods of SAs mainly include instrumental detection methods (such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry), enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) and colloidal gold immunochromatography. Because the sample preparation method of the instrument analysis method is cumbersome, time-consuming, expensive, and requires professional operation, simultaneous detection of a large number of samples cannot be achieved. ELISA is an extremely efficient and sensitive detection method. The purity of the sample is not high and the operation is simple. It is suitable for rapid on-site detection of a large number of samples. However, in order to achieve simultaneous detection of sulfa antibiotics, it is a prerequisite to prepare a monoclonal antibody.
发明内容Summary of the invention
本发明目的是提供一种抗磺胺类抗生素的群选性单克隆抗体杂交瘤细胞株NaN-1,保藏编号为CGMCC No.12028,由该细胞株制备的抗体能识别二十五种SAs,且具有较好的检测灵敏度,可以用来建立SAs的免疫学检测方法。The object of the present invention is to provide a group-selective monoclonal antibody hybridoma cell line NaN-1 resistant to sulfa antibiotics, and the accession number is CGMCC No.12028, and the antibody prepared from the cell line can recognize twenty-five kinds of SAs, and It has good detection sensitivity and can be used to establish immunological detection methods for SAs.
本发明提供的NaN-1细胞株的制备基本步骤为:The basic steps for preparing the NaN-1 cell strain provided by the present invention are as follows:
1)免疫原的合成:500mg氨基噻唑乙酸溶于7.5mL无水甲醇中,在溶液中加入400mg二氯亚砜。反应液回流2h后,旋转蒸发去除挥发物,残余物用饱和碳酸氢钠溶液中和,然后用乙酸乙酯萃取后,旋转蒸发得到中间体Ⅰ,300mg中间体Ⅰ与600mg 4-乙酰氨基苯磺酰氯溶解于15mL吡啶中,在室温下反应3h,将反应液浓缩得到中间体Ⅱ,134mg中间体Ⅱ溶解在5mL、1M氢氧化钠溶液中,加热至80℃,在氮气保护下反应2h。反应结束后蒸发掉挥发物,残余物用盐酸中和,然后用乙酸乙酯萃取两次。将得到的目标液干燥并蒸发掉有机溶
剂,最后在真空下浓缩,得到目标半抗原。1) Synthesis of immunogen: 500 mg of aminothiazoleacetic acid was dissolved in 7.5 mL of anhydrous methanol, and 400 mg of thionyl chloride was added to the solution. After the reaction mixture was refluxed for 2 h, the volatiles were evaporated, evaporated, evaporated, evaporated, evaporated, evaporated. The acid chloride was dissolved in 15 mL of pyridine, and reacted at room temperature for 3 hours. The reaction mixture was concentrated to give Intermediate II. 134 mg of Intermediate II was dissolved in 5 mL of 1M sodium hydroxide solution, heated to 80 ° C, and reacted under nitrogen for 2 h. After the end of the reaction, the volatiles were evaporated, and the residue was crystallised from hydrochloric acid Dry the obtained target liquid and evaporate the organic solvent
The reagent is finally concentrated under vacuum to obtain the target hapten.
半抗原是带有羧基的产物,用碳二亚胺法进行偶联。具体步骤为:称取20mg半抗原溶解于5mLN,N-二甲基甲酰胺中,加入15.1mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与12.8mg N-羟基琥珀酰亚胺,在室温下活化4h;称取50mg牛血清白蛋白溶解于6mL碳酸盐缓冲溶液中,将活化液滴加进蛋白溶液中,室温反应过夜。用0.01M磷酸盐缓冲溶液透析3天去除未偶联上的半抗原,得到完全抗原。The hapten is a product having a carboxyl group and is coupled by a carbodiimide method. The specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8. Mg N-hydroxysuccinimide was activated at room temperature for 4 h; 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution and allowed to react at room temperature overnight. The unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen.
2)小鼠的免疫:完全抗原与等量弗氏佐剂混合乳化后,通过背部皮下注射免疫BALB/c小鼠。第一次免疫用完全弗氏佐剂,之后都用不完全弗氏佐剂。首次免疫与加强免疫之间间隔一个月,加强免疫之间间隔21天。最后一次直接用完全抗原(不含佐剂)冲击免疫,通过间接ELISA检测血清效价和抑制;2) Immunization of mice: After complete emulsification of complete antigen and an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. The last time was directly immunized with complete antigen (without adjuvant), and serum titer and inhibition were detected by indirect ELISA;
3)细胞融合和细胞株的建立:通过聚乙二醇(PEG 4000)法将小鼠脾细胞和小鼠骨髓瘤细胞融合,通过HAT培养基培养,利用间接ELISA检测阳性细胞孔,并进一步利用间接竞争ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆,最终筛选获得杂交瘤细胞株NaN-1。3) Cell fusion and cell line establishment: Mouse spleen cells and mouse myeloma cells were fused by polyethylene glycol (PEG 4000) method, cultured in HAT medium, positive cell wells were detected by indirect ELISA, and further utilized. The inhibitory effect of positive cell wells was determined by indirect competitive ELISA. The positive cells with the best inhibition were subcloned three times by limiting dilution method, and finally the hybridoma cell line NaN-1 was obtained.
本发明还提供一种抗磺胺类抗生素单克隆抗体,它由所述保藏编号为CGMCC No.12028的抗磺胺类抗生素单克隆抗体杂交瘤细胞株NaN-1所分泌产生。所述抗SAs单克隆抗体,对二十五种磺胺类抗生素有较好的检测灵敏度,其中,对磺胺嘧啶(Sulfadiazine,SD)、磺胺甲基嘧啶(Sulfamerazine,SMR)、磺胺二甲氧嘧啶(Sulfadimidine,SDM)及磺胺噻唑(Sulfathizzole,STZ)的IC50分别为:4.51、1.80、1.98及0.53μg/L。The present invention also provides a monoclonal antibody against sulfa antibiotics which is secreted by the anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 of the accession number CGMCC No. 12028. The anti-SAs monoclonal antibody has good detection sensitivity to twenty-five sulfa antibiotics, among which, sulfadiazine (SD), Sulfamerazine (SMR), sulfamethoxine (Sulfamerazine, SMR) The IC 50 of Sulfadimidine, SDM) and Sulfathizzole (STZ) were 4.51, 1.80, 1.98 and 0.53 μg/L, respectively.
所述抗磺胺类抗生素单克隆抗体,可用于食品安全领域,检测食品中磺胺类抗生素的含量。The anti-sulfonamide antibiotic monoclonal antibody can be used in the field of food safety to detect the content of sulfa antibiotics in food.
本发明提供的一株抗磺胺类抗生素单克隆抗体杂交瘤细胞株NaN-1,此细胞株分泌的单克隆抗体,可以识别二十五种磺胺类抗生素,且具有较好的检测灵敏度,其中对磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲氧嘧啶及磺胺噻唑的IC50分别为:4.51、1.80、1.98及0.53μg/L可以用于食品安全中磺胺类抗生素的多残留检测。The invention provides an anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1, and the monoclonal antibody secreted by the cell line can recognize twenty-five sulfa antibiotics, and has good detection sensitivity, wherein The IC 50 of sulfadiazine, sulfamethazine, sulfadimethoxine and sulfathiazole are: 4.51, 1.80, 1.98 and 0.53 μg/L, respectively, which can be used for multi-residue detection of sulfonamides in food safety.
生物材料保藏Biomaterial preservation
单克隆抗体杂交瘤细胞株NaN-1,已于2016年1月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,分类命名为单克隆细胞株,保藏编号为CGMCC No.12028。The monoclonal antibody hybridoma cell line NaN-1 has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, referred to as CGMCC. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China. The Institute of Microbiology, Chinese Academy of Sciences, is classified as a monoclonal cell strain with the accession number CGMCC No.12028.
图1:NaN-1分泌的单克隆抗体对磺胺嘧啶(SD)的抑制标准曲线。Figure 1: Standard curve for inhibition of sulfadiazine (SD) by monoclonal antibodies secreted by NaN-1.
本发明通过将SAs的完全抗原免疫小鼠,通过细胞融合、HAT选择性培养基培养,间接ELISA和间接竞争ELISA筛选细胞上清,最终得到了对SAs有较广的识别性和较好的灵敏度的单克隆抗体杂交瘤细胞株NaN-1。The invention screens cell supernatant by cell fusion, HAT selective medium culture, indirect ELISA and indirect competition ELISA by immunizing mice with complete antigen of SAs, and finally obtains wide recognition and good sensitivity to SAs. Monoclonal antibody hybridoma cell line NaN-1.
实施例1:杂交瘤细胞株NaN-1的制备Example 1: Preparation of hybridoma cell line NaN-1
溶液的配置:Solution configuration:
碳酸盐缓冲液(CBS):称取Na2CO31.59g,NaHCO32.93g,分别溶于少量双蒸水后混合,加双蒸水至约800mL混匀,调pH值至9.6,加双蒸水定容至1000mL,4℃贮存备用;Carbonate buffer (CBS): Weigh 1.59g of Na 2 CO 3 and 2.93g of NaHCO 3 , dissolve in a small amount of double distilled water, mix with double distilled water to about 800mL, adjust the pH to 9.6, add Double distilled water to a volume of 1000mL, stored at 4 ° C for use;
磷酸盐缓冲液(PBS):8.00g NaCl,0.2g KCl,0.2g KH2PO4,2.9g Na2HPO4·12H2O,溶于800mL纯水中,用NaOH或HCl调pH到7.2~7.4,定容至1000mL;Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , 2.9 g Na 2 HPO 4 · 12H 2 O, dissolved in 800 mL of pure water, adjusted to pH 7.2 with NaOH or HCl 7.4, constant volume to 1000mL;
PBST:含0.05%吐温20的PBS;PBST: PBS containing 0.05% Tween 20;
TMB显色液:A液:Na2HPO4·12H2O 18.43g,柠檬酸9.33g,纯水定容至1000mL;B液:60mg TMB溶于100mL乙二醇中。A、B液按体积比1︰5混合即为TMB显色液,现用现混。TMB color developing solution: liquid A: Na 2 HPO 4 · 12H 2 O 18.43 g, citric acid 9.33 g, pure water to 100 mL; B liquid: 60 mg TMB dissolved in 100 mL of ethylene glycol. A and B liquids are mixed with a volume ratio of 1..5 to be a TMB coloring liquid, which is currently used.
1、抗原的合成1. Synthesis of antigen
500mg氨基噻唑乙酸于溶7.5mL无水甲醇中,在溶液中加入400mg二氯亚砜。反应液回流2h后,旋转蒸发去除挥发物,残余物用饱和碳酸氢钠溶液中和,然后用乙酸乙酯萃取后,旋转蒸发得到中间体Ⅰ,300mg中间体Ⅰ与600mg 4-乙酰氨基苯磺酰氯溶解于15mL吡啶中,在室温下反应3h,将反应液浓缩得到中间体Ⅱ,134mg中间体Ⅱ溶解在5mL、1M氢氧化钠溶液中,加热至80℃,在氮气保护下反应2h。反应结束后蒸发掉挥发物,残余物用盐酸中和,然后用乙酸乙酯萃取两次。将得到的目标液干燥并蒸发掉有机溶剂,最后在真空下浓缩,得到目标半抗原。500 mg of aminothiazoleacetic acid was dissolved in 7.5 mL of anhydrous methanol, and 400 mg of thionyl chloride was added to the solution. After the reaction mixture was refluxed for 2 h, the volatiles were evaporated, evaporated, evaporated, evaporated, evaporated, evaporated. The acid chloride was dissolved in 15 mL of pyridine, and reacted at room temperature for 3 hours. The reaction mixture was concentrated to give Intermediate II. 134 mg of Intermediate II was dissolved in 5 mL of 1M sodium hydroxide solution, heated to 80 ° C, and reacted under nitrogen for 2 h. After the end of the reaction, the volatiles were evaporated, and the residue was crystallised from hydrochloric acid The obtained target liquid was dried and evaporated to an organic solvent, and finally concentrated under vacuum to obtain a target hapten.
半抗原是带有羧基的产物,用碳二亚胺法进行偶联。具体步骤为:称取20mg半抗原溶解于5mLN,N-二甲基甲酰胺中,加入15.1mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与12.8mg N-羟基琥珀酰亚胺,在室温下活化4h,称取50mg牛血清白蛋白溶解于6mL碳酸盐缓冲溶液中,将活化液滴加进蛋白溶液中,室温反应过夜。用0.01M磷酸盐缓冲溶液透析3天去除未偶联上的半抗原,得到完全抗原,并用紫外进行表征。The hapten is a product having a carboxyl group and is coupled by a carbodiimide method. The specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8. Mg N-hydroxysuccinimide was activated at room temperature for 4 h, and 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution, and reacted at room temperature overnight. The unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen and characterized by UV.
包被原的合成与免疫原的合成过程类似,只需将载体蛋白换成鸡卵清白蛋白。The original synthesis of the coating is similar to the synthesis of the immunogen, and the carrier protein is simply replaced with chicken egg albumin.
2、动物免疫
2, animal immunity
选择健康的6~8周龄的BALB/c小鼠进行免疫。取完全抗原与等量弗氏佐剂混合乳化后,通过背部皮下注射免疫BALB/c小鼠。第一次免疫用完全弗氏佐剂,之后都用不完全弗氏佐剂。首次免疫与加强免疫之间间隔一个月,加强免疫之间间隔21天。三免后7天采血,使用间接竞争ELISA方法测定小鼠血清效价和抑制,选择效价高且能识别较多SAs的小鼠,在四免后18天冲击免疫,不使用佐剂,腹腔注射。Healthy 6-8 week old BALB/c mice were selected for immunization. After the complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. Blood was collected 7 days after the third immunization, and the serum titer and inhibition of the mice were determined by indirect competitive ELISA. The mice with high titer and more SAs were selected, and the immunization was performed 18 days after the four immunizations without adjuvant. injection.
3、细胞融合3, cell fusion
在冲击免疫3天后,按照常规PEG(聚乙二醇,分子量为4000)方法进行细胞融合,具体步骤如下:(1)无菌取小鼠脾脏,研磨并通过200目细胞筛网得到脾细胞悬液,并进行细胞计数;(2)收集SP2/0细胞,悬浮于RPMI-1640基础培养液中,进行细胞计数;(3)将脾细胞和SP2/0细胞按照计数比1︰10的比例混合,离心后用50%PEG融合,时间1min,之后按照从慢到快,加入RPMI-1640基础培养液,离心后悬浮于含20%胎牛血清、2%的50×HAT的RPMI-1640筛选培养液中,加到96孔细胞培养板,置于37℃、5%CO2的培养箱中培养。After 3 days of shock immunization, cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows: (1) The mouse spleen was aseptically taken, ground and passed through a 200 mesh cell sieve to obtain spleen cell suspension. Liquid, and perform cell counting; (2) collect SP2/0 cells, suspend in RPMI-1640 basal medium for cell counting; (3) mix spleen cells and SP2/0 cells according to a ratio of 1..10 After centrifugation, the cells were fused with 50% PEG for 1 min, then RPMI-1640 basal medium was added from slow to fast, centrifuged and suspended in RPMI-1640 containing 20% fetal bovine serum and 2% 50×HAT. The solution was applied to a 96-well cell culture plate and cultured in an incubator at 37 ° C in a 5% CO 2 solution .
4、细胞筛选与细胞株建立4. Cell selection and cell line establishment
在细胞融合的第3天对融合细胞进行RPMI-1640筛选培养液半换液,第5天进行用含20%胎牛血清、1%的100×HT的RPMI-1640过渡培养液进行全换液,在第7天取细胞上清进行筛选。筛选分两步:第一步先用间接ELISA筛选出阳性细胞孔,第二步选用SD、SMR、SDM及STZ为标准品,用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对这四种标准品均有较好抑制的细胞孔,采用有限稀释法进行亚克隆,用同样的方法进行检测。重复三次,获得细胞株NaN-1。The RPMI-1640 screening medium was semi-exchanged on the third day of cell fusion, and the whole day was changed with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100×HT on the 5th day. The cell supernatant was taken for screening on day 7. The screening is divided into two steps: the first step is to select positive cell wells by indirect ELISA, and the second step is to select SD, SMR, SDM and STZ as standard products, and the inhibition effect of positive cells is determined by indirect competition ELISA. Cell wells with good inhibition of these four standards were selected, subcloned by limiting dilution method, and detected by the same method. This was repeated three times to obtain a cell strain NaN-1.
5、单克隆抗体的制备与鉴定5. Preparation and identification of monoclonal antibodies
取8-10周龄BALB/c小鼠,每只小鼠腹腔注射石蜡油1mL;7天后每只小鼠腹腔注射1×106杂交瘤细胞NaN-1,从第7天开始收集腹水,将腹水通过辛酸-硫酸铵法纯化,获得的单抗置于-20℃保存。8-8 weeks old BALB/c mice were injected with 1 mL of paraffin oil per mouse. After 7 days, each mouse was intraperitoneally injected with 1×10 6 hybridoma cell NaN-1, and ascites was collected from the 7th day. Ascites was purified by caprylic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20 °C.
使用间接竞争ELISA,测定单克隆抗体对二十五种SAs的IC50见表1:Using an indirect competition ELISA, the IC 50 of monoclonal antibodies against twenty-five SAs is shown in Table 1:
表1 NaN-1分泌的单克隆抗体的交叉反应率Table 1 Cross-reaction rate of monoclonal antibodies secreted by NaN-1
药物名称Drug name | IC50(μg/L)IC 50 (μg/L) | 交叉反应率(%)Cross-reaction rate (%) |
磺胺嘧啶(SD)Sulfadiazine (SD) | 4.514.51 | 100.00100.00 |
磺胺甲基嘧啶(SMR)Sulfamethazine (SMR) | 1.81.8 | 250.56250.56 |
磺胺对甲氧嘧啶Sulfamethoxine | 1.091.09 | 413.76413.76 |
磺胺二甲氧嘧啶(SDM)Sulfadimethoxypyrimidine (SDM) | 1.981.98 | 227.78227.78 |
磺胺甲二唑Sulfamethoxazole | 0.150.15 | 3006.673006.67 |
磺胺甲噁唑Sulfamethoxazole | 0.210.21 | 2147.622147.62 |
磺胺噻唑(STZ)Sulfathiazole (STZ) | 0.520.52 | 850.94850.94 |
磺胺喹噁啉Sulfaquinoxaline | 50.1250.12 | 9.009.00 |
磺胺二甲基嘧啶Sulfadimethylpyrimidine | 20.3220.32 | 22.1922.19 |
磺胺索嘧啶Sulfadiazine | 20.220.2 | 22.3322.33 |
磺胺氯哒嗪Sulfachloropyridazine | 0.510.51 | 884.31884.31 |
磺胺甲氧哒嗪Sulfamethoxazine | 1.121.12 | 402.68402.68 |
磺胺多辛Sulfadoxine | 50.350.3 | 8.978.97 |
磺胺吡啶Sulfapyridine | 1.21.2 | 375.83375.83 |
苯酰磺胺Phenyl sulfonamide | 124.29124.29 | 3.633.63 |
磺胺醋酰Sulfaacetamide | 115.95115.95 | 3.893.89 |
磺胺噁唑Sulfaoxazole | 138.21138.21 | 3.263.26 |
磺胺Sulfonamide | 109.19109.19 | 4.134.13 |
磺胺硝苯Sulfanitrobenzene | 149.2149.2 | 3.023.02 |
磺胺苯吡唑Sulfaphenazole | 80.2780.27 | 5.625.62 |
磺胺异恶唑Sulfaisoxazole | 70.6570.65 | 6.386.38 |
柳氮磺胺嘧啶Sulfasalazine | 90.4590.45 | 4.994.99 |
磺胺林Sulfonamide | 85.6985.69 | 5.265.26 |
磺胺脒Sulfaguanidine | 149.13149.13 | 3.013.01 |
酞磺胺噻唑Sulfonamide | 150.03150.03 | 3.633.63 |
6.抗体应用6. Antibody application
将杂交瘤细胞株NaN-1通过体内腹水制备的单克隆抗体应用于几种磺胺类抗生素(SD,SMR,SDM,STZ)的ELISA添加回收试验,具体步骤如下:The monoclonal antibody prepared by the hybridoma cell line NaN-1 by in vivo ascites was applied to the ELISA addition recovery test of several sulfa antibiotics (SD, SMR, SDM, STZ). The specific steps are as follows:
(1)包被:将包被原用0.05M pH 9.6碳酸盐缓冲液从2μg/mL开始倍比稀释,100μL/孔,37℃反应2h;(1) coating: the original was diluted with 0.05M pH 9.6 carbonate buffer from 2μg / mL, 100μL / well, 37 ° C reaction for 2h;
(2)洗涤:将板内溶液倾去,甩干,并用洗涤液洗涤3次,每次3min;(2) washing: the solution in the plate was decanted, dried, and washed 3 times with the washing liquid for 3 min each time;
(3)封闭:拍干后,加入200μL/孔封闭液,37℃反应2h。洗涤后烘干备用;(3) Closure: After pat dry, add 200 μL/well blocking solution and react at 37 ° C for 2 h. Dry after washing;
(4)加样:将抗血清从1︰1000开始倍比稀释,并加入到各稀释度的包被孔中,100μL/孔,37℃反应1h;充分洗涤后,加入1︰3000稀释的HRP-羊抗鼠IgG,100μL/孔,37℃反应1h;(4) Loading: The antiserum was diluted from 1..1000 and added to the coated wells of each dilution, 100 μL/well, and reacted at 37 ° C for 1 h; after washing, add 1..3000 diluted HRP. - goat anti-mouse IgG, 100 μL / well, reacted at 37 ° C for 1 h;
(5)显色:将酶标板取出,充分洗涤后,每孔加入100μL的TMB显色液,37℃避光反应15min;(5) Color development: the enzyme plate was taken out, and after thoroughly washing, 100 μL of TMB color developing solution was added to each well, and the reaction was carried out at 37 ° C for 15 minutes in the dark;
(6)终止和测定:每孔加入50μL终止液以终止反应,然后用酶标仪测定各孔的OD 450值。(6) Termination and measurement: 50 μL of the stop solution was added to each well to terminate the reaction, and then the OD 450 value of each well was measured with a microplate reader.
(7)结果判读:以OD450值大于或等于阴性对照孔的2.1倍(即P/N≥2.1)所对应的血清最高稀释倍数即为血清的ELISA效价;
(7) Results interpretation: The ELISA titer of serum is the highest dilution factor of serum corresponding to 2.1 times of OD 450 value (ie P/N ≥ 2.1).
(8)添加回收及样品前处理:以加入SD的添加回收试验为例,吸取1mL牛奶于15mL离心管中,向牛奶中分别添加20ng、50ng、100ng SD,用0.01M PBS稀释10倍,采用间接ELISA进行添加回收试验。SMR的添加回收试验添加量分别为:1ng、2ng、5ng;SDM的添加量为:10ng、20ng、50ng;STZ的添加量为:1ng、2ng、5ng;NaN-1单克隆抗体对SD、SMR、SDM及STZ四种药物的添加回收实验结果,其回收率见表2。(8) Adding recovery and sample pretreatment: Take the addition and recovery test of adding SD as an example, take 1 mL of milk into a 15 mL centrifuge tube, add 20 ng, 50 ng, 100 ng SD to the milk, and dilute 10 times with 0.01 M PBS. An indirect ELISA was used for the addition recovery test. The addition amount of SMR was 1 ng, 2 ng, 5 ng; the amount of SDM added was: 10 ng, 20 ng, 50 ng; the amount of STZ added was: 1 ng, 2 ng, 5 ng; NaN-1 monoclonal antibody against SD, SMR The results of the addition and recovery of four drugs, SDM and STZ, the recovery rate is shown in Table 2.
表2:Table 2:
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.
Claims (8)
- 一株抗磺胺类抗生素单克隆抗体杂交瘤细胞株NaN-1,已于2016年1月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.12028,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。An anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, with the preservation number CGMCC No.12028, and the deposit address is Beijing. No. 3, Beichen West Road, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences.
- 抗磺胺类抗生素单克隆抗体,其特征在于,它由权利要求1所述保藏编号为CGMCC No.12028的抗磺胺类抗生素单克隆抗体杂交瘤细胞株NaN-1所分泌产生。A monoclonal antibody against sulfa antibiotics, which is produced by the anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 of claim 1 having the accession number CGMCC No. 12028.
- 权利要求2所述抗磺胺类抗生素单克隆抗体的应用,其特征在于,用于制备检测磺胺类抗生素的工具或者直接用于检测磺胺类抗生素。Use of the anti-sulfa antibiotic monoclonal antibody of claim 2, which is useful for preparing a tool for detecting a sulfa antibiotic or for directly detecting a sulfa antibiotic.
- 根据权利要求3所述的应用,其特征在于,用于食品安全中磺胺类抗生素的多残留检测。The use according to claim 3, characterized in that it is used for multi-residue detection of sulfonamides in food safety.
- 根据权利要求3所述的抗磺胺类抗生素单克隆抗体的应用,其特征在于,所述磺胺类抗生素包括磺胺嘧啶、磺胺甲基嘧啶、磺胺对甲氧嘧啶、磺胺二甲氧嘧啶、磺胺甲二唑、磺胺甲噁唑、磺胺噻唑、磺胺喹噁啉、磺胺二甲基嘧啶、磺胺索嘧啶、磺胺氯哒嗪、磺胺甲氧哒嗪、磺胺多辛、磺胺吡啶、苯酰磺胺、磺胺醋酰、磺胺噁唑、磺胺、磺胺硝苯、磺胺苯吡唑、磺胺异恶唑、柳氮磺胺嘧啶、磺胺林、磺胺脒、酞磺胺噻唑。The anti-sulfa antibiotic monoclonal antibody according to claim 3, wherein the sulfa antibiotic comprises sulfadiazine, sulfamethazine, sulfamethoxine, sulfamethoxine, sulfamethoxine Azole, sulfamethoxazole, sulfathiazole, sulfaquinoxaline, sulfamethazine, sulfamethazine, sulfachloropyridazine, sulfamethoxazine, sulfadoxine, sulfapyridine, phenyl sulfonamide, sulfacetamide Sulfaoxazole, sulfonamide, sulfanitrobenzene, sulfaphenazole, sulfisoxazole, sulfasalazine, sulfamethamine, sulfaguanidine, sulfamethoxazole.
- 一种检测磺胺类抗生素的工具,其特征在于,含有权利要求2所述的抗磺胺类抗生素单克隆抗体或者权利要求1所述的抗磺胺类抗生素单克隆抗体杂交瘤细胞株NaN-1。A tool for detecting a sulfa antibiotic, which comprises the anti-sulfa antibiotic monoclonal antibody according to claim 2 or the anti-sulfa antibiotic monoclonal antibody hybridoma cell strain NaN-1 according to claim 1.
- 根据权利要求6所述的一种检测磺胺类抗生素的工具,其特征在于,所述工具基于酶联免疫技术的试剂盒或者基于免疫层析的试纸条。A tool for detecting a sulfa antibiotic according to claim 6, wherein the tool is based on a kit of enzyme-linked immunosorbent technology or a test strip based on immunochromatography.
- 权利要求6或7所述根据在检测食品中磺胺类抗生素含量中的应用。 Use according to claim 6 or 7 in accordance with the detection of the content of sulfa antibiotics in foods.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610843734.5A CN106282125B (en) | 2016-09-23 | 2016-09-23 | One plant of hybridoma cell strain NaN-1 for secreting anti-sulfa antibiotics monoclonal antibody and its application |
CN2016108437345 | 2016-09-23 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018054394A1 true WO2018054394A1 (en) | 2018-03-29 |
Family
ID=57712925
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2017/110882 WO2018054394A1 (en) | 2016-09-23 | 2017-11-14 | Hybridoma cell line nan-1 which secretes sulfonamide antibiotic-resistant monoclonal antibody, and application of hybridoma cell line |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN106282125B (en) |
WO (1) | WO2018054394A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106282125A (en) * | 2016-09-23 | 2017-01-04 | 江南大学 | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain |
CN109022366A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | One plant of hybridoma cell strain for secreting anti-levamisol monoclonal antibody and its application |
CN114594179A (en) * | 2022-03-01 | 2022-06-07 | 农业农村部环境保护科研监测所 | Method for simultaneously and rapidly extracting and detecting multiple antibiotics in soil |
CN114908060A (en) * | 2022-05-12 | 2022-08-16 | 江南大学 | Hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof |
CN114990072A (en) * | 2022-05-05 | 2022-09-02 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody against quinolone antibiotics and application thereof |
CN116699127A (en) * | 2023-02-28 | 2023-09-05 | 中国农业科学院北京畜牧兽医研究所 | Kit for tuberculin titer calibration, calibration method and application thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112379088B (en) * | 2020-12-04 | 2022-05-31 | 福建农林大学 | Detection test paper for detecting lead residue based on gold nanoflower technology |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101113981A (en) * | 2007-04-06 | 2008-01-30 | 江苏省微生物研究所有限责任公司 | Sulfonamides direct-competition ELISA detecting reagent kit |
CN101245338A (en) * | 2008-03-31 | 2008-08-20 | 北京农学院 | Immunity colloidal gold test paper strip for testing sulfalene oxazole relict and manufacture method thereof |
CN101571541A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof |
CN101955536A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Anti-sulfonamides antibody and application thereof |
CN101983972A (en) * | 2010-10-19 | 2011-03-09 | 浙江大学 | Preparation method of anti-sulfanilamide rabbit monoclonal antibody and application thereof |
CN102079788A (en) * | 2009-11-27 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
CN106282125A (en) * | 2016-09-23 | 2017-01-04 | 江南大学 | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain |
-
2016
- 2016-09-23 CN CN201610843734.5A patent/CN106282125B/en active Active
-
2017
- 2017-11-14 WO PCT/CN2017/110882 patent/WO2018054394A1/en active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101113981A (en) * | 2007-04-06 | 2008-01-30 | 江苏省微生物研究所有限责任公司 | Sulfonamides direct-competition ELISA detecting reagent kit |
CN101245338A (en) * | 2008-03-31 | 2008-08-20 | 北京农学院 | Immunity colloidal gold test paper strip for testing sulfalene oxazole relict and manufacture method thereof |
CN101571541A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof |
CN102079788A (en) * | 2009-11-27 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
CN101955536A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Anti-sulfonamides antibody and application thereof |
CN101983972A (en) * | 2010-10-19 | 2011-03-09 | 浙江大学 | Preparation method of anti-sulfanilamide rabbit monoclonal antibody and application thereof |
CN106282125A (en) * | 2016-09-23 | 2017-01-04 | 江南大学 | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain |
Non-Patent Citations (1)
Title |
---|
WANG, ZHANHUI ET AL.: "Investigation of Antigen-Antibody Interactions of Sulfonamides with a Monoclonal Antibody in a Fluorescence Polarization Immunoassay Using 3D-QSA ] Models", INT. J. MOL. SCI., vol. 13, no. 5, 23 May 2012 (2012-05-23), pages 6334 - 6351, XP055502041 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106282125A (en) * | 2016-09-23 | 2017-01-04 | 江南大学 | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain |
CN109022366A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | One plant of hybridoma cell strain for secreting anti-levamisol monoclonal antibody and its application |
CN114594179A (en) * | 2022-03-01 | 2022-06-07 | 农业农村部环境保护科研监测所 | Method for simultaneously and rapidly extracting and detecting multiple antibiotics in soil |
CN114594179B (en) * | 2022-03-01 | 2023-08-18 | 农业农村部环境保护科研监测所 | Method for simultaneously and rapidly extracting and detecting multiple antibiotics in soil |
CN114990072A (en) * | 2022-05-05 | 2022-09-02 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody against quinolone antibiotics and application thereof |
CN114990072B (en) * | 2022-05-05 | 2023-08-22 | 江南大学 | Hybridoma cell strain secreting anti-quinolone antibiotic monoclonal antibody and application thereof |
CN114908060A (en) * | 2022-05-12 | 2022-08-16 | 江南大学 | Hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof |
CN116699127A (en) * | 2023-02-28 | 2023-09-05 | 中国农业科学院北京畜牧兽医研究所 | Kit for tuberculin titer calibration, calibration method and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN106282125A (en) | 2017-01-04 |
CN106282125B (en) | 2019-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018054394A1 (en) | Hybridoma cell line nan-1 which secretes sulfonamide antibiotic-resistant monoclonal antibody, and application of hybridoma cell line | |
CN102482346B (en) | Antibody specific to methicillin resistant staphylococcus aureus, detection method and kit for methicillin resistant staphylococcus aureus using the same | |
CN1908664B (en) | Multiple cluster antigen and its preparation, wide spectrum specific multiple clone antigen and its use | |
US7658922B2 (en) | Monoclonal antibodies, hybridoma cell lines, methods and kits for detecting phytase | |
CN111304174A (en) | Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof | |
CN106153933B (en) | A kind of immunological reagent box of efficient quantitative detection c reactive protein | |
WO2018196573A1 (en) | Flunixin meglumine monoclonal antibody hybridoma cell strain yy and application thereof | |
WO2018196572A1 (en) | Iprodione monoclonal antibody hybridoma cell strain zxl-2 and application thereof | |
CN110117575B (en) | Pyrimidinemonoclonal antibody hybridoma cell strain HFG and application thereof | |
CN109705216B (en) | Monoclonal antibody for resisting bovine skeletal muscle troponin I and application thereof | |
CN104093737A (en) | Antibody specific for trans-resveratrol and use thereof | |
CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
CN111334479A (en) | Chlorhydroxypyridine monoclonal antibody hybridoma cell strain TYL and application thereof | |
CN112266901B (en) | Azoxystrobin monoclonal antibody hybridoma cell strain and application thereof | |
CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
CN115246818A (en) | Hapten, artificial antigen and antibody for detecting imidacloprid as well as preparation methods and applications thereof | |
CN113637642A (en) | Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain | |
WO2019129082A1 (en) | A lincosamide universal monoclonal antibody hybridoma cell strain and application thereof | |
KR20060027404A (en) | Reagents, methods and kits for detecting feed enzymes | |
CN106701686B (en) | hybridoma cell strain 1A5, secreted anti-sulbactam monoclonal antibody and application | |
CN114034869A (en) | Detection method and kit for cardiac troponin I | |
CN106929477B (en) | Anti-prostaglandin F2αSpecific monoclonal antibody hybridoma cell strain WXX-2 and application thereof | |
CN110616194A (en) | Aconitine monoclonal antibody cell strain SJ and application thereof | |
CN111748528A (en) | Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain | |
CN114317450B (en) | Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof |
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: 17852461 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: 17852461 Country of ref document: EP Kind code of ref document: A1 |