Structural and antigenic relationships between avian immunoglobulins. 3. Antigenic relationships of the immunoglobulins of the chicken, pheasant and Japanese quail
G A Leslie, A A Benedict
1970
Journal of Immunology
iii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to the members of the Dissertation Committeefilr their guidance and assistance throughout this study. Particular appreciation is extended to Lene for her unending patience and encouragement during my studies. This study was made possible by USPHS Training Grant #5 TOI Al 00243. ABSTRACT Radioimmunoelectrophoresis (RIE) showed that ringnecked pheasants (Phasianus colchicus) and Japanese quail (Coturnix coturnix) produced
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... G and IgM antibodies in response to a single injection of bovine serum albumin (BSA). All pheasants and approximately 25% of the quail possessed antibody activity after a single injection of BSA. The sedimentation coefficients of purified pheasant and quail IgG (P-IgG, Q-IgG) were 7.1-7.3S and that of IgM of both birds was 26-275 (aggregates) and 15-l6S. Chicken (C-IgG), pheasant and quail IgG reduced with 0.2 M mercaptoethanol (2-ME), alkylated and acidified, yielded ggr~gates, heavy chains and light chains by. gel-filtration in propionic acid. Each fraction was characterized by. ge1diffusion, immunoelectrophoresis (IE), u1tracentrif~gation and urea starch~ge1 electrophoresis. In alkaline buffers, C-~gG and P-IgG required 0.05-0.1 M 2-ME for the release of l~ght chains whereas Q-IgG required 0.2 M 2-ME. About 35% of the IgG's dissociated in alkaline buffers when reduced with 0.2 M 2-ME. Hydrolysis of C-~gG, P-IgG and Q-IgG with papain for 16-18 hours produced fr~gments with a sedimentation coefficient of about 3.5S. Approximately 40-50% of the initial protein was converted into dialyzable pep.tides. The e1ectrophoreticallyfast fr~gment was Fc and the electrophoretically-slow fr~gment was Fab. v Chicken IgG pseudoglobulins were converted into Fab' . . fragments after d,igestion with 1% pepsin, pH 4.5, for 18 hours. After five hours Fab' and some Fc-like fr~gments were present. Digestion of pseudoglobulins for 18 hours at pH 5 allowed the isolation of a small amount of 5.75 (Pep-I) material. Und~gested IgG, Fab' and Fc fr~gments were also present in pH 5 digests. At pH 4.5, euglobulins, gave more Pep-I fr~gments than did pseudoglobulins. Variations in di-. gestion times, temperatures, pH's and method of IgG preparation influenced the types of fragments that were formed. D~gestion of P-IgG with 1% pepsin at pH 4.5 resulted in 5.45 fragments (Pep-I) and und~gested~gG, whereas d~gestion with 2% pepsin for 18 hours at pH 4.5 yielded mainly Pep-I. D~gestion with 1% and 2% pepsin yielded 18 and 36% dia1yzable peptides, respectively. The Pep-I fragment contained no Fc determinants but compared to papain-produced Fab had additional determinants. Quail IgG, d~gested with 1% pepsin for 18 hours at pH 4.5 yielded 37% dialyzable peptides. Of the undia1yzable protein remaining, approximately 39% was 5.45 Pep-I and the remainder was mainly Fab l • Isolated Pep-I was similar, by IE, to papain-produced Fab. The cross-reacti~g ant~genic determinants between chicken, pheasant and quail IgG were found both on the heavy and light chains and both Fab and Fc fr~gments. In. gel-diffusion, chicken and pheasant l~ght chains. gave a line of identity vi with rabbit anti-chicken globulin, anti-pheasant. globulin and anti-chicken l~ght chain antisera. The results of analysis with chains and fragments indicated that C-IgG and P-IgG were ant~genically more similar than was Q-IgG and either of the other two.
pmid:4099923
fatcat:hoyewjjrgjdjdj2u7ijgxkbnri