抗人血清IgD抗体与微球颗粒连接方法的研究

Conjugation of anti-human serum IgD antibody to beads

目的研究抗人血清IgD抗体与微球颗粒连接的方法,为相关产品试剂盒的研发奠定基础。方法微球颗粒表面有许多COOH基团,EDAC与COOH基团反应,形成一个中间产物,S-NHS能与这个中间产物反应,进一步形成一个稳定的能与氨基反应的S-NHS ester中间物,抗人血清IgD抗体上的氨基能与这个S-NHS ester中间物反应,从而将抗人血清IgD抗体共价键连接到微球颗粒上。结果如果抗人血清IgD抗体被连接到微球颗粒上,这种微球颗粒在Luminex200仪器上检测时,平均荧光强度值高,而没有连接抗人血清IgD抗体的微球颗粒,平均荧光强度值低。同时被连接到微球颗粒上的抗人血清IgD抗体仍然能与相对应的抗原发生特异性的结合。结论该方法简单有效且重复性好,能够应用到临床检测及相关诊断试剂盒的开发中。

[ Objective ] To develop a method for anti-human serum IgD antibody conjugation to beads for use in clinical diagnosis. [ Methods ] The surface of the polystyrene beads has carboxyl functional groups which are available to react with the amino groups of antibodies to form amide bonds. This is achieved by reacting EDAC with the carboxyl on the bead to form an unstable reactive intermediate called o-acylisourea ester. This is then semi-stabilised with sulpho-NHS to form an amine-reactive NHS ester. The second intermediate reacts with the amino group of the antibody molecule to form a stable amide bond thus covalently coupling the macromolecule to the bead. [ Resuits ] The efficiency of coupling can be determined by measuring the intensity of signal generated using Luminex 200 which should be many times greater than that of uncoupled beads. Meanwhile the antibodies coated on the beads still can react with antigens. [ Conclusion] This method is simple, effective and reproducible. Conjugated beads can be used to develop diagnostic test kits in clinical evaluation of disease state.