Gradient chromatography of human serum proteins and its application to the examination of “albumin” and “globulin” obtained by ammonium sulfate fractionation

146

The nature of the antibodies produced by the rabbit during the primary and secondary responses to T2 phage, proteins, and the O and H antigens of Salmonella typhosa has been determined. Immune sera have been fractionated by zone electrophoresis, sucrose density ultracentrifugation, and anion exchange chromatography. The resulting fractions have been assayed by phage neutralization or hemagglutination (antisera to proteins) or bacterial agglutination. In confirmation and extension of earlier work from this laboratory, the primary response to these antigens, with the exception of the O antigen of the Salmonella, included the early synthesis of 19S, γ-1 globulin antibody, and the later synthesis of 7S, γ-2 globulin antibody. The primary response to the O antigen consisted of the synthesis of only a macroglobulin agglutinin. The secondary response to the proteins, including the H antigen of the Salmonella, comprised the early synthesis of large amounts of the 7S γ-2 globulin antibody to the same level attained during the primary response. The secondary response to the phage consisted in the synthesis of 7S, γ-2 globulin antibody alone. Treatment of the macroglobulin phage-neutralizing antibody with mercaptoethanol resulted in complete loss of its neutralizing activity. A working hypothesis to explain these observations was presented. A salient feature of this hypothesis was the suggestion that different cells synthesized the two distinct molecular forms of antibody. The significance of the sequential synthesis of the two forms of antibody is not known. It was proposed that the system for synthesis of macroglobulin antibody is an auxiliary system for antibody synthesis, perhaps the first to develop phylogenetically and ontogenetically. It is felt that the present observations indicate a clear-cut qualitative distinction between the primary and secondary responses to immunization whereby these responses might be identified in various experimental situations. It is also felt that these findings with the primary and secondary responses to various antigens in the rabbit may be of widespread occurrence in nature among a variety of species.

Section: Methodsmentioning

confidence: 99%

Section: Zone Electrophoretic Fractionation--mentioning

confidence: 99%

Sequences of Synthesis of Γ-1 Macroglobulin and Γ-2 Globulin Antibodies During Primary and Secondary Responses to Proteins, Salmonella Antigens, and Phage

Bauer

Mathies

Stavitsky

1963

146

“…I131-Labded Proteins.--Mouse "y-globulin (My) was prepared by zone (block) electrophoresis of pooled normal NIH-WS mouse sera (1). Guinea pig 3,-giobulin (GP3') and bovine "yglobulin (Boy) were obtained from the pooled sera of normal animals by the 0.005 M phosphate buffer eluate (pH 8.0) from a diethylaminoethyl cellulose (DEAE) column (13). Human "y-globulin (Hu3') was obtained from a normal serum by the 0.01 u phosphate buffer eiuate (pH 8.0) from a DEAE column.…”

mentioning

confidence: 99%

EVIDENCE FOR SPECIES' DIFFERENCES IN THE EFFECT OF SERUM Γ-Globulin CONCENTRATION ON Γ-Globulin CATABOLISM

Sell

1964

The serum y-globulin level depends on a dynamic equilibrium between synthesis and catabolism. The mechanisms which regulate this equilibrium are not well understood, but a balance must exist between the rate of production and the rate of breakdown in order to maintain a constant serum -/-globulin concentration. Injections of exogenous y-globulin into mice increase the rate of catabolism of isotopically labeled homologous (mouse) y-globulin (1). The rate of catabolism of homologous y-globulin is also more rapid in hyperimmunized mice (1) or mice harboring y-globulin-producing plasma cell tumors (2). On the other hand, germfree mice, which have subnormal serum y-globulin concentrations, have slower rates of y-globulin breakdown than normal mice (3). The rate of T-globulin catabolism also appears to be related to the serum y-globulin concentration in man (4-10).In guinea pigs, however, the rates of homologous y-globulin catabolism are the same in germfree and normal animals in spite of up to 6-fold differences in the serum ")'-globulin levels (11), suggesting that the fractional catabolic rate of homologous y-globulin is independent of the serum level in these animals.Because of this apparent difference between the mouse and guinea pig and the relative ease with which their serum y-globulin levels may be increased by administration of exogenous y-globulins, the present study was planned to determine the relationship between serum y-globulin levels and the fractional catabolic rate of y-globulins in these two species. This was accomplished by raising the serum y-globulin levels of mice and guinea pigs by hyperimmunization or by injections of exogenous y-globulins and following the rate of catabolism of la31-trace labeled serum proteins including: mouse, guinea pig, rabbit, bovine, and human y-gl0bulins, and human serum albumin. The results indicate a species difference in the relationship between the fractional rate of y-globulin catabolism and the serum level of y-globulin in the mouse and guinea pig. In addition, exogenous y-globulins from man, mouse, guinea pig, and rabbit are all equally effective in increasing the fractional rate of y-globulin catabolism in the mouse, but bovine y-globulin is only partially active.

“…Rabbit globulins were prepared by precipitation from normal rabbit serum at 50 per cent saturation with (NH4)~SO4. Rabbit "yG-globullns were purified from normal rabbit serum by chromatography on DEAE cellulose (8). Cohn fractions II and III from rabbit serum were purchased from Pentex Inc.…”

Section: Methodsmentioning

confidence: 99%

“…Treatment of tissue containing antimycobacterial antibodies was also done with these reagents. Chromatography.--Sera were fractionated on DEAE cellulose columns using a concave pH gradient of decreasing pH and increasing molarity (8).…”

Section: Methodsmentioning

confidence: 99%

Cellular Sites of Synthesis of Rabbit Immunoglobulins During Primary Response to Diphtheria Toxoid-Freund's Adjuvant

Schoenberg

Stavitsky

Moore

et al. 1965

The initial immunization of rabbits with many protein, bacterial, and bacteriophage antigens results in the synthesis of ~,M-and 3,G-globulin hemagglutinating, agglutinating, and phage-neutralizing antibodies (1, 2). 1 On the basis of a number of observations it was suggested (1, 2) that different cells synthesize ~/M-and 3,G-globulin antibodies. ~ Further immunofluorescent studies (4, 5) of the spleens of rabbits which had been injected intravenously with diphtheria toxoid and Freund's adjuvant provided evidence for the participation of two types of cells in the synthesis of these two molecular species of antibody. Non-phagocytic mononuclear cells in the walls of the sinusoids of the red pulp contained antidiphtheria toxoid during the time when only "yM-hemagglutinating antidiphtheria toxoid was found in the serum of these animals. Later plasma cells in the non-follicular white pulp of the spleen contained antidiphtheria toxoid and the rabbits' sera had both "yM-and 3,G-globulin hemagglutinating antidiphtheria toxoid. It was, therefore, tentatively concluded that the mononuclear cells produced 3'M-globulin antitoxin and the plasma cells synthesized 7G-globulin antitoxoid.