利用人免疫球蛋白构建免疫磁珠富集金葡菌条件优化

Condition Optimization of Building Immunomagnetic Bead Using Human Immunoglobulin G for Enrichment Staphylococcus aureus

DOI：10.3969/j.issn.1673-1689.2013.03.006

中文关键词: 金黄色葡萄球菌 免疫磁法 人免疫球蛋白 富集

英文关键词: Staphylococcus aureus Immunomagnetic bead-based separation Human immunoglo-bulin G Concentration

基金项目:国家自然科学基金项目(30972287);国家“十二五”支撑计划项目(2012BAD29B06);广东省教育厅及广东海洋大学引进人才基金项目(0812311);广东省科技厅省部级产学研项目(2011B090400154)

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中文摘要:

利用人免疫球蛋白制备富集金黄色葡萄球菌的免疫磁珠及优化条件;利用化学键法制备免疫磁珠,并用矩阵法和单因素法优化免疫磁珠制备条件,通过磁珠对抗体饱和吸附量及加入量相关性评价磁珠与抗体固定化程度,根据免疫磁珠的特异性、灵敏性及饱和捕捉量来考察IMBS.aureus对金葡菌的富集能力;结果显示采用MB-M型磁珠,加入质量浓度均为5 mg/L的活化剂EDC和NHS,于24℃偶联人IgG,24 h得到IMBS.aureus,其饱和吸附量为393.27μg,磁珠与人IgG加入量具有相关性,IMBS.aureus特异性好、灵敏度高(26 CFU/10 mL),0.2 mL IMBS.aureus饱和捕捉菌液浓度为106CFU/mL;通过优化,磁珠装载人IgG分子的能力提高了5倍,检测限提高了4倍,富集金葡菌的效率大大增强,成本降低,为人免疫球蛋白磁珠富集和分离金黄色葡萄球菌实际应用创造了条件。

英文摘要:

The aim of this study is to determine the building conditions for developing immunomagnetic bead using human immunoglobulin G enrichment of Staphylococcus aureus.For this,the preparation conditions of immunomagnetic beads was optimized by matrix method and the single factor,and the the degree of fixed antibody to immunomagnetic bead was evulated by the amount of saturated adsorption capacity and correlation.Inspect enrichment ability of immunomagnetic bead to Staphylococcus aureus according to specificity,sensitivity and saturation catches.Aas a result,the homemade IMBS.aureus was obtained.The bead was adopted the type of MB-M,added activator combination EDC and NHS(5 mg/L),then incubated 24 h with human immunoglobulin G at 24 ℃;Adsorption capacity of IMBS.aureus was 393.27 g;There was correlation between the contents of beads and human immunoglobulin G;IMBS.aureus has high specificity,sensitivity(26 CFU/10 mL) and catch capability.The aboved results demonstrated that Homemade IMBS.aureus exhibited the characters of stable property,strong accumulation ability and low costs.This manuscript provided a novel way to deal with rapid detection about food-borne pathogens.

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