Development of an oxyntomodulin/glicentin C-terminal radioimmunoassay using a "thiol-maleoyl" coupling method for preparing the immunogen.
Oxyntomodulin (OXM) and glicentin, two peptides processed from proglucagon, both contain the glucagon sequence and a C-terminal basic octapeptide, KRNRNNIA extension. A method to produce antibodies, directed specifically toward the C-terminal extension of these two peptides, was developed; it consisted of the use of thioled bovine serum albumin conjugated with the synthetic N-maleoyl C-terminal octapeptide as the immunogen. Three rabbits (FAN, LEG, and PIP) generated antisera with affinity constants close to 5 X 10(10) M-1. In the radioimmunoassay system, these antisera showed a 100% cross-reactivity with OXM, partially purified rat and human glicentin, and the C-terminal 19-37 OXM fragment. They displayed no cross-reactivity toward the glucagon molecule. The cross-reactivity of C-terminal fragments of OXM demonstrated that the epitope involves the C-terminal hexapeptide and that the two last amino acid residues are essential for the binding. The high-performance liquid chromatography elution profiles of human jejunum or rat intestinal extracts obtained by radioimmunoassay with LEG antiserum showed two major peaks which had the same retention times as OXM and glicentin markers. Thus, the major end products in the human and rat small intestine are OXM and glicentin. In human or rat pancreas, the two main peaks detected were glucagon and the C-terminal hexapeptide of OXM/glicentin. Small amounts of OXM were also found in pancreas, whereas no significant quantities of glicentin could be detected. The "thiol-maleoyl" coupling method described here, and applied to produce C-terminal OXM/glicentin specific antisera, might be of general use to obtain antibodies against a well-defined epitope.
Blache
P
P
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Kervran
A
A
Martinez
J
J
Bataille
D
D
D000818:Animals; D000915:Antibody Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D005768:Gastrointestinal Hormones / Q000032:analysis*; D053771:Glicentin; D005934:Glucagon / Q000032:analysis*; D004763:Glucagon-Like Peptides / Q000032:analysis*; D007422:Intestines / Q000032:analysis; D008297:Male; D053772:Oxyntomodulin; D010179:Pancreas / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis*; D011498:Protein Precursors / Q000032:analysis*; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains
D005768:Gastrointestinal Hormones; D053772:Oxyntomodulin; D010446:Peptide Fragments; D011498:Protein Precursors; D004763:Glucagon-Like Peptides; D053771:Glicentin; D005934:Glucagon