INTERFACIAL BINDING OF HUMAN GASTRIC LIPASE TO LIPID MONOLAYERS, MEASURED WITH AN ELISA

TWO sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mA bs). These assays were adapted to the monomolecular film technique use d previously for measuring lipase kinetics. HGL and the two anti-HGL m Abs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin-peroxidas e conjugate as tracer. The detection limit was 25 and 85 pg in the cas e of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for t he first time to measure the enzymatic activity of HGL on 1,2-didecano yl-sn-glycerol (dicaprin) monolayers as well as to determine the corre sponding interfacial excess of the enzyme. The HGL turnover number inc reased steadily with the lipid packing. The specific activities determ ined on dicaprin films spread at 35 mN . m(-1) were found to be in the range of the values measured under optimal bulk assay conditions, usi ng tributyrin emulsion as a substrate [i.e., 1000 mu mol/(min . mg of enzyme)]. At a given Lipase concentration in the water subphase, the i nterfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidyl choline (egg PC) monolayers was found to be 10 times lower than that i n the case of dicaprin monolayers. Given the low tensioactivity of the mAbs of the IgG isotype [Ivanova et al. (1993) Colloids Surf. BI, 17- 22], we also investigated the effects of five anti-HGL mAbs (mAbs 4-3, 25-4, 35-2, 83-15, and 218-13) on the catalytic activity as well as o n the interfacial binding of HGL to lipid/water interfaces. Four out o f these five mAbs (mAbs 4-3, 25-4, 35-2, and 83-15) were found to sign ificantly reduce the lipolytic activity of HGL. Moreover, three of the four inhibitory mAbs (mAbs 4-3, 25-4, and 35-2) were found to reduce the specific activity of HGL, while mAb 83-15 had no effect on the spe cific activity. These results clearly indicate that the latter mAb (83 -15) complexed with HGL mainly affects the binding of the enzyme to th e lipid/water interface, while the other three inhibitory mAbs (mAbs 4 -3, 25-4, and 35-2) affect both the binding and the catalytic steps of HGL.