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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >Enzyme-linked immunosorbent fluorescence assay and high-pressure liquid chromatography for analysis of humoral immune responses to Coxiella burnetti proteins.
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Enzyme-linked immunosorbent fluorescence assay and high-pressure liquid chromatography for analysis of humoral immune responses to Coxiella burnetti proteins.

机译：酶联免疫吸附荧光测定法和高压液相色谱法用于分析对柯氏杆菌Burnetti蛋白的体液免疫应答。

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摘要

A microtiter enzyme-linked immunosorbent fluorescence assay based on alkaline phosphatase conjugate and 4-methylumbelliferyl phosphate as fluorogenic substrate was developed and adapted to quantitatively analyze immunoglobulin G subclass 1 (IgG1) and IgG2 responses of vaccinated and infected cattle to proteins of Coxiella burnetii. The enzyme-linked immunosorbent fluorescence assay surpassed the conventional enzyme-linked immunosorbent assay with a 50-fold-higher sensitivity and a broader range of linear dose-response signals. Antigens of C. burnetii were purified by sodium dodecyl sulfate extraction and molecular-sieve high-pressure liquid chromatography. The purified 14-, 27-, and 30-kilodalton proteins were used as antigens without any further treatment. Vaccination with either chloroform-methanol-extracted cell residues of C. burnetii or the 27-kilodalton major surface protein evoked an early IgG2 response to the 27-kilodalton protein (2 weeks after immunization), whereas IgG2 to lipopolysaccharides of C. burnetii was detected only in the late phase (13 weeks after immunization). These results may have implications for the serodiagnosis of acute and chronic Q fever. IgG1 against these antigens was induced solely by naturally occurring C. burnetii infections, indicating that infected cattle can be distinguished from vaccinated cattle by using the enzyme-linked immunosorbent fluorescence assay and SP27 antigen.
机译：建立了一种基于碱性磷酸酶偶联物和4-甲基伞形磷酸酯作为荧光底物的微量滴定酶联免疫吸附荧光测定方法，该方法适用于定量分析接种和感染牛对柯氏杆菌的免疫球蛋白G亚类1（IgG1）和IgG2的应答。酶联免疫吸附荧光测定法比常规的酶联免疫吸附测定法灵敏度高50倍，线性剂量反应信号范围更广。通过十二烷基硫酸钠萃取和分子筛高压液相色谱法纯化伯氏梭菌的抗原。纯化的14-，27-和30-千达尔顿蛋白未经任何进一步处理即可用作抗原。用氯仿-甲醇提取的伯氏梭状芽胞杆菌细胞残基或27公斤的主要表面蛋白进行疫苗接种可引起对27公斤的蛋白质的早期IgG2反应（免疫后2周），而检测到的IgG2对伯氏梭状芽孢杆菌的脂多糖则为。仅在晚期（免疫后13周）。这些结果可能对急慢性Q热的血清诊断有影响。针对这些抗原的IgG1仅由自然发生的伯氏梭状芽胞杆菌感染诱导，这表明可以通过使用酶联免疫吸附荧光测定法和SP27抗原将感染的牛与接种的牛区分开。

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