从无细胞培养滤液中预纯化和检测葡萄球菌肠毒素B的改进方法。

Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate.

作者信息

Dainiak Maria, Hedström Martin, Galaev Igor Yu, Mattiasson Bo

机构信息

Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, 221 00, Lund, Sweden.

出版信息

Biotechnol Prog. 2005 Jul-Aug;21(4):1347-51. doi: 10.1021/bp050099j.

DOI:10.1021/bp050099j

PMID:16080722

Abstract

An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.

摘要

本文介绍了一种改进的酶联免疫吸附测定（ELISA）方法，用于检测蛋白A制剂中的葡萄球菌肠毒素B（SEB）。通过用木瓜蛋白酶消化固定在琼脂糖4B上的与SEB结合的抗SEB IgG获得Fab片段。抗SEB和来自二抗的过氧化物酶标记的Fab片段成功用于蛋白A制剂中SEB的改良ELISA。SEB-琼脂糖通过木瓜蛋白酶消化反复用于生产抗SEB Fab片段，而不会丧失亲和力。此外，为了从粗培养滤液中纯化SEB，提出了一个初始步骤，即利用加热和pH联合处理去除大量污染蛋白，而不损失毒素活性。考虑到时间缩短和总回收率提高，这一预处理步骤在进一步的下游加工中产生了积极效果。