Cross protection between an encapsulated strain of Staphylococcus hyicus and encapsulated strains of Staphylococcus aureus

Strain ST67P of Staphylococcus hyicus grew diffusely in regular serum-soft agar. With the addition of rabbit antisera prepared with Staph. aureus strains, Smith, NS58D or NS41D, capsular type A, B or C, respectively, the organisms converted to compact type growth. Mice immunized with heat-killed vaccine of strain ST67P showed significant resistance against either homologous or heterologous strains, Smith, NS58D and NS41D. Passive protective activities in rabbit antisera prepared with strains Smith, NS58D and NS41D were absorbed out with either homologous cell surface polysaccharide fraction or cell surface polysaccharide fraction extracted from strain ST67P. Well-defined large capsules were observed in ultra-thin sections treated with rabbit antiserum prepared with homologous strain conjugated with ferritin. Also, the capsule surrounded by ferritin granules was shown in ultra-thin sections treated with ferritin conjugated with antisera prepared with those heterologous strains although the capsular size was significantly smaller than those observed by homologous antiserum.     

Cross protection between an encapsulated strain of Staphylococcus hyicus and encapsulated strains of Staphylococcus aureus

Strain ST67P of Staphylococcus hyicus grew diffusely in regular serum-soft agar. With the addition of rabbit antisera prepared with Staph. aureus strains, Smith, NS58D or NS41D, capsular type A, B or C, respectively, the organisms converted to compact type growth. Mice immunized with heat-killed vaccine of strain ST67P showed significant resistance against either homologous or heterologous strains, Smith, NS58D and NS41D. Passive protective activities in rabbit antisera prepared with strains Smith, NS58D and NS41D were absorbed out with either homologous cell surface polysaccharide fraction or cell surface polysaccharide fraction extracted from strain ST67P. Well-defined large capsules were observed in ultra-thin sections treated with rabbit antiserum prepared with homologous strain conjugated with ferritin. Also, the capsule surrounded by ferritin granules was shown in ultra-thin sections treated with ferritin conjugated with antisera prepared with those heterologous strains although the capsular size was significantly smaller than those observed by homologous antiserum.     

Precise localization of antigens on follicular dendritic cells

Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA bovine serum albumin - BSA-gold BSA conjugated to colloidal gold particles - FDC follicular dendritic cells     

Antibodies to cell-surface antigens of plant protoplasts

Both mouse and rabbit polyclonal antibodies to plant cell-surface antigens were developed by immunization with cell membrane material from oat (Avena sativa L. cv. Garry) roots. We were able to quickly assess the activity of antisera by monitoring the degree of protoplast agglutination and by using an indirect immunofluorescence assay. Using polyclonal antibodies to cell-surface antigens, we have found that oat root protoplasts share common surface antigens with protoplasts from other plant tissues and species. From experiments with antisera treated with excess oat leaf or oat root protoplasts before our immunoassays, we have obtained evidence for the existence of organ-specific cell-surface antigens in higher plants.     

Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.     

Cross-reactivity of birch anthers and leaves with birch pollen antigens and allergens. A fine-structural immunocytochemical study using the post-embedding protein-A-gold technique

Ultra-thin sections of vegetative tissues from birch (anthers and leaves) were labeled for pollen antigens and allergens using a commercial rabbit IgG antibody preparation directed against birch pollen antigens and allergens. Antibody binding sites were visualized using the protein A-gold technique. Specific labeling occurred in anther tissue (tape-tum cells, anther wall cells) as well as in the birch leaf (assimilation parenchyma). In both types of tissue, antigens and allergens were detected throughout the living protoplast (including cell organelles such as nuclei, mitochondria, and plastids). The cellulose cell walls were always free from anti-birch-pollen IgG-binding sites. The immunological controls (normal rabbit IgG) showed a low degree of nonspecific labeling. In plant tissues belonging to genera quite different from birch (tulip anther, rhododendron leaves), after incubation with the specific IgG weak labeling was observed. The immunological basis for these results is discussed.     

Toxoplasma gondii: ultrastructure and antigenicity of purified tachyzoite pellicle.

When purified Toxoplasma gondii tachyzoites were treated with hemolysin, DNase, and RNase, the organisms yielded a three-component system containing the outer membrane (pellicle), microtubules, and conoid in relatively normal morphological configuration. Further treatment of this preparation with protease digested all but the pellicle which was more collapsed in appearance. These two preparations were used in rabbit anti-toxoplasma and goat anti-rabbit ferritin labeling experiments. The three-component system showed ferritin label on the conoid and equal ferritin label on the outer and inner surfaces of the pellicle. The microtubules were unlabeled. The pellicle after protease treatment was labeled equally on its outer and inner surfaces, which indicated that the rabbit anti-toxoplasma serum contained antibodies against antigens on the outer and inner surfaces of the pellicle.     

Localization of prostaglandin on the plasmalemma of rabbit sperm.

Prostaglandin and prostaglandin receptors were localized on unwashed ejaculate rabbit sperm utilizing ferritin conjugated antibody to prostaglandin. Reaction was obtained over the acrosomal portion of the plasma membrane, with a greater aggregation over the apex of the head. The remainder of the head plasmalemma and flagellum showed no deposits of of reaction product. The reaction did not occur if the sperm were washed prior to incubation with the labelled antisera. However enhanced binding of ferritin was demonstrated if washed sperm were pre-incubated in PGE1. No reaction was seen in similar studies with human sperm.     

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus

E. YOSHIDA, Y. ICHIMAN, M. SUGANUMA AND K. YOSHIDA. 1991. Repeated subculture at 42°C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsules surrounded by ferritin granules were demonstrated by electron microscopy. In variants ST67L and ST67S, but not ST67C medium size capsules surrounded by ferritin granules and only ferritin granules located around the cell wall, respectively, were observed.     

MEMBRANE LESIONS IN IMMUNE LYSIS : Surface Rings, Globule Aggregates, and Transient Openings

It is known that there are 100 Å-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 Å-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K⁺, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.     

Combined Use of Immunoferritin and Immunocolloidal Gold for the Simultaneous Demonstration of Two Antigens by Immuno-Electron Microscopy

In this report we describe a double-label bridging technique for the simultaneous visualization of two cell surface antigens by immuno-electron microscopy. Mouse IgG-labeled horse spleen ferritin and rabbit IgG-labeled gold sols served as the electron dense marker-conjugates. The technique was found to discriminate reproducibly two cell surface antigens simultaneously in two separate murine systems, one an in vivo cell line and the other an in vitro cell line. The technique described in this report differs from other double-labeling procedures in two notable ways: (1) the electron dense markers are easily distinguished from one another, and (2) the primary and link antibodies are not modified.     

Steric hindrance as a factor in the reaction of labeled antibody with cell surface antigenic determinants.

The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding.     

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus

Repeated subculture at 42 degrees C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsule surrounded by ferritin granules were demonstrated by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

Summary ¹²⁵I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 11.4, contained over 90% ferritin-IgG monomers; 70–90% of the ¹²⁵I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.Dr. Rudick is the recipient of Teacher-Investigator Development Award PHS 1 KO7 NS 00791-01     

Intracellular labeling with ferritin conjugates. A specificity problem due to the affinity of unconjugated ferritin for selected intracellular sites.

Nonspecific binding of ferritin to chromatin and the cytoplasmic aspect of the nuclear envelope was observed when nonantigenic, serum-washed hepatocyte nuclei were incubated in ferritin-antibody conjugates. This labeling was duplicated when nuclei from a wide range of species and cell types were exposed to unconjugated ferritin. Unconjugated ferritin binding to nuclei did not depend on a subpopulation of denatured molecules or on the ferritin purification procedure. Binding occurred equally on unfixed and formaldehyde-fixed nuclei, but no ferritin bound to glutaraldehyde-fixed nuclei. Inconjugated ferritin also bound to the cytoplasmic aspects of the rough endoplasmic reticulum and the plasma membrane. The tracer did not bind to lysosomes, mitochondria, Golgi vesicles, the extracellular surface of plasma membranes, or the intracisternal surfaces of ruptured nuclear envelopes. The addition of 0.4 M KCl or 0.7 M NaCl to ferritin solutions and washing media at neutral pH reduced the binding of conjugated and unconjugated ferritin to nuclei to about 3% of that seen in 0.10 M phosphate buffer alone. The added salts caused little extraction of nuclear contents from formaldehyde-fixed nuclei. The use of one of these salts in ferritin conjugates should considerably improve the specificity of intracellular labeling.     

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.     

Subcellular localization of cellulases in auxin-treated pea

Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Gogli complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.