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At least 19 recordsLinked to original sources
PubMed1975

Demonstration and assaying of IgG antibodies in tissues and on cells by labeled staphylococcal protein A.

Staphylococcal protein A (SpA) labeled with FITC was used to detect IgG antibodies to tissue and cell surface antigens. Radioiodinated SpA was employed in a radioimmuno technique to assay IgG antibodies which had reacted with cell membrane antigens. Red blood cells coated with SpA were used as an indicator system in the mixed haemadsorption technique to detect IgG antibodies on tissue culture cells. The results showed that SpA was highly specific in the various systems tested, although it was not as sensitive in the immunofluorescence test as FITC anti-IgG. It was confirmed that SpA reacts with IgG antibodies of human, monkey, rabbit, sheep and mice origin. The reactivity with human and monkey IgG was stronger than with IgG of the other species. We consider labeled SpA as an alternative to monospecific anti-IgG for detection and assaying of IgG antibodies to tissue and cell surface antigens.

Adrenal Glands↗
PubMed1992

Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.

Amino Acid Sequence↗
PubMed1992

Production and secretion of a bifunctional staphylococcal protein A::antiphytochrome single-chain Fv fusion protein in Escherichia coli.

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.

Animals↗
PubMed1992

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.

Amino Acid Sequence↗
PubMed1978

The binding of murine immunoglobulins to staphylococcal protein A.

The binding of murine IgG1, IgG2a, IgG2b, IgG3 protein molecules to Staphylococcal A protein is presented. Differential elution by sodium thiocyanate gradients is described. Proteins of the IgG2 class (either a or b subclass) require between 1.5 and 2.0 M for complete elution, whereas, IgG1 proteins are fully eluted with only 0.5 M NaSCN. These differential elution patterns can be utilized to distinguish between, or prepare proteins of different Ig classes. It is proposed that this quantitative rather than qualitative distinction between Ig subclass binding to Staph A, indicates the existence of multiple binding sites per molecule, with different markers of such sites being present on the heavy chain of the different Ig classes.

Animals↗
PubMed1992

An unexpected complication following immunoadsorption with a staphylococcal protein A column.

Extracorporeal immune adsorption with staphylococcal protein A (SPA) columns can remove immune complexes and immunoglobulins in the treatment of a variety of diseases. We present the case of an elderly man with neuropathy associated with monoclonal gammopathy, treated by 3 on-line SPA procedures. At the completion of these treatments his neuropathy relapsed, progressing to near-total paralysis. Return to a baseline clinical status required several months. The reason for this severe relapse is not clear. Possible explanations include SPA activation of T-lymphocytes, with release of gamma interferon and increased antigen recognition, or removal of an antiidiotype control mechanism. We advise caution in the application of immunoadsorption to conditions in which it has not yet been evaluated.

Hereditary Sensory and Motor Neuropathy↗
PubMed1992

Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A.

A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.

Amino Acid Sequence↗
PubMed1976

Immunological studies on LATS-immunogloblin by the reaction with staphylococcal protein A.

The reaction of LATS activity with Staphylococcal Protein A, a specific binding protein with the Fc part of human IgG(1), IgG(2) and Ig(4), was examined. When IgG(1), IgG(2) and Ig(4) subclasses were removed from LATS positive sera or LATS-IgG fractions by affinity chromatography on Protein A-Sepharose, LATS activity decreased. Almost all LATS activity was found in the fraction that reacted with Protein A. It is suggested that LATS has an expression of a very distinct immunoglobulin G structure, and that LATS activity is distributed mainly in the fraction containing IgG(1), Ig(2) and Ig(4) in LATS positive serum;

Bacterial Proteins↗
PubMed1992

Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A.

In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage lambda pR promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42 degrees C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved.

Amino Acid Sequence↗
PubMed1979

The use and limitation of labeled staphylococcal protein A for study of antineutrophil antibodies.

Antineutrophil antibodies can be detected following their attachment to neutrophils by employing labeled staphylococcal protein A (SPA). Radiolabeled SPA provides a sensitive means for identifying the presence of IgG restricted to subclasses IgG1, IgG2, and IgG4 that will specifically bind to neutrophils and that are found in the serum of patients with isoimmune and autoimmune neutropenia. However, SPA bound to the Fc region of IgG does not interfere with the attachment of IgG to the Fc domain of the neutrophil. Fluorescein-labeled SPA, in turn is useful in monitoring the functional consequences of antibody attachment to the surface of polymorphonuclear leukocytes (PMN). Both heterologous and isoimmune antisera induced lateral movement of surface antigens into polar-capped pseudopodia. The formation of such pseudopodia may facilitate leukoagglutination and the subsequent removal of sensitized cells from the circulation.

Animals↗
PubMed1979

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.

Animals↗
PubMed1979

The use of staphylococcal protein A in the mixed agglutination test.

A mixed agglutination (MA) test employing staphylococcal protein A (SpA) instead of an antiglobulin reagent has been developed for the detection of IgG antibodies bound to cell-surface antigens. In the SpA MA test, a single indicator system may be used for the detection of IgG antibodies from several mammalian species. The sensitivity of this test compares favorably with that of the conventional MA test in the detection of most mammalian IgG. The main advantage of the conventional SpA MA test is its usefulness in the study of antigens on cells because of adherence of antibody-coated indicator erythrocytes.

Animals↗
PubMed1992

Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.

The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1978

Use of staphylococcal protein A as an immunological reagent.

This brief review summarises the major uses of staphylococcal protein A in immunology. Protein A is covalently linked to the cell wall of most strains of Staphylococcus aureus, and binds immunoglobulin molecules with high affinity. The principal molecule bo-nd is IgG, although in many cases binding is restricted to certain IgG subclasses. Some IgM and IgA binds in certain species. This property allows rapid, simple and economical methods for the purification and analysis of immunoglobulins, and the fractionation of subclasses which are difficult to separate by other means. Fractionation on protein A affinity columns is a simple and efficient way of separating immunoglobulin F (ab) and F (ab')2 from Fc fragments. Intact staphylococci are useful as a solid phase adsorbent for isolating antigen-antibody complexes, membrane antigens and receptors, and to replace 'second antibody' in radioimmunoassay. Finally, protein A has proven useful for the study of antigens and receptors on the surface of intact cells, and for the detection of antibody-secreting cells. Thus, the use of protein A is now the method of choice for many preparative and analytical purposes in immunology.

Animals↗
PubMed1979

[Use of staphylococcal protein A for the detection of rubella virus IgM antibodies].

The ability of Staphylococcus aureus protein A to bind human serum immunoglobulin of different classes and the conditions which allow a selective reduction of IgG in comparison to other immunoglobulin classes were preliminarly studied. Working under standardized conditions, a recovery of 3.5% of the initial value for IgG, of 40-50% for IgM and of 70-75% for IgA was obtained. Rubella HI antibodies, before and after staphylococcus treatment, were titred in 88 sera collected from adult women at different times from hexantema and in 210 sera of 109 subjects exposed to eventual contamination. The results were compared with the ones obtained by means of sucrose density gradient centrifugation followed by titration of rubella HI antibodies in the fractions. The tests proved that HI antibodies titration, before and after serum treatment with staphylococcal protein A, can be used, as presumptive test, for the research of rubella specific IgM antibodies. In fact, all the sera, on which the presence of rubella IgM antibodies was proved by sucrose density gradient centrifugation, were also positive after staphylococcus treatment. In 15% of sera resulting positive for HI rubella antibodies after staphylococcus treatment, usually at low titer, the presence of specific IgM antibodies was not detected by serum fractionation on sucrose density gradient ("false positive" cases). The results of serological diagnosis, carried out by using treatment with staphylococcal protein A and serum fractionation on sucrose density gradient, matched the clinical-epidemiological data. Twenty-seven women, out of 28 examined during the first three months of pregnancy and for whom serological diagnosis of a "recent" rubella virus infection was negative, had normal children at normal time. Therefore only in one case a spontaneous abortion occurred for uncertain causes. Rubella virus was isolated from the embryo of a woman who interrupted pregnancy because asymptomatic rubella virus infection was serologically diagnosed in the second month.

Antibodies, Viral↗
PubMed1992

Efficient thyroid hormone formation by in vitro iodination of a segment of rat thyroglobulin fused to Staphylococcal protein A.

A polypeptide of 224 amino acids from the C terminus of rat thyroglobulin fused to Staphylococcal protein A (TgC 224), containing 3 tyrosines which have been shown to be hormonogenic in vivo (Tyr-2555, -2569 and -2748), forms thyroid hormones with relatively high efficiency upon in vitro enzymatic iodination using, most likely, the hormonogenic Tyr-2555 and Tyr-2569. Acetylcholinesterase, which has sequence and structural homology with the C terminus of the thyroglobulin molecule and bovine serum albumin, used as control proteins, formed thyroid hormones with lower efficiency. These results validate our experimental approach to define the structural requirements for thyroid hormone formation using thyroglobulin fragments.

Amino Acid Sequence↗
PubMed1977

Failure of the reaction of beta2-microglobulin with staphylococcal protein A.

Because beta2-microglobulin is structurally similar to IgG, the reaction of beta2-microglobulin with Staphylococcal Protein A, which is known to react with the Fc region of IgG, was examined. 125I-beta2-microglobulin did not bind to Protein A. This may due to the difference in the amino acid sequence between beta2-microglobulin and the Fc region of IgG.

Beta-Globulins↗