Showing papers on "Protein A published in 1984"

Region X, the cell-wall-attachment part of staphylococcal protein A

Abstract: The sequence of region X of staphylococcal protein A has been determined. The hypothesis has been put forward that this region spans the Staphylococcus aureus cell wall and is responsible for the binding to the peptidoglycan. The primary amino acid sequence of region X was determined for two strains exhibiting cell-wall-bound protein A, Cowan I and 8325–4. The sequence determination of the Cowan I material is partial and was performed by Edman degradation, in contrast to the sequence of the 8325–4 material which was completely analyzed by nucleotide sequencing of the corresponding gene. The region consists of two structurally different domains, a highly repetitive region (Xr), with an octapeptide structure repeated approximately 12 times, and a C-terminal domain (Xc) with an unique sequence. A comparison between the two strains reveals a high mutual homology as well as a high internal homology between the octapeptide structures. Six out of eight amino acids are identical in the repetition of this structure throughout region Xr in both proteins and the other two are changed in a rather regular pattern.

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes.

Abstract: Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A.

Abstract: The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.

Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci.

Abstract: Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676. Images

Staphylococcal protein A binding to the Fab fragments of mouse monoclonal antibodies.

Abstract: Two mouse IgG1 monoclonal antibodies specific for the Lewis(a) human blood group antigen were purified on protein A-Sepharose by using buffers of decreasing pH for elution. Unlike other IgG1 antibodies that eluted at pH 7.0 to 6.0, these antibodies could only be eluted at pH 4.0 to 3.0. The Fab and F(ab')2 fragments of these antibodies also eluted at pH 4.0 to 3.0, although the Fc fragment of one eluted at pH 6.0. This interaction of protein A with Fab was not due to anti-protein A antibody activity, because the presence of Lewis(a) trisaccharide did not prevent the binding of Fab to protein A-Sepharose and because Fab that had bound to solid phase hapten could still be recognized by protein A. Thus, certain mouse IgG1 antibodies possess determinants in their Fab portion recognized by protein A, allowing for the purification of such Fab fragments on protein A-Sepharose.

Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens

Abstract: The use of two new reagents, biotin-avidin conjugates and colloidal gold particles, for ultrastructural immunocytochemistry has been reviewed, and their potential for immunocytochemistry in the central nervous system has been discussed. Although these compounds have been used very little in the C.N.S. in the past, with their high sensitivity, versatility, and potential for simultaneous localization of two antigens with two electron dense markers, these reagents may prove very useful for answering a number of neural questions. Colloidal gold adsorbed to immunoglobulins can be used for light microscopic observation, and owing to its high electron density and ability to readily adsorb to immunoglobulins, protein A, and antigen-carrier complexes, it also serves as an excellent immunomarker at the ultrastructural level. Several procedures have been described for the use of pre- and post-embedding immunostaining with colloidal gold particles. Different size gold particles, adsorbed to different antibodies can be used for pre- and post-embedding staining of different antigens in the same tissue. While at the light microscopic level only the large particles are visible as a pink haze, a fluorescent step can be added to allow easy identification and characterization of neurones labelled with any size colloidal gold, even if the pink colour of the gold is not visible. These same sections can subsequently be studied ultrastructurally. Unlike enzymatic methods, gold particles have less tendency to obscure ultrastructure. Biotin can be easily conjugated to immunoglobulins and, due to its high affinity for avidin (10(15) M-1) which has four biotin binding sites, can be localized with avidin markers, or biotin-avidin-marker complexes. Some procedures such as the avidin-biotin-peroxidase complex are extremely sensitive. Biotin and avidin can be bound to a number of different markers including peroxidase, colloidal gold, and ferritin. Photomicrographs from our hypothalamic work have been used to illustrate the histological results of some of the procedures described.

Process for binding IgG to protein A

Abstract: Unusually strong binding of IgG to protein A is achieved by contacting these components in the presence of a medium containing a high concentration of salt. Although such binding is of general utility, a particularly useful application is the purification of monoclonal antibodies from ascites fluid by affinity chromatography.

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder II. Identification of the cellular target structures by immunoprecipitation and SDS-PAGE analysis

Abstract: The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads. The precipitates were separated by sodium dodecyl sulfate- gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. The antibodies 4B5, 7E9, and 14B11 have previously been found to react in a similar way with TCC-targets and some non-TCC tumor cells, but not with normal urothelial cells or cells of hematopoietic origin. When tested with lysates of a TCC-cell line (TCCSuP) a strong 92K band and a weak 23K band were precipitated with any one of these antibodies. These polypeptides were expressed on the cell surface and were not linked by disulfide bonds. Depletion experiments confirmed that the three antibodies recognized the same antigens. Another antibody (4E8) probably directed to a differentiation antigen present on both urothelial and melanoma cells detected two high molecular polypeptides, 190K and 170K. Antibodies from the S2C6 hybridoma, which displayed a distinct dual specificity for TCC- targets and for malignant or transformed cells of B cell origin, precipitated a 50K component from extracts of either TCC- or B cell-derived cell lines. Antibodies produced by the S2A9 hybridoma were shown to bind to a framework epitope of the HLA-A, B, C heavy chain.

Two-dimensional electrophoresis of cerebrospinal fluid and ventricular fluid proteins, identification of enriched and unique proteins, and comparison with serum.

Abstract: The proteins of cerebrospinal fluid (CSF) and ventricular fluid have been analyzed by two-dimensional electrophoresis (2DE) and the patterns compared with autologous serum. Fourteen proteins were specifically identified by immunoprecipitation followed by 2DE, or by blotting 2DE gels to nitrocellulose and detection by peroxidase staining. Proteins in CSF and serum with high and low affinity for the ligands, protein A, Cibacron Blue, and concanavalin A, were also characterized by 2DE. The 2DE profiles of CSF and serum proteins were similar and indicated that a relatively nonselective filtration mechanism based on protein size is the major determinant for the overall pattern of CSF proteins. The classic CSF-enriched or CSF-specific proteins, beta-trace, prealbumin, transferrin, and beta-2-microglobulin, were identified according to 2DE coordinates. Charge differences between CSF and serum for transferrin and prealbumin were identified. In addition, a large number of additional CSF-enriched or CSF-specific proteins of high, intermediate, and low molecular weight, all predominantly anodic in mobility, were identified. Three acidic protein complexes, heterogeneous in charge and molecular weight, were characterized as constituents of normal CSF, and two of these are increased in patients with inflammatory diseases of the CNS. All three proteins and several other proteins unique to CSF bound to Cibacron Blue-Sepharose. The use of 2DE in conjunction with affinity chromatography and sensitive protein stains enlarged the number of proteins previously identified as unique to CSF. By a modified 2DE and silver staining procedure, most of these proteins were visible without prior concentration of CSF.

Nucleic acid-protein conjugate used in immunoassay

Abstract: A protein is covalently coupled to a 3'terminal end of a nucleic acid which carries several labels. In an assay the protein will specifically recognize some component of a test system; in an immunoassay the protein can be Protein A which will recognize the FC portion of IgG which is bound to an unknown antigen if present in the test sample.

Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins.

Abstract: Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.

Generation of human C3a, C4a, and C5a anaphylatoxins by protein A of Staphylococcus aureus and immobilized protein A reagents used in serotherapy of cancer.

Abstract: Protein A (SpA) alone or immobilized on bacteria (e.g., Cowan strain I), collodion charcoal, or on Sepharose have been used in serotherapy of cancer in humans and experimental animals. Because SpA forms complexes with IgG that can activate complement, and the physiologic response during treatment often involves hypocomplementemia and reactions that are similar to those induced by anaphylatoxins, we used sensitive and specific radioimmunoassays to test the ability of SpA reagents to generate C3a, C4a, and C5a from human serum. The yield of anaphylatoxins depended on the dose of SpA, with the maximum generation of C3a (47 to 55 micrograms/ml) and C5a (1.4 to 1.9 micrograms/ml) being produced with levels of SpA that were maximally precipitated from serum. Maximum C4a levels (up to 15 micrograms/ml) were obtained at concentrations of SpA equal to or greater than the dose required to give optimal precipitation. The maximum concentrations of anaphylatoxins correspond to essentially quantitative conversions of C3 to C3a, C4 to C4a, and 40% of C5 to C5a after correction for levels found in serum incubated in pyrogen-free saline. Preformed insoluble complexes prepared from either serum or monomeric IgG also were capable of generating anaphylatoxins in fresh whole serum up to levels approximately equal to those observed in serum treated directly with an optimal amount of SpA. The preformed complexes from serum or IgG generated similar high concentrations of anaphylatoxins when carried through four sequential incubations with fresh serum, and complexes that contained approximately 1 microgram SpA were still active. Preincubating the insoluble complexes with chicken anti-SpA serum did not alter their activity. Incubation of serum with collodion charcoal coated with SpA, in a system that models the perfusion technique used to treat cancer, produced complexes that generated significant levels of C3a compared with levels found in serum passaged over albumin charcoal or in untreated serum. The C3a levels in serum from the albumin collodion charcoal were not significantly different from those found in untreated serum. Similar amounts of C3a, C4a, or C5a were observed in serum incubated with differing numbers of bacteria representing a strain of S. aureus rich in cell bound SpA (Cowan strain I) or a strain (Wood 46) deficient in SpA. This suggests that in intact bacteria, cell wall factors other than SpA (e.g., peptidoglycan) are predominantly responsible for generating anaphylatoxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Precipitation of the Thyrotropin Receptor and Identification of Thyroid Autoantigens using Graves' Disease Immunoglobulins

Abstract: The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor.

Immunoglobulin G- and immunoglobulin E-mediated airway smooth muscle contraction in the guinea pig.

Abstract: Guinea-pig antibody to ovalbumin was separated into two pools using Protein A Sepharose. The antibody pool retained by the Protein A Sepharose [immunoglobulin (Ig) G pool] was heat stable and persisted in skin sites of the guinea pig less than 7 days. The antibody pool which did not bind to Protein A Sepharose (IgE pool) was heat labile and persisted in guinea-pig skin at least 10 days. Both antibody pools were capable of mediating contraction of isolated guinea-pig tracheal and lung parenchymal strips using airway strips passively sensitized in vivo or in vitro. Although the majority of the skin sensitizing ability of the IgE pool was destroyed by heating at 56 degrees C for 4 hr, its ability to mediate airway contraction was only partially inhibited. Experiments examining [125I]Protein A binding of the antibody pools indicated that the contractions seen in airways sensitized with IgE pool was not due to contamination with IgG. These studies provide a system for examining antagonism of and mediators responsible for IgG-vs. IgE-mediated airway contraction and for evaluating the apparently heat stable properties of IgE.

Tumoricidal response following perfusion over immobilized protein A: identification of immunoglobulin oligomers in serum after perfusion and their partial characterization.

Abstract: Previously, we showed that perfusion of plasma from hosts bearing breast adenocarcinoma over immobilized staphylococcal protein A resulted in objective tumor regressions. In the present study, sera perfused in vitro over immobilized staphylococcal protein A were analyzed by physicochemical and immunochemical methods to characterize newly formed products. Sera from normal and breast adenocarcinoma-bearing dogs showed increased levels of C1q-binding IgG after perfusion over a strain of staphylococcus that is protein A rich (Cowan I), but not protein A deficient (Woods 46). C1q binding levels were also increased in normal and tumor-bearing canine or human sera which were perfused over purified protein A immobilized in collodion charcoal (PACC), and this increase was localized in sucrose density gradient fractions ranging from 7S to 19S. Polyacrylamide gel electrophoresis analysis of the high-molecular-weight fraction in postperfusion canine sera, isolated by G-200 fractionation and immunoaffinity chromatography, showed predominantly heavy and light immunoglobulin chains of canine IgG. Furthermore, protein A was released from PACC after perfusion with serum or solutions containing IgG or albumin from humans, dogs, and chickens. After serum perfusion over PACC, protein A was identified in the effluent by additional studies as follows: ( a ) polyacrylamide gel electrophoresis analysis showed that eluted 125I-protein A comigrated with the protein A marker; ( b ) postperfusion C1q-binding complexes, isolated by gel filtration under dissociating conditions and affinity chromatography on IgG-Sepharose showed a single precipitin band with normal human (protein A reactive) but not chicken (protein A unreactive) serum. Protein A released from PACC which appeared in postperfusion sera was associated with immunoglobulins in macromolecular complexes since ( a ) eluted 125I-protein A was largely (NH4)2SO4 and polyethylene glycol precipitable, whereas free protein A was not, and it sedimented in sucrose density gradient fractions distributed beyond the 7S marker, compared to free protein A which localized below 7S; ( b ) radiolabeled protein A eluting from PACC after serum perfusion showed 8-fold greater binding to C1q-coated tubes compared to free protein A; and ( c ) increased C1q-binding IgG in postperfusion sucrose density gradient fractions corresponded to the appearance of protein A in parallel gradient fractions. In addition to protein A, analysis of the components of postperfusion complexes in human and canine sera bound to solid-phase C1q using radiolabeled monospecific F(ab′)2 fragments demonstrated IgG and IgM in canine complexes, while human complexes contained IgG, IgA, IgM, and C3. Human and canine serum containing a trace amount of 131I-IgG were passaged over PACC containing 125I-protein A, and the effluents were chromatographed on Sepharose 6B. By use of molecular weight standards, the molecular weight of complexes ranged from 600,000 to 2,000,000. Hence, perfusion of normal or tumor-bearing sera over Staphylococcus aureus Cowan I strain or PACC results in the formation of C1q binding- and polyethylene glycol-precipitable oligomers composed predominantly of IgG and protein A. These complexes may activate cellular and humoral inflammatory systems after entering the circulation and contribute to the tumoricidal effects and toxicity previously observed after administration of plasma perfused over immobilized staphylococcal protein A.

An immunological method for detecting gene expression in yeast colonies.

Abstract: A method for detection of cloned, expressed genes in yeast colonies has been developed. The 70-kilodalton (kDa) mitochondrial outer membrane protein of yeast was used as a model protein. Transformation of a strain deficient in the gene for the 70-kDa protein was performed, and transformed colonies were detected with the antibody decoration technique. This technique is based upon gentle lysis of yeast colonies that have been grown on nitrocellulose filters such that the yeast proteins remain bound in discrete spots after lysis. The lysis is carried out by alkaline conditions in the presence of 2-mercaptoethanol and sodium dodecyl sulfate. After lysis, empty sites on the nitrocellulose filter are blocked to eliminate nonspecific binding of proteins by either 0.5% bovine serum albumin or 0.05% Tween 20. Decoration with antibody is visualized by using 125I-labeled protein A or peroxidase-conjugated second antibody. Antigens amounting to less than 0.1% of the total protein in the cell can be readily detected by the assay. The sensitivity of the assay enables detection of 1 positive colony per plate containing about 1000 colonies.

Macrophage Fc receptors control infectivity and neutralization of canine distemper virus-antibody complexes.

Abstract: Dogs that are persistently infected or that become moribund after exposure to canine distemper virus (CDV) have antibody that neutralized CDV when tested in dog lung macrophage cultures but failed to neutralize CDV when tested in epithelial, fibroblastic, or lymphatic cells. The antibody attached to protein A and was found in the immunoglobulin G fraction. The antibody bound complement and lysed CDV-infected target cells. The neutralizing activity in macrophages could be abolished (i) by pepsin digestion and removal of Fc portions from the antibody, (ii) by blocking the Fc receptors of macrophages with heat-treated normal dog serum, and (iii) by binding of protein A to Fc portions of the antibody. It was concluded that attachment of the CDV-antibody complex to Fc receptors of macrophages was essential for virus neutralization. If this attachment was hindered, the CDV-antibody complex became infectious for macrophages. In contrast, serum from recovering dogs neutralized CDV when tested in epithelial, fibroblastic, or lymphatic cells as well as in macrophages.

Antitumor activity with nontoxic doses of protein A.

Abstract: This report confirms our previous observation that IV inoculation of purified protein A causes regression of rat mammary adenocarcinomas. In treated tumors, we have obtained histological evidence of changes indicating tumor cell destruction. Protein A treatment does not cause reduction in the body weight or organ weights of rats; nor does it cause any decrease in activity of the enzymes of the microsomal mixed function oxidase system in the liver. Protein A stimulates peripheral white cell counts in normal rats, but not in tumor-bearing rats. We found that protein A infusion reduced (P<0.0005) the level of circulating plasma immune complex concentration. A homing study with 125I-labeled protein A indicated that liver, spleen, and kidney tissues are the major sites of protein A accumulation. Therefore, protein A seemed to exert its antitumor effects without causing any generalized toxicity to the system. It is postulated that the action of protein A may be related to its ability to cause a drastic reduction in circulating plasma immune complex concentration, thus potentiating the immune reactivity of the host observed earlier.

Neurotoxic and haemolytic activity of a protein isolated from Bacillus thuringiensis var. israelensis crystals

Abstract: Two proteins (protein A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. Protein A was found to have haemolytic and neurotoxic activities. Inhibition of nerve conduction in the sixth abdominal ganglion of the American cockroach was observed following application of protein A. Protein A was found to be haemolytic to human and rabbit erythrocytes. Protein B did not exhibit any of these activities.