Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies.
Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase-dependent lipase activity. The dissociation constant of the inactive antibody-antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody-antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid).
Dezan
C
C
Laboratoire de Biochimie Case 65, Faculté des Science St Charles, Marseille, France.
Daniel
C
C
Hirn
J
J
Sarda
L
L
Bellon
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D003089:Colipases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D003429:Cross Reactions; D004792:Enzyme Precursors; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006736:Horses / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010179:Pancreas / Q000201:enzymology*; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity; D013552:Swine / Q000276:immunology
D000911:Antibodies, Monoclonal; D003089:Colipases; D004792:Enzyme Precursors; D000939:Epitopes; D007074:Immunoglobulin G; D011498:Protein Precursors; C037107:procolipase