Development of monoclonal antibodies against bovine mucin core 2 beta6 N-acetylglucosaminyltransferase.
Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 beta6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M(-1)) for these MAbs range from 5.4 x 10(7) to 1.2 x 10(9). These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, these antibodies should be useful for further study of bovine and human core 2 enzymes.
Li
C M
CM
Department of Pediatrics, University of North Carolina, Chapel Hill, USA.
Joshee
N
N
Adler
K B
KB
Cheng
P W
PW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006639:Histidine; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000276:immunology*; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase