Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation.
Sequential sublining was used in combination with enzyme-linked immunosorbent assays to isolate mu----gamma isotype switch variants of the rat IgM secreting mouse-rat B cell hybridoma line BA1.8. Switch variants to all four subclasses of IgG were obtained. The variant antibodies retained the antigen specificity of the parental IgM for the O18 (lipopolysaccharide) antigen of Escherichia coli. In sodium dodecyl sulfate-polyacrylamide gels the apparent molecular mass of the gamma heavy chains decreased in the order gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. IgM, IgG1, IgG2a, IgG2b and IgG2c of the BA1.8 variant family and IgG2b, IgE and IgA of the previously described BA1.2 family were used for a comparative analysis of the capacity of rat Ig to activate complement. Efficient lysis of sheep erythrocytes coated with the O18 antigen was observed with IgM and all IgG subclasses, but no lysis was triggered by IgE or IgA. One hundred to 1000 IgG molecules were required to mediate the same hemolytic activity as one IgM molecule. The four IgG subclasses were equally efficient at mediating lysis by rat or human complement, while IgG2a was less efficient with guinea pig complement than the other three IgG subclasses. Antibody-triggered binding of C3 to pathogenic O18:K1 E. coli bacteria was measured using serum containing 125I-labeled C3. K1-encapsulated strains did not fix C3 efficiently in the absence of specific antibodies while acapsular mutants fixed C3 via the alternative pathway. IgM and all IgG subclasses triggered C3 binding to the K1 encapsulated bacteria. The capacity of IgM to mediate C3 fixation was not greater than that observed with IgG.
Pluschke
G
G
Basal Institute for Immunology, Switzerland.
Bordmann
G
G
Daoudaki
M E
ME
Lambris
J D
JD
Achtman
M
M
Neibert
M
M
D000818:Animals; D002469:Cell Separation; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D006386:Hemagglutination Tests; D006825:Hybridomas / Q000378:metabolism*; D007070:Immunoglobulin A / Q000502:physiology; D007073:Immunoglobulin E / Q000502:physiology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D051379:Mice; D011817:Rabbits; D051381:Rats
D003176:Complement C3; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D007073:Immunoglobulin E