Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate.
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.
Dainiak
Maria
M
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, 221 00, Lund, Sweden.
Hedström
Martin
M
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D002138:Calibration; D002474:Cell-Free System; D004768:Enterotoxins / Q000032:analysis* / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000378:metabolism; D010206:Papain / Q000737:chemistry
D004768:Enterotoxins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; C031818:enterotoxin B, staphylococcal; D010206:Papain