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A rapid, sensitive and versatile two-site immunoradiometric assay for insulin
P C Guest1, C Lowing, S D Arden
1Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Insights
A new, rapid, and highly sensitive two-site immunoradiometric assay for insulin was developed. This assay accurately measures insulin levels in various biological samples, including plasma and islet secretions.
Area of Science:
- Endocrinology
- Immunoassay Development
- Biochemistry
Background:
- Accurate insulin measurement is crucial for diagnosing and managing diabetes and other metabolic disorders.
- Existing assays may lack the required speed, sensitivity, or specificity for certain applications.
Purpose of the Study:
- To develop and validate a novel two-site immunoradiometric assay for insulin.
- To assess the assay's sensitivity, specificity, reproducibility, and applicability to biological samples.
Main Methods:
- Utilized a two-site immunoradiometric assay (IRMA) employing a 125I-labelled monoclonal antibody against human proinsulin.
- Employed immunoprecipitation with an immunoadsorbent from polyclonal antisera against bovine insulin for binding assessment.
- Tested reactivity with various insulin types and related peptides, and assessed reproducibility using human insulin standards.
Main Results:
- Achieved a rapid processing time of 60 minutes and a low detection limit of 2 pM.
- Demonstrated similar reactivity for human, rat, bovine, and porcine insulins.
- Showed high reproducibility with coefficients of variation of 2.3% and 5.5% at different concentrations.
- Observed varying but significant reactivity with human insulin-like peptides.
Conclusions:
- The developed IRMA is both rapid and highly sensitive for insulin quantification.
- The assay exhibits broad applicability across different species and sample types.
- This assay is suitable for clinical and research applications, including plasma analysis and in vitro secretion studies.
Abstract:
A two-site immunoradiometric assay for insulin is described which is both rapid (processing time 60 min) and highly sensitive (lower detection limit 2 pM). Insulin is bound by a 125I-labelled mouse monoclonal antibody raised against human proinsulin and binding assessed by immunoprecipitation with an immunoadsorbent prepared from guinea pig polyclonal antisera raised against bovine insulin. Human, rat, bovine and porcine insulins (10-600 pM) showed similar reactivities in the assay. The human insulin-like peptides, proinsulin, des-31,32-proinsulin and des-64,65-proinsulin (25 pM) had reactivities which were 44.7%, 63.2% and 73.4% of that of insulin, respectively. The assay was highly reproducible with a coefficient of variation of 2.3% for the highest human insulin standard (1000 pM) and 5.5% for the lowest (2 pM). The assay was suitable for determining the concentration of insulin in plasma of fasting human subjects, in normal and tumour-bearing rats and for in vitro studies of insulin secretion from rat pancreatic islets.
