Werner Schaeg

A radioimmunoassay for tetanus antibodies using protein A - containing Staphylococcus aureus.

To measure tetanus antibodies a trace amount of125I-labeled tetanus toxin is mixed with appropriate dilutions of human serum or blood. The labeled antigen-antibody complexes are adsorbed to heat-killed staphylococci (Cowan I) via their surface protein A. The radioactivity of the washed solid phase is a function of the initial antibody concentration.The test allows the measurement of 6×10−0 U of tetanus antitoxin in a volume of 0.03 ml. In order to avoid possible interferences, serum has to be diluted 20-fold before use. Taking that into account, the real border limit of sensitivity is 4×10−3 U/ml serum. Antibodies may be measured in serum, in plasma, and even in heparinized blood.As to its sensitivity, the test compares well with the toxin neutralization procedure. It is superior to the previous radioimmunologic, enzymoimmunologic, and hemagglutination techniques with respect to sensitivity and reproducibility. It reflects the values obtained in the toxin neutralization test better than the other in vitro procedures, as shown by parallel assays of 17 sera.

Purification of penicillinase (β-lactamase) and acid phosphatase from Staphylococcus aureus in one procedure

Abstract Penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) and acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) from Staphylococcus aureus were purified in one procedure. The enzymes were selectively removed from the culture supernatant by adsorptions onto glass beads and cellulose phosphate. Subsequent elution and gel filtration on Sephadex G-100, superfine, allowed the separation of the two enzymes in one step. The enzymatic activities of penicillinase and acid phosphatase were not markedly inhibited by their respective antibodies.

Determination of IgG, IgM, and IgA antibodies toMycoplasma pneumoniae by an indirect staphylococcal radioimmunoassay

An indirect staphylococcal radioimmunoassay (SRIA) has been developed for determination ofM. pneumoniae antibodies. This test allows the detection of antibodies in various immunoglobulin (Ig) classes similar to the previously described radioimmunoprecipitation test (RIP). SRIA has two advantages over RIP: first, it uses 100-fold less anti-Ig reagents than RIP; second, bound can be separated from unbound antigen more easily by the relatively heavy staphylococci. SRIA antibodies, belonging to the IgA class of Ig, could be detected in nasal secretions of volunteers infected intranasally with ts H43 ofM. pneumoniae. In sera of patients withM. pneumoniae pneumonia antibodies to the IgG or the IgM class of Igs could be determined separately. This is especially important for an early diagnosis ofM. pneumoniae disease.

A staphylococcal radioimmunoassay for detection of antibodies toMycoplasma pneumoniae

A radioimmunoassay (RIA) which depends on the property of protein A ofStaphylococcus aureus to combine with the Fc-fragment of immunoglobulins was developed.This technique was employed to measure antibodies in human and various animal sera. It could be demonstrated that the staphylococcal RIA was at least as sensitive as the previously described radioimmunoprecipitation technique in detecting antibodies toM.pneumoniae in human sera. In addition, antibodies toM.pneumoniae could be demonstrated in sera of hamsters intranasally inoculated with the organisms. Antibodies could also be demonstrated in rabbit sera after immunization withM.pneumoniae. The test proved to be considerably more sensitive than conventional tests for detection of antibodies to the organisms. The test requires only small amounts of reagents and is relatively inexpensive.

Antiphagocytic factors of Staphylococcus aureus

Abstract Several staphylococcal substances could interfere with phagocytosis, imparting a definite advantage on Staphylococcus aureus in the initial phase of infection. Leukocidins were shown to damage mainly granulocytes and macrophages. Clumping-factor, by direct reaction with fibrinogen, induced clumping of the staphylococci in plasma. This impaired phagocytosis. The increased virulence of encapsulated staphylococci was caused by a delay in chemotaxis and phagocytosis. Apparently encapsulation prevented activation of C3, by the staphylococci.

Protein A-Aktivität von Staphylococcus hyicus im Vergleich zu Protein A von Staphylococcus aureus

Abstract Protein A (PA)-Activity was demonstrated in 44 (93.6%) of 47 Staphylococcus hyicus -cultures. PA from S. hyicus , as well as PA from S. aureus , could be released by extraction with concentrated formic acid or by treatment of the staphylococci with lysostaphin. PA was also demonstrable in the culture medium. Purification of PA from S. hyicus and S. aureus could be achieved by affinity-chromatography on IgG-Sepharose®. In Ouchterlony-tests both PA-preparations gave single lines of identity with normal sera from man, pig, dog and guinea pig (Fig. 1). Immunoelectrophoretic analysis indicated a significantly faster migration towards the anode for PA from S. hyicus than PA from S. aureus (Fig. 2). Isoelectric focusing revealed maximal activity for PA from culture supernatant of S. hyicus about pH 4.3 and for that of S. aureus at pH 5.0 (Fig. 3). Gelchromatographic experiments indicated a lower molecular weight for PA from S. hyicus than for PA from S. aureus (Fig. 4).

Characterization of immunoglobulin G binding to Staphylococcus aureus strain Wood 46

Protein A (PA)-activity was detected in Staphylococcus aureus strain Wood 46 which had been considered to be PA-negative. This staphylococcal strain bound 28% of 125I-labelled IgG, compared with 89% by strain Cowan I. The binding activities of both S. aureus strains were saturable, time-dependent and specific. The dissociation constants of 1.6 X 10(-9) M for Wood 46 and 9.3 X 10(-8) M for Cowan I indicated a similar affinity for human IgG in both strains. The number of IgG-binding sites were estimated to be 16,970 for Wood 46 and 41,200 for Cowan I. Exposure to heat and ultrasonication reduced PA-activities of strain Cowan I, but not that of strain Wood 46. Extraction of the staphylococci with guanidine and formic acid resulted in a reduction of IgG-binding activities only in strain Wood 46. Photooxidation, trypsinization and lysozyme treatment also diminished IgG-binding of strain Wood 46 to a larger extent than that of strain Cowan I. Extracellular PA from S. aureus strains Wood 46 and Cowan I could be purified by affinity chromatography on IgG-sepharose. The purified PA preparations gave single protein bands upon SDS-polyacrylamide gel electrophoresis. Their molecular weights were 42,000 and their isoelectric points approximated 5.0.

Purification of oligomeric staphylococcal α-toxin by affinity chromatography on digitonin-sepharose

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

Release of some staphylococcal enzymes and toxins under the influence of size of inoculum and time of incumbation

Abstract With increasing size of inoculum (0.025, 0.25, 2.5 g of wet Staphylococcus aureus per 100 ml broth) maximal coagulase-activity (256–512 U/ml) was detected in the supernatant after 30 min-2 hr of incubation at 37°C. Alpha- and beta-hemolysins appeared later (after 8–24 hr of incubation). Acid/alkaline phosphatase could be demonstrated from the beginning of incubation, but their maximal activity in culture supernatant could be demonstrated after 8–24 hr of incubation (Table 1). Thus, size of inoculum and time of incubation have a greater significance in the optimal production of some staphylococcal substances than generally assumed.

Lipase und Phospholipase C von Staphylococcus aureus verschiedener Herkunft I. Nachweis und vorkommen

Abstract Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin-and lecithinagar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenylpalmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).