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Related Experiment Videos

ELISA for human proinsulin.

S G Hartling, B Dinesen, A M Kappelgård

    Clinica Chimica Acta; International Journal of Clinical Chemistry
    |May 15, 1986
    PubMed
    Summary
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    A new micro enzyme-linked-immunosorbent-assay (ELISA) effectively monitors human proinsulin (hPI) in serum. This assay is valuable for diagnosing insulinoma and assessing beta-cell function, though not sensitive enough for IDDM patients.

    Area of Science:

    • Biochemistry
    • Immunology
    • Endocrinology

    Background:

    • Circulating human proinsulin (hPI) is a key biomarker in metabolic and endocrine disorders.
    • Accurate monitoring of hPI levels is crucial for understanding beta-cell function and diagnosing conditions like insulinoma.
    • Existing immunoassays may lack the sensitivity or specificity required for comprehensive hPI analysis.

    Purpose of the Study:

    • To develop and validate a sensitive micro enzyme-linked-immunosorbent-assay (ELISA) for quantifying circulating human proinsulin (hPI).
    • To establish the assay's performance characteristics, including detection limit, operating range, and specificity.
    • To evaluate the assay's utility in clinical settings, such as diagnosing insulinoma and assessing beta-cell function.

    Main Methods:

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  • A micro ELISA was developed using guinea pig anti-insulin antibody-coated plates and peroxidase-labelled F(ab')2-fragments of guinea pig anti-human C-peptide as the detection system.
  • Assay sensitivity, specificity, and intra-assay variation were determined using buffer and serum samples.
  • Cross-reactivity studies were performed with insulin, human C-peptide, and proinsulins from other species.
  • The assay was applied to samples from healthy subjects, pancreatectomized patients, and an insulinoma patient.

    • Main Results:

    • The assay demonstrated a detection limit of 1.2 pmol/l in serum (50% dilution) with a standard operating range of 0-160 pmol/l.
    • Interassay variation was 9%, and significant cross-reactivity was not observed with insulin, human C-peptide, or porcine/bovine proinsulins.
    • Reference ranges in healthy subjects were 1.2-13 pmol/l (median 4.1 pmol/l), while pancreatectomized patients showed values below the detection limit, and an insulinoma patient had 263 pmol/l.

    Conclusions:

    • The developed micro ELISA is a sensitive and specific method for monitoring circulating human proinsulin (hPI).
    • The assay is useful for diagnosing insulinoma and characterizing beta-cell function in stimulated conditions.
    • While not sensitive enough for fasting IDDM patients, it offers valuable clinical insights in other endocrine contexts.