Immunogenicity of the  -Haemolysin of Staphylococcus Aureus

Abstract

PLATE XIXAT T E M P,T s by several investigators have failed to demonstrate the immunogenicity of the 6-haemolysin of StaphyZococcus aureus. Gladstone and Yoshida (1967) did not convincingly demonstrate that 8-lysin was immunogenic and they showed that inhibitors of its haemolytic activity were present in serum. Recent evidence suggests that these serum inhibitors are lipoproteins (Donahue, 1969;Kantor, Temples and Shaw, 1972).In this study, the immunogenicity of 8-lysin has been demonstrated by the use of purified antibody preparations. MATERIALS AND METHODSPreparation of 6-lysin The strain employed, methods of production and purification of 6-lysin were those of Caird and Wiseman (1970). Purified S-lysin was also kindly sent to us by Dr Arnold Kreger. Haemolysin titrations were performed as described by Wiseman and Caird (1972). Buffers were prepared according to Gomori (1955) and, where required, 0 . 0 1~ phosphate buffer at pH 7 was supplemented with 0.85% NaCl. Nitrogen content was assayed by the microKjeldahl technique as described by Markham (1942) and an estimate of protein content was derived from the assay value multiplied by 6.25.Preparation of u-, ,8-and 7-lysins u-and y-Haemolysins were prepared and purified as described elsewhere (Fackrell and Wiseman, 1974; Wiseman, Caird and Fackrell, 1975). Purified ,!I-lysin was used in one immunodiffusion study and was prepared by a modification of the method of Wiseman and Caird (1967) in which the haemolysin was passed through Sephadex G-75 equilibrated with Hallander's (1963) buffer. The active lysin was pooled when removed from the column, dialysed against distilled water for 48 hours, centrifuged and the supernatant fluid lyophilised.Serological procedures Immunodi'usion, immunoelectrophoresis and quantitative precipitin methods were those of Campbell et al. (1970). The blood agarose required in some immunodiffusion tests was prepared by the addition of 1.0 ml of packed, washed human group-0 RBC to 100 ml of a 1 % (w/v) solution of Difco agarose in phosphate-buffered saline held at 50°C.Production of antisera. Antibodies to the haemolysins were prepared in 2-month-old New Zealand white rabbits purchased from the Canadian Research Animal Farm, Bradford,

Immunogenicity and molecular characterisation of staphylococcal haemolysin (Plate XIII)

“…Anti-a haemolysin was not detectable in any of the sera tested, in contrast to the report of Fackrell and Wiseman (1974) that 6 haemolysin purified by the procedure of Kreger et al (1971) induced anti-a haemolysin in rabbits. Immunisation may provide a sensitive test for the presence of low levels of contamination in an immunising preparation; exclusion of the biologically potent a haemolysin is important when the biological properties of 6 haemolysin are being considered.…”

“…Plommet and Bouillane (1966), Gladstone and Yoshida (1967), Hallander (1968) and Kantor, Temples and Shaw (1972) were unable to demonstrate an immune response to S haemolysin. More recently, antigenic and haemolysin-inhibitory activity has been demonstrated in IgG fractions of immune sera by Fackrell and Wiseman (1974), Birkbeck and Whitelaw (1976) and Heatley (1977). This paper describes properties of antisera to 6 haemolysin and some molecular measurements of 6 haemolysin.…”

“…Rabbits were immunised as described by Fackrell and Wiseman (1974) except that in some animals antigen was injected intravenously in physiological saline or subcutaneously in Freund's Complete Adjuvant (Difco).…”

Partitioning of staphylococcal haemolysin

“…In addition, the chromatographic procedures employed frequently yielded multiple peaks of 6 haemolysin, while crude preparations yielded only one peak of S haemolysin when tested electrophoretically and by molecular sieving. These findings, as well as those previously reported (Fackrell and Wiseman, 1974;Lee et al, 1976) clearly indicate the need to renew efforts to develop methods for the purification of 6 haemolysin.…”

Properties of the Gamma Haemolysin of Staphylococcus aureus 'Smith 5R'

“…They also found that the LDao of delta lysin for mice was 125 mg, although such a large LD, is meaningless except to suggest that the lysin is relatively non-toxic for mice. Calculations based on the data of Kreger et al (1971) give an LDm of 2 mg of delta lysin for mice and 7.2 mg for guinea pigs, but contamination of their preparations with alpha lysin at a concentration of one part in 2000 or less would account for these findings (see Fackrell & Wiseman, 1974).…”