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Specificity of two-site immunometric assays.

B Perry1, R S Chapman, M G McConway

  • 1Institute of Biochemistry, Royal Infirmary, Glasgow, UK.

Annals of Clinical Biochemistry
|January 1, 1991
PubMed
Summary
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Assessing assay specificity requires careful validation. Standard substitution methods can be misleading, as demonstrated by cross-reactivity in a human growth hormone assay, highlighting the need for robust recovery studies.

Area of Science:

  • Biochemistry
  • Immunology
  • Assay Development

Background:

  • Specificity assessment in two-site immunometric assays is challenging.
  • Conventional methods using standard substitution with cross-reactants can produce inaccurate results.
  • An initial assessment suggested no cross-reactivity in an in-house immunoradiometric assay (IRMA) for human growth hormone (hGH).

Purpose of the Study:

  • To investigate the true specificity of an in-house immunoradiometric assay (IRMA) for human growth hormone (hGH).
  • To evaluate the reliability of conventional cross-reactivity assessment methods.
  • To establish improved criteria for validating assay specificity.

Main Methods:

  • Detailed cross-reactivity studies involving substitution of hGH standards with human placental lactogen (hPL).

Related Experiment Videos

  • Addition of hPL to hGH standards to assess its impact.
  • Recovery experiments of added hGH from sera containing endogenous cross-reactants (pregnant subjects).

    • Main Results:

    • Conventional single-point substitution tests were misleading.
    • Significant cross-reactivity of hPL was detected in the hGH assay through detailed studies.
    • The assay showed substantial interference from hPL, contrary to initial findings.

    Conclusions:

    • Standard substitution experiments are insufficient for validating assay specificity.
    • Robust validation requires demonstrating quantitative recovery of analytes from serum with varying endogenous cross-reactants.
    • Improved methodologies are crucial for accurate assessment of immunometric assay specificity.