Allo-A Sepharose 4B亲和层析分离血清糖缺失转铁蛋白

发布时间：2018-08-29 11:00

【摘要】： 目的：建立Allo-A Sepharose亲和层析技术分离血清中Tf和CDT，然后用直接夹心ELISA分别测定Tf和CDT水平。方法 1．建立转铁蛋白直接夹心ELISA测定技术。用抗人Tf IgG和HRP标记羊抗人Tf抗体建立直接夹心ELISA用于Tf和CDT的定量测定。以重复率和回收率评价所建立的直接夹心ELISA。 2．Allo-A Sepharose 4B亲和层析分离CDT。用Allo-A和CNBr活化的Sepharose 4B制备Allo-A Sepharose 4B亲和层析柱，用于血清Tf和CDT的分离。建立的Allo-A Sepharose 4B亲和层析技术用固相pH梯度等电聚焦电泳(IPG IEF)和Western blot评价。 3．人血Tf和CDT的分离和测定要测定的血清先由亲和层析分离Tf和CDT，然后测定用直接夹心法测定洗脱液中的Tf和CDT。结果 1．建立转铁蛋白直接夹心ELISA测定技术鼠抗人Tf IgG包被浓度初步确定为2.5μg／ml。确定HRP标记羊抗人Tf抗体最适稀释度初步确定为1／2000。Tf的浓度与450nm光密度极为相关(R~2=0.9845)。不同浓度的人Tf标准品及血清样品的变异系数(CV％)为1.8％～7.52％。所观察的4份酗酒者血清，回收率为96％～105.7％。 2．Allo-A Sepharose 4B亲和层析分离CDT。 2．1 Allo-A与CNBr-activated Sepharose 4B的偶联，共进行了4次偶联实验，偶联率为80.6～91.4％，CV=4.9％。 2．2 Allo-A Sepharose 4B亲和层析分离CDT Allo-A Sepharose 4B亲和层析图谱显示2个洗脱峰，CDT存在于第一洗脱峰，，Tf存在于第二沈脱峰。 2．3 Tf和CDT的鉴定等电聚焦电泳和免疫印记结果表明，在本实验确定的分离条件下，Allo-A Sepharose 4B亲和层析可有效分离CDT和Tf。 3．人血Tf和CDT的测定Tf水平与OD450nm呈线性关系(R~2=0.9917)。正常人血清CDT平均水平为27.02μg／ml，99％可信限为23.1～30.9μg／ml；Tf平均水平为1998.0μg／ml，99％可信限为1606.1～2389.9μg／ml，CDT与Tf比值为1.45％。酒精性肝病患者血清CDT平均水平为36.9μg／ml，高于正常人血清CDT水平。Tf平均水平为720.7μg／ml，显著低于正常人水平。CDT与Tf比值为5.1％。结论 1．本实验建立的Tf直接夹心ELISA方法具有较理想的灵敏度、精密度和可靠性。 2．本实验建立的Allo-A Sepharose 4B亲和层析技术适用于血清Tf和CDT分离。从Allo-A Sepharose 4B层析柱的制备到Tf和CDT的分离，操作简便易行，不需昂贵设备，因而可作为常规技术广泛应用。 3．通过Allo-A Sepharose 4B亲和层析技术联合使用Tf直接央心ELISA方法对20例正常人和27例酒精性肝病患者的初步观察，发现酒精性肝病患者血清CDT水平高于正常人，而Tf水平则低于正常人。CDT／Tf比值在酒精性肝病患者也升高，比值的升高主要由Tf水平降低所致。 4．根据对正常人和酒精性肝病患者的观察结果，Allo-A Sepharose 4B亲和层析技术联合使用Tf直接夹心ELISA方法可作为常规检测技术，用于酒精性肝病的诊断。
[Abstract]:OBJECTIVE: To establish an Allo-A Sepharose affinity chromatography method for the separation of Tf and CDT in serum, and to determine the levels of Tf and CDT by direct sandwich ELISA.
Method
1. to establish a direct sandwich ELISA assay for transferrin.
A direct sandwich ELISA for the quantitative determination of Tf and CDT was established by labeling sheep anti-human Tf antibody with anti-human Tf IgG and HRP.
Separation of CDT. by affinity chromatography with 2.Allo-A Sepharose 4B
Allo-A Sepharose 4B affinity chromatography column was prepared with Allo-A and CNBr activated Sepharose 4B for the separation of Tf and CDT in serum.
Separation and determination of Tf and CDT in 3. human blood
The Tf and CDT in the eluent were separated by affinity chromatography and then determined by direct sandwich method.
Result
1. establishment of direct sandwich ELISA for transferrin detection
The optimum dilution of HRP-labelled sheep anti-human Tf antibody was determined to be 1/2000. The concentration of Tf was highly correlated with 450 nm optical density (R~2=0.9845). The coefficient of variation (CV%) of different concentrations of human Tf standard and serum samples was 1.8%-7.52%. The four alcoholic sera were observed and returned to the study. The yield is 96% ~ 105.7%..
Separation of CDT. by affinity chromatography with 2.Allo-A Sepharose 4B
2.1 The coupling experiments of Allo-A and CNBr-activated Sepharose 4B were carried out four times. The coupling rates were 80.6-91.4% and CV=4.9%.
2.2 The affinity chromatogram of CDT Allo-A Sepharose 4B showed that there were two elution peaks, CDT existed in the first elution peak and Tf existed in the second one.
Identification of 2.3 Tf and CDT by isoelectric focusing electrophoresis and immunoblotting showed that the affinity chromatography of Allo-A Sepharose 4B could effectively separate CDT and Tf under the conditions determined in this experiment.
3. There was a linear relationship between Tf and OD450nm in human serum (R~2=0.9917). The average level of CDT in normal subjects was 27.02 ug/ml, the 99% confidence limit was 23.1-30.9 ug/ml, the average level of Tf was 1998.0 ug/ml, the 99% confidence limit was 1606.1-2389.9 ug/ml, and the ratio of CDT to Tf was 1.45%. The average level of Tf was 720.7 ug/ml, which was significantly lower than the normal level. The ratio of CDT to Tf was 5.1%.
conclusion
1. the Tf direct sandwich ELISA method established in this experiment has better sensitivity, precision and reliability.
2. The affinity chromatography technique of Allo-A Sepharose 4B established in this experiment is suitable for the separation of Tf and CDT in serum.
3. By using Allo-A Sepharose 4B affinity chromatography combined with Tf direct central ELISA, the serum CDT levels in 20 normal subjects and 27 patients with alcoholic liver disease were higher than those in normal subjects, while Tf levels were lower than those in normal subjects. It is mainly caused by the decrease of Tf level.
4. According to the observation of normal people and alcoholic liver disease patients, Allo-A Sepharose 4B affinity chromatography combined with Tf sandwich ELISA can be used as a routine detection technique for the diagnosis of alcoholic liver disease.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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