Van Rensburg E, Du Preez J C, Ellis C E
Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa.
Onderstepoort J Vet Res. 2006 Dec;73(4):241-50.
An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.
开发了一种间接酶联免疫吸附测定法（ELISA），用于定量无血清培养基中溶血曼氏杆菌恒化器培养物产生的细胞外白细胞毒素（LKT）。用制备性SDS-PAGE纯化的白细胞毒素用于制备鸡多克隆抗体（PAb），作为主要检测抗体。从分析性SDS-PAGE凝胶中切下LKT蛋白是一种有效的毒素纯化技术。因此，以这种方式纯化的102 kDa LKT多肽用作参考毒素，并使用四参数逻辑拟合对所得校准曲线进行建模，以将吸光度与LKT蛋白浓度相关联。检测下限对应于14.5 ng ml(-1)的LKT浓度。SDS、血清白蛋白和包被pH的存在对间接ELISA的吸光度值有明显影响。
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