Inhibition of pancreatic colipase by antibodies and Fab fragments. Selective effects of two fractions of antibodies on the functional sites of the cofactor.
Rabbit antiserum was raised against porcine pancreatic colipase and Fab fragments were prepared by papain digestion of purified antibodies followed by purification on protein A-Sepharose. Fab fragments showed inactivation toward porcine colipase activity similar to that of antiserum and purified antibodies. From inactivation studies carried out by incubating porcine colipase and lipase with Fab fragments in the absence of lipid or in the presence of triolein and sodium deoxycholate, it could be concluded that polyclonal antiporcine colipase antibodies contain fractions that bind specifically to epitopes at or near the functional regions of the porcine cofactor. Studies with an enzyme-linked immunosorbent assay showed that cross-reactivity of horse or chicken colipase with antiporcine colipase antiserum was lower than that of the human or porcine protein. Results of immunoactivation kinetic studies performed with the same proteins, fully confirmed these observations. Partial cross-reactivity between porcine and chicken colipases allowed us to fractionate antibodies by immunoaffinity chromatography on immobilized chicken colipase. Fraction I contains antibodies absorbed on porcine colipase not accessible when the cofactor is bound to lipid. Antibodies of fraction II, nonadsorbed on chicken colipase, inactivate porcine colipase preincubated with triolein/deoxycholate. Lipase had a protective effect against inactivation. Antibodies of fraction II bind likely to epitopes close to the specific region of colipase interacting with lipase. Our conclusions are in good agreement with analysis of the sequence of porcine, equine and human colipases by calculating local hydrophilicity indices.
Bosc-Bierne
I
I
Perrot
C
C
Sarda
L
L
Rathelot
J
J
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003089:Colipases / Q000037:antagonists & inhibitors* / Q000276:immunology; D003429:Cross Reactions; D000939:Epitopes; D006736:Horses; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D011506:Proteins / Q000037:antagonists & inhibitors*; D013552:Swine
D003089:Colipases; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011506:Proteins