Production, characterization, and application of monoclonal antibodies which distinguish four glucosyltransferases from Streptococcus sobrinus.
A 1,3-alpha-glucan synthase (GTF-I), a highly branched 1, 6-alpha-glucan synthase (GTF-U) and a 1,6-alpha-glucan synthase (GTF-T) were purified to near homogeneity from the culture fluid of Streptococcus sobrinus strain B13N (serotype d) and characterized. In addition, a crude preparation of a recombinant oligo-isomaltosaccharide synthase (rGTF-S) was prepared from a cell-free extract of Escherichia coli MD124 transformant. Using four homogeneous GTF preparations including previously purified rGTF-S as antigens for immunization, 11 murine hybridomas producing a monoclonal antibody (MAb) were established through the fusion of myeloma cells (P3X63-Ag8-U1) and spleen cells of immunized BALB/c mice. When the immunoreactivities of the resultant MAbs were tested, all five MAbs raised against GTF-I, all three MAbs raised against GTF-T, and two of three MAbs raised against GTF-U reacted specifically with the homologous enzyme alone, while one MAb (B86) raised against GTF-U cross-reacted strongly with all GTFs. Although no MAb monospecific for rGTF-S was obtained, precise recognition of GTF-S was possible using the nonspecific B86 antibody together with the MAbs monospecific for the three glucan synthases. Thus, a set of four typical MAbs (B17, B76, B19 and B86) were successfully used for the identification of gene products expressed in 24 previously constructed E. coli phage clones, and the findings suggested that six phage clones might express a gtfU gene encoding GTF-U which has not been hitherto isolated.
Nanbu
A
A
Department of Microbiology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Hayakawa
M
M
Takada
K
K
Shinozaki
N
N
Abiko
Y
Y
Fukushima
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins; D015153:Blotting, Western; D003090:Coliphages / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D015151:Immunoblotting / Q000379:methods; D051379:Mice; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D016986:Streptococcus sobrinus / Q000201:enzymology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011506:Proteins; C067118:GTF-I protein, Streptococcus; D005964:Glucosyltransferases; C489364:glucosyltransferase I