白喉产毒培养基中铁离子磺基水杨酸分光光度检测方法的建立及验证  

Development and verification of a sulfosalicylic acid spectrophotometric method for determination of iron ion in medium for producing diphtheria toxin

作　　者：温嘉纳[1] 李婧妍 姬光[1] 廖宏玮 李燕[1] 段维国[1] 何春艳[1] 孙明波[1] 顾琴[1] WEN Jiana;LI Jingyan;JI Guang;LIAO Hongwei;LI Yan;DUAN Weiguo;HE Chunyan;SUN Mingbo;GU Qin(Institute of Medical Biology of Chinese Academy of Medical Science&Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research&Development on Severe Infectious Diseases,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院,医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2023年第10期1242-1247,共6页Chinese Journal of Biologicals

基　　金：云南省科技厅科技计划(202002AA100009);昆明市科技局重点项目(2019-1-N-25318000003332)。

摘　　要：目的 建立测定白喉产毒培养基中微量铁离子的磺基水杨酸分光光度法,并进行验证及初步应用。方法 全光谱扫描铁-磺基水杨酸络合物最大吸光度;以铁磺基水杨酸络合物吸光度与铁离子含量进行线性回归,建立培养基中铁离子检测方法,并对方法 进行稳定性、准确性和精密性验证;考察Ca^(2+)、Mg^(2+)、K^(+)、Na^(+)以及显色反应物对方法 的影响;采用磺基水杨酸分光光度法测定自制及市售白喉产毒培养基中微量铁离子;分别采用磺基水杨酸分光度法及常用方法 亚铁嗪比色法、邻菲啰啉分光光度法检测两种培养基(牛肉胰酶消化液、5%多聚蛋白胨)中铁含量,比较3种方法 的检测结果。结果 铁-磺基水杨酸络合物在波长425 nm处有最大吸光度;铁离子浓度在1~0.05μg/mL范围内与吸光度存在良好的线性关系,检测限为0.05μg/mL,标准曲线方程为:Y=0.027 9X+0.046 1,R^(2)>0.99;每间隔5 min测定1次A425值,各浓度标准品A_(425)值的变异系数(CV)均小于5%;低(0.05μg/mL)、中(0.5μg/mL)、高浓度(1μg/mL)Fe^(3+)标准品溶液连续测定3次,每个浓度平行测定9组的CV均<5%,回收率均>95%;Ca^(2+)、Mg^(2+)、K^(+)、Na^(+)、15μL磺基水杨酸(20%)和50μL氢氧化氨(1:1)不对检测方法 构成干扰;产毒培养基测定结果 与白喉细菌产毒结果 一致;3种检测方法 结果 一致。结论 建立的磺基水杨酸分光光度法能够准确、有效地测定白喉产毒培养基中微量铁离子含量。Objective To develop and verify a sulfosalicylic acid spectrophotometric method for the determination of trace iron ions in diphtheria toxin medium,and apply it preliminarily.Methods The maximum absorbance of the complex of iron and sulfosalicylic acid was scanned by full spectrum;A method for the determination of iron ion in culture medium was developed by linear regression between the absorbance of the complex and the content of iron ion,and the stability,accuracy and precision of the method were verified.The effects of Ca~(2+),Mg~(2+),K^(+),Na^(+) and reactants on the method were investigated.Spectrophotometric method with sulfosalicylic acid was used to determine trace iron in the self-made and commercial medium for diphtheria toxin production.Sulfosalicylic acid spectrophotometry,ferrizine colorimetry and o-phenanthroline spectrophotometry were used to detect iron content in two kinds of culture media(beef trypsin digestion liquid and 5% polypeptone),and the detection results of the three methods were compared.Results The complex of iron and sulfosalicylic acid showed the maximum absorbance at the wavelength of 425 nm;There was a good linear relationship between the absorbance and concentration of iron ion in the range of 1~0.05 μg/mL,the detection limit was 0.05 μg/mL,and the standard equation was:Y=0.027 9 X+0.046 1,R^(2) >0.99;The coefficients of variation(CVs) of A_(425) value of each concentration of standards measured every 5 min were less than 5%;Low(0.05 μg/mL),medium(0.5 μg/mL)and high concentration(1 μg/mL) of Fe~(3+) standard solutions were continuously determined for 3 times.The CVs of 9groups of each concentration measured in parallel were all less than 5% and the recovery rates were higher than 95%;Ca~(2+),Mg~(2+),K^(+),Na^(+),15 μL of sulfo salicylic acid(20%) and 50 μL of ammonia hydroxide(1:1) showed no interference in the method;The results of toxin-producing medium were consistent with those of diphtheria bacteria;The results of the three detection methods were consistent.Conclusion

关 键 词：白喉 白喉毒素 铁离子 磺基水杨酸 分光光度法 

分 类 号：R331[医药卫生—人体生理学]