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 (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (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--peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined 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, using tributyrin emulsion as a substrate [i.e., 1000 mumol/(min.mg of enzyme)]. At a given lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Aoubala
M
M
UPR 9025 de l'IFRC, Laboratorie de Lipolyse Enzymatique du CNRS, Marseille, France.
Ivanova
M
M
Douchet
I
I
De Caro
A
A
Verger
R
R
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin; D004075:Diglycerides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D005750:Gastric Juice / Q000201:enzymology; D006801:Humans; D008049:Lipase / Q000276:immunology / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D004075:Diglycerides; D000939:Epitopes; D010713:Phosphatidylcholines; C044297:1,2-didecanoylglycerol; D001710:Biotin; D008049:Lipase