| 1. |
- Carlson, M, et al.
(författare)
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Human eosinophil peroxidase : purification and characterization
- 1985
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 134:3, s. 1875-1879
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Tidskriftsartikel (refereegranskat)abstract
- Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.
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| 2. |
- Nilsson, B, et al.
(författare)
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Anti-idiotypic antibodies in antisera against human C3 and factor H and their application in the enrichment of antibodies specific for H-binding domains of C3.
- 1987
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 138:6, s. 1858-1863
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Tidskriftsartikel (refereegranskat)abstract
- Antisera separately raised against C3 and factor H should contain antibodies against the binding domains by which these factors interact. Because these interacting domains are likely to be sterically complementary to each other, antibodies specific for these domains should also be sterically complementary or of anti-idiotypic specificity. To test this hypothesis, rabbit anti-human C3 IgG antibodies which bound to Sepharose-coupled rabbit anti-factor H IgG (anti-H-binding anti-C3) were separated by affinity chromatography. In a control experiment, the anti-human factor H column was found to bind 10 times more anti-C3 antibodies than a Sepharose column coupled with nonimmune rabbit IgG. The binding specificity of the control eluate was indistinguishable from the original anti-C3 preparation, whereas the anti-H-binding anti-C3 was shown to be enriched in regard to antibodies directed against the 42 kd fragment of the C3 alpha-chain of C3c, as demonstrated by immunoblotting of electrophoretically separated polypeptides of C3c and C3d in polyacrylamide gels. In competition binding studies, factor H was 25 times more potent as inhibitor of the anti-H-binding anti-C3 than of the original anti-C3 preparation. This similarity in binding specificity also paralleled a functional similarity in that anti-H-binding anti-C3 in the presence of factor I effected a 20% degradation of the alpha'-chain of C3b into fragments of 65, 45, and 42 kd which are the normal degradation products in the presence of factors I and H. These results suggest that the affinity procedure described resulted in the enrichment of anti-C3 antibodies with selective specificities for factor H-binding domains of C3, from a polyclonal antibody source.
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| 3. |
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| 4. |
- Sjöbring, U, et al.
(författare)
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Ig-binding bacterial proteins also bind proteinase inhibitors
- 1989
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Ingår i: Journal of immunology. - 0022-1767. ; 143:9, s. 54-2948
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Tidskriftsartikel (refereegranskat)abstract
- Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.
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| 5. |
- Sjöbring, U, et al.
(författare)
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Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G
- 1988
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Ingår i: Journal of immunology. - 0022-1767. ; 140:5, s. 9-1595
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Tidskriftsartikel (refereegranskat)abstract
- Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.
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