氟喹诺酮多组分残留检测试纸条的研制
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摘要
氟喹诺酮类(fluoroquinolones,FQNS或FQS)药物是一类广谱高效抗菌药,几乎适用于临床常见的各种细菌感染性疾病。但由于广泛的应用,且常伴有不合理用药和滥用药情况的发生,因FQNS残留而产生的不良反应问题越来越多,一些国家、地区或组织对FQNS的使用和最高残留限量(MRL)都做了规定,因此,本研究旨在应用胶体金免疫层析原理,研制氟喹诺酮多组分残留检测试纸条。
本研究根据FQNS的结构特性,选取最接近其母核结构的4种药物——诺氟沙星、环丙沙星、达氟沙星和沙拉沙星,运用ELISA法筛选能与多种同类药物发生交叉反应的抗血清,筛选出抗沙拉沙星血清用于建立FQNS残留的检测方法。获得多克隆抗体,亲和纯化后,采用胶体金免疫层析法,制备氟喹诺酮多组分残留检测试纸条。采用柠檬酸三钠还原法制备胶体金,经紫外分光光度计鉴定制备的金颗粒直径在40nm左右,满足试纸条的检测要求。将FQNS多克隆抗体标记在胶体金结合垫上,SALX-OVA抗原和羊抗兔IgG二抗包被在硝酸纤维素膜上,其中,SALX-OVA作为检测线,羊抗兔IgG二抗作为质控线,制备氟喹诺酮多组分残留检测试纸条。
通过对试纸条的优化,结果表明:采用Sartorius CN140的硝酸纤维素膜,抗原、金标抗体浓度分别为1mg/mL和30μg/mL,其检测灵敏度为50ng/mL。
Fluoroquinolones (FQNS) is the third generation quinolones antibiotics, it has high antibacterial activity to Gram-positive bacteria, Gram-Negative bacillus,coccobacteria and gonococci; it also has powerful inhibiting effect to some of the chlamydia, sprirochete, mycoplasma and anaerobic bacterium, it almost applies to all the regular bacteria-infected diseases. On the other hand, because of the widespread application of FQNS, often accompanied with drug-abuse and irrationality of applying, the adverse reactions (ADR) of FQNS are gaining more and more attention. Some of the countries, regions and organization have made stipulation to the using of FQNS and the Maximum Residue Limit (MRL), so it is imperative under the situation to build effective, fast, high-sensitivity methods of detecting the residues of FQNS.
Our research, based upon the chemical structure of FQNS, experiments the Sarafloxacin (SALX), Ciprofloxacin (CPLX), Danofloxacin (DFLX) and Norfloxacin (NFLX), which have similar structure with it. We try to find a kind of medicine that can detect the residues of various FQNS and find out the detecting method by making use of the immunology principle and testing the medicine specificity. The objective of this study was to develop a rapid and simple colloidal gold immunochromatographic assay (GICA) for the detection of FQNS。
FQNS is a kind of small molecule material, belonging to a hapten, and only has reactionogenicity, no immunogenicity. We can perform immunological experiment only under the situation that the preparation of complete antigen is reached by coupling it with biomacromolecule. Firstly, using the N-hydroxysuccinimideeater method (NHS), we prepare immunizing antigen by coupling the four medicines respectively with bovine serum albumin (BSA), coating antigen by coupling with ovalbumin (OVA). Thus, we get kinds of complete antigen. When we scan the complete antigens, the four medicines, BSA and OVA with ultraviolet light, whose wave-length is between 200～400nm. The result shows that the medicine has coupled with carrier protein successfully. At the same time, we prepare antiserum by immunizing mice and rabbits with immunizing antigen, then perform ELISA test on antiserum by coating antigen coating ELISA plate. The results shows that all the titer of the four antiserum reach above 1:32000, this indicates that the coupling is successful when identify the antiserum in immunology.
Proceed the ci-ELISA experiments on the four antiserum we have got, we have tested specificity (cross reaction rate) of antiserum with the same kind medicine and find that the antiserum of SALX has the most cross reaction, especially with NFLX, CPLX and DFLX. Thus, we select SALX for further study.
Passes through the ammonium sulfate to purify the antibodies thickly first, then HiTrap Protein G HP affinity column further purifies antibodies IgG. Then the colloidal gold immunochromatographic assay can be used for the fluoroquinolones multicomponent Residues
Using the citric acid reducing process preparation colloid gold particles, observes its dispersion degree and the uniformity result under the ultraviolet light scanning appraisal. UV spectrum scanning demonstrated the diameter of colloidal gold particle was around 40nm. Then the purified antibody was labeled with Colloidal gold and was identified by UV spectra scanning to find the wave of maximal absorbed apex had changed after colloidal gold was labeled. This demonstrated the label was successful.
Colloidal gold Immunochromatographic test strip consists of conjugate releasing pad, nitrocellulose membrane, sample pad, wicking paper and adhesive plastic back. Gold-antibody conjugate were immobilized onto theconjugate releasing pad, and the SALX-OVA and goat-anti- rabbit antibody were immobilized onto a nitrocellulose membrane as test line and controlline respectively. The concentration of colloidal gold-labeled antibody and SALX-OVA were optimized by the test,we select the Sartorius CN140 nitrocellulose membrane,30ug/mL colloidal gold-labeled antibody and 1mg/mL SALX-OVA as the reaction condition. Results demonstrated that the detection limit at lab condition was 50ng/mL, which should be suitable for the detection of FQNS.
The major advantages of the one step strip test were that results could be obtained within 10 min and that all needed reagents were included in the strip. The strips could be used to detect the Fluoroquinolones residue. The method producing the one step strip which is used for the detection of FQNS residues, but also supply the effective detecting measure for the supervising departments who are in charge of the medicine residues in animal food. At the same time, it has also explored the ways for the study of the methods of detecting some kind of medicine residues.
本研究根据FQNS的结构特性,选取最接近其母核结构的4种药物——诺氟沙星、环丙沙星、达氟沙星和沙拉沙星,运用ELISA法筛选能与多种同类药物发生交叉反应的抗血清,筛选出抗沙拉沙星血清用于建立FQNS残留的检测方法。获得多克隆抗体,亲和纯化后,采用胶体金免疫层析法,制备氟喹诺酮多组分残留检测试纸条。采用柠檬酸三钠还原法制备胶体金,经紫外分光光度计鉴定制备的金颗粒直径在40nm左右,满足试纸条的检测要求。将FQNS多克隆抗体标记在胶体金结合垫上,SALX-OVA抗原和羊抗兔IgG二抗包被在硝酸纤维素膜上,其中,SALX-OVA作为检测线,羊抗兔IgG二抗作为质控线,制备氟喹诺酮多组分残留检测试纸条。
通过对试纸条的优化,结果表明:采用Sartorius CN140的硝酸纤维素膜,抗原、金标抗体浓度分别为1mg/mL和30μg/mL,其检测灵敏度为50ng/mL。
Fluoroquinolones (FQNS) is the third generation quinolones antibiotics, it has high antibacterial activity to Gram-positive bacteria, Gram-Negative bacillus,coccobacteria and gonococci; it also has powerful inhibiting effect to some of the chlamydia, sprirochete, mycoplasma and anaerobic bacterium, it almost applies to all the regular bacteria-infected diseases. On the other hand, because of the widespread application of FQNS, often accompanied with drug-abuse and irrationality of applying, the adverse reactions (ADR) of FQNS are gaining more and more attention. Some of the countries, regions and organization have made stipulation to the using of FQNS and the Maximum Residue Limit (MRL), so it is imperative under the situation to build effective, fast, high-sensitivity methods of detecting the residues of FQNS.
Our research, based upon the chemical structure of FQNS, experiments the Sarafloxacin (SALX), Ciprofloxacin (CPLX), Danofloxacin (DFLX) and Norfloxacin (NFLX), which have similar structure with it. We try to find a kind of medicine that can detect the residues of various FQNS and find out the detecting method by making use of the immunology principle and testing the medicine specificity. The objective of this study was to develop a rapid and simple colloidal gold immunochromatographic assay (GICA) for the detection of FQNS。
FQNS is a kind of small molecule material, belonging to a hapten, and only has reactionogenicity, no immunogenicity. We can perform immunological experiment only under the situation that the preparation of complete antigen is reached by coupling it with biomacromolecule. Firstly, using the N-hydroxysuccinimideeater method (NHS), we prepare immunizing antigen by coupling the four medicines respectively with bovine serum albumin (BSA), coating antigen by coupling with ovalbumin (OVA). Thus, we get kinds of complete antigen. When we scan the complete antigens, the four medicines, BSA and OVA with ultraviolet light, whose wave-length is between 200～400nm. The result shows that the medicine has coupled with carrier protein successfully. At the same time, we prepare antiserum by immunizing mice and rabbits with immunizing antigen, then perform ELISA test on antiserum by coating antigen coating ELISA plate. The results shows that all the titer of the four antiserum reach above 1:32000, this indicates that the coupling is successful when identify the antiserum in immunology.
Proceed the ci-ELISA experiments on the four antiserum we have got, we have tested specificity (cross reaction rate) of antiserum with the same kind medicine and find that the antiserum of SALX has the most cross reaction, especially with NFLX, CPLX and DFLX. Thus, we select SALX for further study.
Passes through the ammonium sulfate to purify the antibodies thickly first, then HiTrap Protein G HP affinity column further purifies antibodies IgG. Then the colloidal gold immunochromatographic assay can be used for the fluoroquinolones multicomponent Residues
Using the citric acid reducing process preparation colloid gold particles, observes its dispersion degree and the uniformity result under the ultraviolet light scanning appraisal. UV spectrum scanning demonstrated the diameter of colloidal gold particle was around 40nm. Then the purified antibody was labeled with Colloidal gold and was identified by UV spectra scanning to find the wave of maximal absorbed apex had changed after colloidal gold was labeled. This demonstrated the label was successful.
Colloidal gold Immunochromatographic test strip consists of conjugate releasing pad, nitrocellulose membrane, sample pad, wicking paper and adhesive plastic back. Gold-antibody conjugate were immobilized onto theconjugate releasing pad, and the SALX-OVA and goat-anti- rabbit antibody were immobilized onto a nitrocellulose membrane as test line and controlline respectively. The concentration of colloidal gold-labeled antibody and SALX-OVA were optimized by the test,we select the Sartorius CN140 nitrocellulose membrane,30ug/mL colloidal gold-labeled antibody and 1mg/mL SALX-OVA as the reaction condition. Results demonstrated that the detection limit at lab condition was 50ng/mL, which should be suitable for the detection of FQNS.
The major advantages of the one step strip test were that results could be obtained within 10 min and that all needed reagents were included in the strip. The strips could be used to detect the Fluoroquinolones residue. The method producing the one step strip which is used for the detection of FQNS residues, but also supply the effective detecting measure for the supervising departments who are in charge of the medicine residues in animal food. At the same time, it has also explored the ways for the study of the methods of detecting some kind of medicine residues.
引文
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[2] 徐子刚,郑琳,吴清洲.环丙沙星荧光特性的研究.药物分析杂志,2003,(23):41～43
[3] 管绍光.新型广谱抗微生物药物—氟喹诺酮类.河北农业大学学报,1994,17(增刊):227～233
[4] 顾成词,高树宇,张大鹏,等.动物用氟喹诺酮类药物的研究.辽宁畜牧兽医,2004,(10):27～29
[5] 汪雪雁,祁克宗,朱良强.氟喹诺酮类药物残留分析研究进展.安徽农业科学,2004,32(5):1021～1023
[6] 魏东,李振华,张乃生.氟喹诺酮类药物的不良反应.动物医学进展,2006,27(7):105～107
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