Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure.
Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.
Aoubala
M
M
Laboratoire de Lipolyse Enzymatique du Centre National de la Recherche Scientifique, Marseille, France.
Douchet
I
I
Laugier
R
R
Hirn
M
M
Verger
R
R
De Caro
A
A
D002846:Chromatography, Affinity / Q000379:methods; D004386:Duodenum / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D005750:Gastric Juice / Q000201:enzymology*; D006801:Humans; D008049:Lipase / Q000276:immunology / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology; D011199:Potentiometry; D012680:Sensitivity and Specificity
D008049:Lipase