Development of an assay for bioactive insulin

Area of Science:

• Endocrinology
• Biochemistry
• Cell Biology

Background:

• Assessing the biological activity of serum insulin is critical for understanding insulin's function and metabolic effects.
• Traditional immunological assays measure total insulin but do not differentiate between biologically active and inactive forms.

Purpose of the Study:

• To develop and validate a novel assay for quantifying biologically active insulin in serum.
• To assess the utility of this assay in cases of mutant insulin variants.

Main Methods:

• A bioactive insulin assay was developed using Chinese hamster ovary (CHO) cells overexpressing human insulin receptors.
• Serum was added to cells, followed by lysis, and insulin receptor autophosphorylation was measured via a two-site immunofluorometric assay.
• Detection utilized monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies.

Main Results:

• The assay demonstrated a detection limit of 1 microU/ml of insulin with acceptable inter- and intra-assay variations.
• Results correlated strongly with a traditional immunological insulin assay (r = 0.94, P < 0.001).
• In a case study with mutant insulin (Wakayama subject), biologically active insulin constituted 9% of immunologically reactive insulin.

Conclusions:

• The developed bioactive insulin assay accurately measures the functional capacity of serum insulin.
• This assay is valuable for monitoring biologically active insulin levels, especially in patients with insulin resistance or mutant insulin forms.