Human Pancreatic Lipase

Abstract

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Human Pancreatic Lipase: An Exposed Hydrophobic Loop from the C-terminal Domain May Contribute to Interfacial Binding

“…Anti-HPL Antibodies. Anti-HPL pAb and mAb's (numbered 81-23, 146-40, 315-25, and 320-24) were produced and characterized at our laboratory (20).…”

“…ELISA Tests. To test whether the mAb's (81-23, 315-25, and 320-24) recognized the colipase binding site on HPL, a double sandwich ELISA test was performed (20). A second ELISA test was performed in which microtiter plates were also coated with pure anti-HPL pAb (250 ng/well, captor antibody), and HPL was added (2.5 ng/well) to the PVC-coated pAb.…”

“…The residual activity of the HPL/mAb complexes was measured potentiometrically using a pH-stat (716 DMS Titrino, Metrohm). Assays on TC8 and TC4 were carried out under standard conditions (20,28), respectively. TC3 was used as the substrate for measuring the interfacial activation of HPL ( 29) and of the HPL/mAb complex.…”

“…Monoclonal antibodies (mAb's) are useful tools for mapping structural domains and for probing the functional features of multidomain enzymes such as lipases. Four anti-HPL mAb's (81-23, 146-40, 315-25, and 320-24) have been produced (20) at our laboratory and used to carry out epitope mapping on HPL. Four peptides obtained by performing limited chymotryptic cleavage were characterized by Western blotting and N-terminal amino acid sequence analysis.…”

Plant Products and Their Inhibitory Activity Against Pancreatic Lipase

“…1, this conformational change makes the enzyme "open," and the two domains rotate slightly at the hinge region, so the N-terminal domain interacts with colipase. This study was made possible by using monoclonal antibodies technique (Aoubala et al 1995).…”