Antigen specificity and cross-reactivity of fifteen monoclonal antibodies against porcine pancreatic alpha-amylase II and its AB and C domains.
Highly productive hybridoma secreting mabs specific for porcine alpha-pancreatic amylase II were established. Fifteen clones were selected. The mabs produced (KD = 1.68-11.2 nM) were checked for cross-reactivity with six heterologous antigens, namely porcine pancreatic alpha-amylase I, barley amylase, human pancreatic alpha-amylase, Taka amylase and triose phosphate isomerase, using direct ELISA assay; mabs were classified within seven groups: in a few groups mabs cross-reacted with a single heterologous antigen either porcine pancreatic amylase I (6 mabs) or barley amylase (2 mabs) or human pancreatic amylase (3 mabs). Two other groups cross-reacted with two heterologous antigen either porcine I and human or porcine I and barley. Only one mab out of fifteen cross-reacted in direct ELISA binding to all amylases and triose phosphate isomerase. Using sandwich ELISA test only three mabs were found to bind porcine amylase II present at high concentration. Results consistent with direct porcine amylase binding were obtained from binding inhibition assays. Analysis by the additivity test allowed to find that 3 mabs, B10.10, B1.11, C6.4 recognize distinct epitopes while the epitopes for the other pairs tested are either overlapping or at least close to each other. Finally mabs binding specifically either to the AB or to the C domain fragment or to both fragments have been obtained.
Fueri
J
J
Laboratoire de Biochimie et de Biologie Moléculaire de la Nutrition, Faculté des Sciences, Université d'Aix-Marseille III, France.
Fueri
C
C
Ferrey
G
G
Chaix
J C
JC
Marchis-Mouren
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D013552:Swine / Q000276:immunology*; D000516:alpha-Amylases / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D000516:alpha-Amylases