Immunochemical determination of porcine pancreatic colipase: differentiation between procolipase and its trypsin-activated form.
A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative determination of porcine pancreatic colipase. Calibration curves were established by coating polystyrene immunoplates with pure procolipase or its trypsin-activated derivative. Bound antigen was detected with antiporcine procolipase polyclonal antibodies. Under optimizing conditions, the minimal detectable amount of porcine colipase was 0.1 ng, which is about 1,000 times less than the minimal amount that can be assayed titrimetrically. The useful range of the immunoassay was between 0.1 to 1 ng (2-20 micrograms/L). Under standard assay conditions, no distinction can be made between the precursor and activated forms of the cofactor. Results of immunochemical determinations of colipase in porcine pancreatic juice and tissue extract were in good agreement with those obtained with the potentiometric method. The specific determination of activated colipase in pancreatic juice was performed by coating the immunoplates with antigen in solution in PBS with 0.5 g/L of Tween 20. The detergent selectively impaired the binding of procolipase to the plate. Determination of colipase in human pancreatic juice carried out under the same experimental conditions showed that the minimal amount of human cofactor detectable with ELISA was 1 ng due to partial immunological crossreactivity of the human and porcine proteins. Immunoassay performed with antiporcine procolipase monoclonal antibodies (Mab) showed lower sensitivity than that performed with polyclonal antibodies. However, Mab 72.11, a monoclonal antibody that reacted only with porcine procolipase, allowed specific detection and differential determination of the precursor form of porcine colipase in pancreatic juice. ELISA performed with pure human colipase indicated that no antiporcine procolipase monoclonal antibodies cross-reacted with the human cofactor.
de La Fournière
L
L
Laboratoire de Biochimie, Institut de Chimie Biologique, Faculté St Charles, Marseille, France.
Forte
G
G
Rathelot
J
J
Piéroni
G
G
Julien
R
R
Sarda
L
L
D000818:Animals; D000911:Antibodies, Monoclonal; D003089:Colipases / Q000032:analysis*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010179:Pancreas / Q000201:enzymology*; D010189:Pancreatic Juice / Q000201:enzymology; D011498:Protein Precursors / Q000032:analysis*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003089:Colipases; D004792:Enzyme Precursors; D011498:Protein Precursors; C037107:procolipase; D014357:Trypsin