Aims: 

The peptide hormone - called thrittene - has the same amino acid sequence as somatostatin-28 N-terminal (1–13). It was detected in mammalian gut and plasma by immunochemistry and subtraction radioimmunoassay (RIA). Our research group working on diabetes and obesity, considered that it is important to develop a direct thrittene RIA method that is specific and sensitive enough for measurement of thrittene.

Methods: 

The antiserum (TH3), sensitive to the C-terminal region of peptide was produced by subcutan immunization of rabbits administering thrittene-bovine serum albumin antigen. To show the antibody attachment we used iodogen method labeled mono-¹²⁵I-Tyr⁽⁰⁾-thrittene as RIA tracer. Thrittene was used as standard for the RIA determination in a range of 0-1000 fm/ml.

Results: 

After ten measurements the D₅₀ value of the calibration curves was 8.61±1.22 fmol/ml. The detection limit of our method was 0.2 fmol/ml thrittene. The cross-reactivity studies showed that the antiserum used in our RIA method has a limited cross-reactivity with other peptides with similar structure. By determining the thrittene content of the rat gastrointestinal tract tissue the highest concentration was measured in duodenum followed by jejunum and ileum; however, all the examined tissues contained highly enough thrittene for the measurement. Eventually, the receptor of the thrittene in rat brain tissue was identified using receptor binding assay (B_max: 0,23±0,05 fmol/mg protein and K_d: 9,50±1,81 nmol).

Conclusion: 

The thrittene RIA method developed in our laboratory has high sensitivity and peptide specificity. It is suitable for determining tissue and plasma concentrations and it helps to get further information about the biological role of the peptide hormone.

Support: 

Hungarian Reseach Founds GOP-1.1.2-07/1-2008-0004 and OTKA 75965