Use of staphylococcal protein A as an immunological reagent

Conjugation of antibodies with fluorochromes: modifications to the standard methods.

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent.

The adaptive immune system (AIS) in mammals, which is centred on lymphocytes bearing antigen receptors that are generated by somatic recombination, arose approximately 500 million years ago in jawed fish. This intricate defence system consists of many molecules, mechanisms and tissues that are not present in jawless vertebrates. Two macroevolutionary events are believed to have contributed to the genesis of the AIS: the emergence of the recombination-activating gene (RAG) transposon, and two rounds of whole-genome duplication. It has recently been discovered that a non-RAG-based AIS with similarities to the jawed vertebrate AIS — including two lymphoid cell lineages — arose in jawless fish by convergent evolution. We offer insights into the latest advances in this field and speculate on the selective pressures that led to the emergence and maintenance of the AIS.

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Origin and evolution of the adaptive immune system: genetic events and selective pressures

The evolution of adaptive immune systems.

The reaction between protein A of staphylococci and the Fc portion of γG globulins has been studied using representative sera and isolated γ globulins from species representing seven classes and 30 orders of living vertebrates. No reactions with immunoglobulins derived from either the primitive or later evolving fishes were noted. No reactions were recorded among Amphibia and Reptilia as well as all birds tested, except with serum from the primitive flightless bird Rhea americana . All members of the class Mammalia, including egg-laying monotremes, showed reactions with protein A except for the American oppossum. A comparison of patterns of reactivity between protein A and isolated human rheumatoid factors showed that in the majority of instances different specificities were involved. Protein A may provide a useful tool in the study of the evolution of γG globulin.

Phylogenetic insight into evolution of mammalian Fc fragment of gamma G globulin using staphylococcal protein A.

1. Studies of the immune response have been carried out in more than 1700 lampreys representing three stages in the life cycle of these animals. 2. Lampreys used in this study were unable to clear certain soluble protein antigens and bacteriophage and were unable to make antibodies to these antigens. Hemocyanin was cleared from the circulation. 3. The immune responses demonstrated in lampreys include the production of specific antibody to killed Brucella cells, the rejection of skin homografts, and the development of a delayed allergic response to old tuberculin. 4. A responsive proliferation of lymphoid cells occurred in the protovertebral arch following antigen-adjuvant stimulation. 5. Electrophoretic and immunoelectrophoretic analysis of lamprey serum revealed gamma globulin. Ultracentrifugal analysis of serum revealed proteins with sedimentation coefficients of 17S, 8S, 7S, and 3S. 6. The antibodies thus far observed in the lamprey are of relatively high molecular weight and destroyed by 2-mercaptoethanol. 7. In the lamprey it would appear that there is reflected the coordinate evolution of a primitive thymus, primitive spleen containing lymphoid foci, a family of lymphocytes in the peripheral blood and capacity for gamma globulin synthesis and expression of adaptive immunity.

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The evolution of the immune response iii. immunologic responses in the lamprey

A purified cobra venom factor with C-inhibiting activity also promotes lysis of erythrocytes in fresh mammalian serum. Lysis-inducing activity of purified cobra venom factor was found in sera of lower vertebrates including the cyclostome hagfish and in invertebrates. Lysis-inducing activity was most effective with frog serum. Frog serum was found to be more hemolytic for E(s) in the presence of CVF than when cells were sensitized with hemolysin. The hemolysis induced by CVF with frog serum, as in the higher vertebrates, was inhibited when sera were pretreated with known C inhibitors including heat, chelators, endotoxin, immune complexes, and CVF itself. Complexes formed with CVF and either frog serum or invertebrate hemolymph promoted lysis of indicator cells in the presence of frog serum in EDTA. This lysis was most marked when the starfish-CVF complex was used and was C-dependent. Conversely, complex formed with frog serum and CVF promoted lysis of E in the presence of invertebrate hemolymph (Limulus) in EDTA. Hence, serum components were to some degree at least interchangeable between vertebrate sera and invertebrate hemolymph. Lysis-inducing activity of purified CVF occurs in a wide range of species, has revealed activities resembling those of terminal C-components in lower vertebrates and invertebrates, and provides one means for study of C and C-like activities in primitive species.

https://rupress.org/jem/article-pdf/132/5/941/1083908/941.pdf

Complement and complement-like activity in lower vertebrates and invertebrates

1. The California hagfish, Eptatretus stoutii, seems to be completely lacking in adaptive immunity: it forms no detectable circulating antibody despite intensive stimulation with a range of antigens; it does not show reactivity to old tuberculin following sensitization with BCG; and gives no evidence of homograft immunity. 2. Studies on the sea lamprey, Petromyzon marinus, have been limited to the response to bacteriophage T(2) and hemocyanin in small groups of spawning animals. They suggest that the lamprey may have a low degree of immunologic reactivity. 3. One holostean, the bowfin (Amia calva) and the guitarfish (Rhinobatos productus), an elasmobranch, showed a low level of primary response to phage and hemocyanin. The response is slow and antibody levels low. Both the bowfin and the guitarfish showed a vigorous secondary response to phage, but neither showed much enhancement of reactivity to hemocyanin in the secondary response. The bowfin formed precipitating antibody to hemocyanin, but the guitarfish did not. Both hemagglutinating and precipitating antibody to hemocyanin were also observed in the primary response of the black bass. 4. The bowfin was successfully sensitized to Ascaris antigen, and lesions of the delayed type developed after challenge at varying intervals following sensitization. 5. The horned shark (Heterodontus franciscii) regularly cleared hemocyanin from the circulation after both primary and secondary antigenic stimulation, and regularly formed hemagglutinating antibody, but not precipitating antibody, after both primary and secondary stimulation with this antigen. These animals regularly cleared bacteriophage from the circulation after both the primary and secondary stimulation with bacteriophage T(2). Significant but small amounts of antibody were produced in a few animals in the primary response, and larger amounts in the responding animals after secondary antigenic stimulation. 6. Studies by starch gel and immunoelectrophoresis show that the hagfish has no bands with mobilities of mammalian gamma globulins; that the lamprey has a single, relatively faint band of this type; and that multiple gamma bands are characteristic of the holostean, elasmobranchs, and teleosts studied. By this method of study, the bowfin appeared to have substantial amounts of gamma(2) globulin. 7. We conclude that adaptive immunity and its cellular and humoral correlates developed in the lowest vertebrates, and that a rising level of immunologic reactivity and an increasingly differentiated and complex immunologic mechanism are observed going up the phylogenetic scale from the hagfish, to the lamprey, to the elasmobranchs, to the holosteans, and finally the teleosts.

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Evolution of the immune response. i. the phylogenetic development of adaptive immunologic responsiveness in vertebrates.

Highly purified preparations of radiolabeled lamprey immunoglobulin have been employed to study the structure of the immunoglobulin of this primitive vertebrate. The identity of the labeled material with that of the native immunoglobulin has been established. The lamprey immunoglobulin appears to be unique among vertebrate immunoglobulins since it has a unique molecular weight, electrophoretic mobility, and sedimentation velocity. It is also extremely labile and dissociates in solvents in a complex manner. The observations presented show that this molecule lacks counterparts of heavy and light chains and does not possess interchain disulfide linkages between its major subunits. A structural model based on its dissociation behavior is presented and the implications of such a structure in terms of molecular function and phylogenetic development are discussed.

Joanne Finstad

Papers

Phylogenetic insight into evolution of mammalian Fc fragment of gamma G globulin using staphylococcal protein A.

The evolution of the immune response iii. immunologic responses in the lamprey

Complement and complement-like activity in lower vertebrates and invertebrates

Evolution of the immune response. i. the phylogenetic development of adaptive immunologic responsiveness in vertebrates.

The Evolution of the Immune Response VIII. Structural Studies of the Lamprey Immunoglobulin