Evaluation of new enzyme-linked immunosorbent assay based on a supernatant containing Staphylococcus aureus alpha-toxin produced by Bacillus subtilis.
The gene encoding alpha-toxin from Staphylococcus aureus was cloned into a Bacillus subtilis expression vector (pEF 231/alpha-Tox). The protease-deficient B. subtilis strain DB 104 transformed with pEF 231/alpha-Tox expressed and secreted 5 mg of alpha-toxin per liter into the growth medium. The alpha-toxin-containing supernatant was diluted 200-fold and used as coating antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of septicemia and endocarditis caused by S. aureus. Paired sera from patients in acute and convalescent stages of S. aureus and non-S. aureus infections were used to evaluate this ELISA. To evaluate the effectiveness of the crude preparation, the results were compared with those of an ELISA based on a commercially available alpha-toxin. Similar rises in serum titers were obtained with either type of alpha-toxin preparation. This is the first time a crude supernatant without any further purification has been used as an ELISA coating antigen. We therefore conclude that B. subtilis is a suitable host organism for cheap and simple production of prokaryotic recombinant antigens to be used in serodiagnosis.
Egnell
P
P
Center for Biotechnology, Karolinska Institute Novum, Huddinge, Sweden.
Christensson
B
B
Möllby
R
R
Flock
J I
JI
D000907:Antibodies, Bacterial / Q000032:analysis*; D001412:Bacillus subtilis / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D011994:Recombinant Proteins / Q000276:immunology*; D012698:Serologic Tests; D013203:Staphylococcal Infections / Q000175:diagnosis*; D013211:Staphylococcus aureus / Q000276:immunology*
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C021091:staphylococcal alpha-toxin