Showing papers on "Protein A published in 1987"

Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.

Abstract: Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.

Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.

Abstract: The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus

Abstract: The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.

A model of protein-colloidal gold interactions.

Abstract: We prepared homogeneous populations of colloidal gold particles of various sizes. These were analyzed for size distribution and number of particles per unit volume. On exposure to increasing concentrations of insulin, myoglobin, protein A, peroxidase, serum albumin, galactosylated serum albumin, lactoferrin, transferrin, catalase, low-density lipoprotein, ferritin, and polymeric IgA, protein binding was a saturable process. Using serum albumin, we verified that a reversible equilibrium was reached within 15 minutes. Scatchard analysis of the interactions between all of these proteins and the gold particles resulted in a single component, linear relation. For a given particle size, the number of binding sites for various proteins was inversely proportional to their molecular weight. Conversely, when the size of particles was varied, the number of binding sites was directly proportional to the average area of each gold particle. All results are compatible with a monomolecular shell of protein surrounding the particle at saturation, the binding capacity being inversely proportional to the projection area of the protein. We present direct morphological evidence for this model. The affinity of the various proteins for the colloid also increased with molecular weight, and was not related to the protein isoelectric point. For globular proteins, the monomolecular shell model makes possible prediction of the number of molecules that will saturate a gold particle, if the average diameter of the gold particles and the molecular weight of the protein are known.

Secretion of a chimeric T-cell receptor-immunoglobulin protein.

Abstract: To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable.

The 87-kDa protein, a major specific substrate for protein kinase C: purification from bovine brain and characterization

Abstract: The 87-kDa protein, a major specific substrate for protein kinase C, has been purified 500-fold to apparent homogeneity from bovine forebrain supernatant. The purification procedure included batch adsorption to DE-52 (DEAE-cellulose), (NH4)2SO4 precipitation, and chromatography on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Sephacryl S-400, and fast protein liquid chromatography ProRPC. The amino acid composition was notable for its high proportion of alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline (11.3 mol%). The partial specific volume was 0.702 ml/g; the Stokes radius and sedimentation coefficient were 85 A and 2.11 S, respectively. Although the relative molecular mass of the protein on NaDodSO4/8% PAGE was 87-90 kDa, the molecular mass as determined from the above values was 68 kDa. The frictional ratio was 3.2, and the axial ratio was 60, indicating that the 87-kDa protein is an extremely elongated monomer. The purified 87-kDa protein was phosphorylated by purified protein kinase C to a stoichiometry of 2.2 mol of 32P per mol of 87-kDa protein (calculated using a value of 68 kDa for the molecular mass). Phosphorylation was exclusively on serine residues.

Protection of infant rats from Haemophilus influenzae type b infection by antiserum to purified outer membrane protein a.

Abstract: Protein a (46,000 molecular weight [46K]) was purified from outer membranes of Haemophilus influenzae type b by a relatively simple procedure. Spontaneously shed outer membranes from a 24-h, 12-liter culture of an unencapsulated variant of strain Eag were combined with outer membranes released from the cells by Tris buffer and extracted with the nonionic detergent octylpolyoxyethylene. The extract was then subjected to open column chromatography on Sephacryl S-200 and Trisacryl-carboxymethyl to yield 7.5 mg of protein a from 180 mg of outer membrane protein. Approximately 99% of the protein in this preparation was protein a; in addition, the preparation contained 1.25% (wt/wt) lipopolysaccharide and had a residual detergent/protein ratio of 1.6:1 (wt/wt). Antibodies to the preparation were induced in rabbits by using alum as an adjuvant. As determined by immunoblotting, the great preponderance of antibodies induced were specific for protein a. However, very low levels of antibodies to several other outer membrane components, which were not apparent on gels of the pure preparation of protein a, were also induced. Preimmune and postimmune sera, after depletion of antibodies to capsular polysaccharide and lipopolysaccharide, were tested for biological activity against H. influenzae type b. Compared with preimmune serum, postimmune serum was bactericidal in vitro against strain Eag (the only strain tested) and offered significant protection (P less than 0.01) to infant rats against infection by all four strains tested, two of which had a protein a that was larger (47K) than the 46K protein a in the preparation. These results indicate that protein a should be considered as a vaccine to prevent H. influenzae type b disease.

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

Abstract: We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction of the protein G-gold complex for high-resolution immunocytochemistry

Abstract: Protein G, an immunoglobulin G-binding molecule, was tagged with colloidal gold particles and was applied in immunocytochemistry. The present study reports the conditions required for the preparation of the protein G–gold complex and for its application in electron microscopy. Once used in combination with specific primary antibodies, the protein G–gold complex was able to yield highly specific labelings over particular tissue compartments. Various control experiments confirmed the specificity of the labelings obtained. Since the IgG-binding properties of protein G are superior to those displayed by protein A, the protein G–gold appears a far better tool for high-resolution immunocytochemistry.

Analysis by gel electrophoresis, Western blot, and peptide mapping of protein A heterogeneity in Staphylococcus aureus strains.

Abstract: To evaluate potential differences in protein A among Staphylococcus aureus strains, lysostaphin-solubilized cell wall proteins from 12 serologically distinct strains were analyzed by 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Seven presumptive protein A variant identified in the 45- to 57-kilodalton range were then studied for qualitative binding affinities to nonimmune mouse and rabbit immunoglobulin G (IgG) by enzyme-linked immunoelectrotransfer blot. Essentially, all presumptive protein A variants demonstrated binding to both nonimmune rabbit and mouse IgG and had differential binding to mouse monoclonal IgG1 at pH 8.2 than at 5.5. Because of Fc-binding properties and molecular weight similarity to the well-characterized Cowan I protein A, these proteins appeared to represent protein A variants. Amino sugar analysis (less than 1%) by reverse-phase high-pressure liquid chromatography suggested that the apparent molecular weight differences in protein A were not due to associated mucopeptides. Further differences in protein A variants were studied by peptide mapping. Each of the seven protein A variants, distinguishable on SDS-PAGE, also produced distinct peptide cleavage patterns. In addition, two protein A variants indistinguishable on SDS-PAGE could be further subdivided by peptide mapping. These results suggest that SDS-PAGE analysis of protein A, particularly in conjunction with peptide mapping, may be useful in distinguishing distinct strains of S. aureus. Different protein A variants may also have unique functional or immunologic capabilities. Images

Monoclonal antibodies as probes for processing of the mosquito yolk protein; a high-resolution immunolocalization of secretory and accumulative pathways.

Abstract: A library of monoclonal antibodies (mAB) directed against yolk polypeptides of the mosquito Aedes aegypti was utilized to visualize the secretory pathway of these polypeptides in the fat body and their accumulative pathway in developing oocytes. Single and double immunolabelling using mABs and colloidal gold of different sizes confirmed biochemical observation that 200 ± 5 and 65 ± 3 kDa polypeptides represent subunits of the yolk protein. This immunocytochemical analysis showed that, in trophocytes of the fat body, both the subunits of the yolk protein were routed simultaneously through the Golgi complex into secretory granules and were subsequently secreted. The yolk protein subunits were also directed together through all the steps of the accumulative pathway in the oocyte. Double immunogold labelling revealed that the subunits were present together during their binding to the oocyte membrane, transportation into and accumulation in the transitional yolk body, and, finally, crystallization in the mature yolk body. Electron microscopical immunocytochemistry also confirmed immunofluorescent data and showed that mABs directed against different steps in the biosynthetic processing of the yolk protein in the fat body, as well as in its accumulative pathway in oocytes.

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice.

Abstract: Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.

Antibody-directed Targeting of Liposomes to Human Cell Lines: Role of Binding and Internalization on Growth Inhibition

Abstract: Small unilamellar liposomes containing methotrexate or methotrexate-gamma-aspartate were conjugated to Staphylococcus aureus protein A and were thus able to bind cell-specific immunoglobulins for targeting to malignant human B- and T-cell lines. We were able to demonstrate enhanced protein A liposome uptake and growth inhibition by targeting with an anti-major histocompatibility complex class II antibody recognizing two different B-cell lines. The enhanced growth inhibition was specific for the targeting antibody and amounted to a 2- to 3-fold lowering of the concentration of drug required to inhibit cell growth by 50% as compared to nontargeted liposomes or liposomes targeted with an antibody not recognizing a cell surface antigen. A strong association between enhanced growth inhibition and liposome internalization as assessed by fluorescent-activated cell sorter analysis of carboxyfluorescein containing protein A liposomes was seen. By contrast, specific enhancement of growth inhibition was not seen with several anti-idiotype antibodies or antibodies to T-cell differentiation antigens. Liposome internalization did not occur with these antibodies. Failure of growth inhibition and PA liposome internalization could not be explained by differences in cell binding of the antibody PA liposomes or the degree of protein A binding of the targeting antibody. Although the ability of the targeting antibody to bind to the cell and to protein A are important, these factors alone are not sufficient to guarantee internalization and growth inhibition. Variations in rates of internalization of various cell surface antigen-antibody complexes may account for different protein A liposome mediated cytotoxicities.

Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method

Abstract: We describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein-conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification. In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC-conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies (mAb) against H-2K and H-2D, respectively. When RDM4 cells were treated with anti-H-2K mAb followed by FITC-conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively. Using the liposome conjugates, we were also able to detect with ease the 2,600 binding sites for the anti-H-2D mAb, whereas the FITC conjugate failed completely to resolve specific binding from background. We estimate conservatively that by using this methodology, it will be possible to detect as few as 800 binding sites percell.

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Abstract: The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the π protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, π of R6K or protein RepH encoded by and iniating the replication of pC194.

Protein a domain mutants

Abstract: The subject invention concerns novel protein A or protein A-like molecules that can be coupled to other materials through a single, defined site on the protein A molecule. Specifically exemplified is Cysteinyl-rProtein A™. The compounds of the invention, for example, Cysteinyl-rProtein A™, can be used in processes wherein protein A is used.

Surface modification with protein A for uniform binding of monoclonal antibodies

Abstract: We describe optimal conditions for immobilization of two monoclonal antibodies to progesterone for solid-phase assays. Polystyrene surfaces are refined with Protein A to achieve uniform, reproducible, stable, and sterically accessible immobilization of immunoglobulins (IgG). To this end, we optimized the amount of immobilized Protein A, the pH of the medium for immobilization, the concentration of antibody, and the polystyrene surface. We also investigated three carriers for solid-phase assays: 12 X 75 mm polystyrene test tubes, Macrowells (Skatron, Inc.; suitable for processing with multiple pipettors), and microwell strips (Immulon II, Dynatech Inc.). Immunoglobulin does not appreciably dissociate from any of these solid matrices, even if the assay procedure takes several hours. Therefore, we postulate that more than one molecule of immobilized Protein A binds to IgG, or that there is an additional interaction between the antibody and the polymer surface.

Presence of fucolipid antigens with mono- and dimeric X determinant (Lex) in the circulating immune complexes of patients with adenocarcinoma.

Abstract: A series of fucosylated glycosphingolipids with the Lewisx (Lex) determinant (Galβ1→4[Fucα1→3]GlcNAc) have been shown to accumulate in human adenocarcinomas. Lex glycolipids were eluted from Protein A-silica columns over which plasma from patients with adenocarcinoma had previously been perfused. The fact that Protein A has strong affinity for IgG and IgG-immune complexes suggested that the Lex antigens isolated from Protein A eluates were complexed with IgG. Lewisx antigen eluted from Protein A columns banded in the immune complex-enriched region (below IgG) of neutral sucrose density gradients. A modified Raji cell assay and an anticomplement C1q enzyme-linked immunosorbent assay were also used for measurement of Lex antigen associated with C3- and C1q-CIC, respectively. Following affinity purification of Lex-IgG complexes and subsequent dissociation of these immune complexes, human antibodies were isolated which reacted with purified glycosphingolipids containing Lex. Levels of Lex-IgG complexes were found to be 2- to 5-fold higher in eluates of Protein A-silica columns perfused with plasma from adenocarcinoma patients compared to eluates from columns perfused with plasma from healthy individuals and patients with other cancers. These assays may prove to be of diagnostic and/or prognostic significance in adenocarcinoma.

Cross-reactivity of surface-exposed epitopes of outer membrane antigens of Haemophilus influenzae type b.

Abstract: The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.