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Determination of assay validation

Determination of assay validation. Lab 2. Assay validation. Evaluating diagnostic tests. Key words Sensitivity Specificity Positive predictive value Negative predictive value. Definitions. True positive: Sick people correctly diagnosed as sick

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Determination of assay validation

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  1. Determination of assay validation Lab 2

  2. Assay validation

  3. Evaluating diagnostic tests Key words Sensitivity Specificity Positive predictive value Negative predictive value

  4. Definitions • True positive: Sick people correctly diagnosed as sick • False positive: Healthy people incorrectly identified as sick • True negative: Healthy people correctly identified as healthy • False negative: Sick people incorrectly identified as healthy • Sensitivity: a measure of the proportion of actual positives which are correctly identified as such • Specificity: a measure of the proportion of negatives which are correctly identified • Positive predictive value: is the proportion of patients with positive test results who are correctly diagnosed • Negative predictive value: is the proportion of patients with negative test results who are correctly diagnosed

  5. Example • You got 110 samples.. 100 -ve and 10 +ve • You correctly diagnosed 95 out of the 100 negative sample and 8 out of the 10 +ve. • Sensitivity=? • Specificity=?

  6. The answer

  7. Assay methods • Need sensitive and specific assays to achieve accurate diagnosis • Most diagnostic tests at the immunology lab utilize antibodies. • Antibodies can be monoclonal or polyclonal Common types of assay • Radioimmunoassay (RIA) • Immunoradiometric assay (IRMA) • Enzyme-labelled immunosorbent assay (ELISA)

  8. Antibodies-Polyclonal Induced in an animal by conventional immunisation A mixture of antibodies recognising many epitopes (sites) on the antigen molecule Antigen Polyclonal antibody

  9. Antibodies-Monoclonal Hybrid cell lines produced by fusing B lymphocyte from immunised animal with myeloma cell line. Fused cells cloned and cultured. A monoclonal antibody is the product of a single cell line and is highly specific, recognising a single epitope

  10. Radioimmunoassay (RIA) • Based on the reaction of a limited concentration of antibody with varying concentrations of antigen (in standards and patient samples). • Radiolabelled antigen is used as a detection system (usually 125I). • Competitive binding assay.

  11. Radioimmunoassay (RIA) Antigen Radiolabelled antigen Antibody Mix and react Free fraction Bound fraction Separation Count radioactivity

  12. Immunoradiometric assay (IRMA) • Antibody is present in excess. • ‘Two-site’ (sandwich) method has radiolabelled antibody (monoclonal) and different antibody (monoclonal or polyclonal) bound to solid phase.

  13. Immunoradiometric assay (IRMA) Solid phase antibody Radiolabelled antibody (monoclonal) Antigen Mix and react Separate and count bond fraction

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