Physico‐ and Immunochemical Properties of Staphylococcal Protein A Extracellularly Produced by a Set of Mutants from <i>Staphylococcus aureus</i> Cowan I

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“…Protein A. Protein A was isolated from the culture medium of the Cowan I strain by affinity chromatography on IgG-Sepharose (20) and dissolved in physiologial saline.…”

Section: Methodsmentioning

confidence: 99%

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Nakano

Toyoda

Tanabe

et al. 1980

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“…The 376 strains were subjected to the detection of SpA producibility on heart infusion agar containing 1% normal dog serum (D 100 Plate) (15,16). After overnight incubation at 37 C on the plates, the SpA producibility of these strains was investigated ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Staphylococcus aureus Cowan I strain and its SpA-deficient mutant were used as positive and negative reference strains for quantitation of cell-bound SpA, respectively (13). A mutant strain which releases SpA molecules in soluble form was used as soluble SpA producer (15)(16)(17).…”

Section: Methodsmentioning

confidence: 99%

Simple Method for Quantitation of Cell‐Bound Protein A on Staphylococcus aureus Cells by Means of Hemagglutination with Sheep Erythrocytes Differentially Sensitized with Rabbit Antibody and Its Clinical Application

Hwang

Seki

Murai

et al. 1989

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A simple method for quantitation of cell-bound protein A (SpA) on organisms of Staphylococcus aureus was successfully devised by using hemagglutination between staphylococcal organisms and a series of sheep erythrocyte suspensions sensitized with different amounts of and sheep erythrocyte rabbit antiserum. The validity of the principle and the reproducibility of the method presented here were precisely analyzed and the details of the method are presented. The hemagglutination was quantitatively inhibited both by normal rabbit serum and by soluble SpA. Using the method presented here, 376 strains of S. aureus freshly isolated from clinical materials were subjected to SpA quantitation in cell-bound form. According to the results, there was an unexpected distribution profile in the amounts of cellbound SpA among the clinical isolates, which showed a two-peak pattern. A possible usefulness of the method presented here in clinical investigations is briefly discussed also.

“…We have studied the mode of reaction between immunoglobulins and SpA molecules produced by mutants derived from the Cowan I strain which releases SpA into culture medium (10,11). These soluble SpA molecules were assumed to have different valencies for the binding to immunoglobulins in proportion to their molecular weights.…”

mentioning

confidence: 99%

“…The mutants, UV-2, LH-IV, and UV-1, were isolated previously (11). For the present investigation, V-11 and V-21 were newly isolated by the cosedimentation technique from independent bacterial clones (9).…”

mentioning

confidence: 99%

Hemagglutination Test with Sheep Erythrocytes Sensitized with Antisera from Several Mammalian Species for the Investigation of Biological Reactivities of Staphylococcal Protein A

Nishihara

Seki

Etani

et al. 1986

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Staphylococcal protein A (SpA) is a cell-bound protein which has a high affinity for the Fc portion of immunoglobulins from various mammalian species (7), and many researchers have been using it as a useful tool in immunoglobulin-associated experiments. We have studied the mode of reaction between immunoglobulins and SpA molecules produced by mutants derived from the Cowan I strain which releases SpA into culture medium (10, 11). These soluble SpA molecules were assumed to have different valencies for the binding to immunoglobulins in proportion to their molecular weights. It was considered interesting to investigate the mode of binding of these SpA molecules to mammalian immunoglobulins by hemagglutination test which might reflect differences in the number of binding sites or steric structure of SpA molecules.The hemagglutination test with sheep erythrocytes (SRBC) sensitized with rabbit antiserum has been considered one of the most sensitive methods for the detection of soluble SpA (1, 4, 6,13,14). In the present communication we describe hemagglutination patterns of SRBC sensitized with antisera from several mammalian species by soluble SpA which has possibly two, three, or four binding sites to immunoglobulins in one molecule. We also propose a new hemagglutination test using SRBC sensitized with guinea pig antiserum, which is more sensitive and stable in detecting SpA activity than using SRBC sensitized with rabbit antiserum.The SpA molecules were purified from the culture supernatant of mutants (UV-2, LH-IV, UV-1, V-11, and V-21) by a column of human IgG-conjugated Sepharose CL 4B according to the procedure of Hjelm et al (5) with a slight modification. The mutants, UV-2, LH-IV, and UV-1, were isolated previously (11). For the present investigation, V-11 and V-21 were newly isolated by the cosedimentation technique from independent bacterial clones (9). As previously reported V-11 and V-21 (V type mutants) were easily distinguished from usual LH mutants on an agar plate containing dog serum because V type mutants develop vague halos compared with usual LH mutants. The molecular weights of UV-2, LH-IV, UV-1, V-11, and V-21 SpA molecules were estimated to be 41