Enzyme-linked sandwich immunoassay for insulin using laser fluorimetric detection

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Accurate measurement of human insulin levels is crucial for diagnosing and managing diabetes and other metabolic disorders.
• Existing methods like radioimmunoassay (RIA) have limitations including radioactive waste and specialized handling requirements.
• Development of sensitive, non-radioactive immunoassays is essential for routine clinical laboratory use.

Purpose of the Study:

• To develop and validate a novel enzyme-linked sandwich immunoassay for the quantitative determination of human insulin in serum samples.
• To establish the assay's sensitivity, precision, and correlation with established methods like RIA.
• To provide a robust and accessible alternative for clinical insulin measurement.

Main Methods:

• Human serum samples were analyzed using a sandwich immunoassay format.
• Horseradish peroxidase (HRP) was employed as the enzyme label for antibodies.
• Enzyme activity was quantified via a fluorogenic substrate (p-hydroxyphenylacetic acid) and detected using a helium/cadmium laser and reverse-phase high-performance liquid chromatography (RP-HPLC).

Main Results:

• The developed immunoassay demonstrated a detection limit of 30 picomolar (pM) for insulin, equivalent to 5 microunits/ml.
• The assay achieved a high correlation coefficient (r = 0.95) when compared to radioimmunoassay.
• A 90-minute incubation period was found to be optimal for the assay.

Conclusions:

• The enzyme-linked immunoassay provides a highly sensitive and specific method for human insulin quantification in serum.
• This non-radioactive assay demonstrates excellent agreement with RIA, making it a suitable replacement for clinical applications.
• The method's high sensitivity and correlation support its utility in diagnosing and monitoring conditions related to insulin levels.