Human C-peptide. Part I: Radioimmunoassay

Area of Science:

• Endocrinology
• Immunochemistry
• Analytical Chemistry

Background:

• Human C-peptide is a biomarker for insulin production and is vital in diabetes mellitus diagnosis and management.
• Accurate quantification of C-peptide is essential for assessing pancreatic beta-cell function.
• Existing methods may have limitations in sensitivity or specificity.

Purpose of the Study:

• To develop and validate a highly sensitive radioimmunoassay (RIA) for synthetic human C-peptide.
• To compare different labeling methods and antibody production techniques for optimal assay performance.
• To establish the specificity and sensitivity of the developed RIA for clinical application.

Main Methods:

• Synthetic human C-peptide was labeled with 125iodine using chloramin T or hydrogen peroxide/lactoperoxidase.
• Antiserum was generated in guinea pigs and goats against synthetic human C-peptide.
• Radioimmunoassay standard curves were established, and the hook effect was investigated.
• Anion exchange resin (Amberlite) was used for separating free and antibody-bound C-peptide.
• Assay sensitivity, intra-assay, and inter-assay variability were determined.

Main Results:

• Goat antiserum demonstrated superior antibody production compared to guinea pig antiserum.
• The radioimmunoassay exhibited a sensitivity of approximately 0.3 ng C-peptide/ml serum.
• Intra- and inter-assay variability were consistently below 10%.
• Human proinsulin was identified as the only substance showing cross-reactivity with the antiserum.
• The anion exchange method effectively excluded non-specific binding in diabetic serum samples.

Conclusions:

• A sensitive and specific radioimmunoassay for human C-peptide has been successfully developed.
• The developed RIA is suitable for quantifying C-peptide in serum, with potential applications in diabetes research and clinical practice.
• The method demonstrates good reproducibility and minimal cross-reactivity, ensuring reliable C-peptide measurements.