Transcript Immunoassay
MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Immunoassays 1 General Considerations • In an immunoassay, an antibody molecule recognizes and binds to an antigen. • Binding is related to: – Concentration of each reactant – Specificity of antibody for antigen – Affinity & avidity for pair – Environmental conditions • Temperature • pH: best in the neutral range of 6.0-7.5 • Time: need adequate time for complete binding of antibody & antigen General Considerations • Meet the antibody – Protein – IgG important in chemistry – Produced in response to foreign invaders • Humoral • Acquired 3 Antibodies • Monoclonal – Arise from one cell line – Provide high specificity • Polyclonal – Arise from many cell lines 4 General Considerations • the antigen – Elicits an antibody response – Has multiple sites (epitopes)to bind antibodies 5 General Considerations • An antigen-antibody reaction – Requires affinity and avidity – Determined by Law of Mass Action Antigen + Antibody Antigen-Antibody Complex 6 Affinity • Attraction between the antibody and antigen As antigens and antibodies come together, a chemical bond forms Stability depends on the “fit” of the connection • Most antibodies have a high affinity for their antigens • Property of the antigen 7 Avidity • Overall strength of antigen/antibody bond formed • Property of the antibody • The greater the avidity- the less likely to have crossreactivity 8 Law of Mass Action • Governs the reversibility of the antigen-antibody reaction. • Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation. • As affinity and avidity increases, strengthens reaction. Zone of Equivalence: Antigen-Antibody Complexes • Become visible when antibody/antigen concentrations are in the Zone of Equivalence • Zone of Equivalence – Optimal ratio of concentration of antibody to concentration of antigen that results in maximal precipitation – 2-3 Antibodies: 1 Antigen 10 Prozone – Antibody excess – No precipitation 11 Postzone • Antigen excess – No precipitation 12 General Principles of Immunoassays • Immunoassay Labels • Competitive Immunoassays • Noncompetitive Sandwich Immunoassays 13 Labeled Immunoassays • Antigen or antibody is labeled ( tagged ) with a substance that can be detected later on and allows for the detection of an antibody – antigen reaction • Types of tags Radioactive isotopes (RIA) Enzymes (EMIT, ELISA) Fluorescent molecules Luminescent labels 14 Fluorescence Polarization Immunoassay • Competitive Immunoassay • Homogenous assay – No separation step required • Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient ) compete for specific antibody (reagent) in a cuvet • The cuvet is exposed to polarized fluorescent light • Large molecules ( tagged - antigen – antibody complexes ) emit polarized light, where as smaller molecules ( free tagged antigens ) do not • The amount of polarized light emitted is inversely proportional to the concentration of patient ( untagged ) antigen • Fluorescence Polarization is used by the ABBOTT TDX analyzer, commonly used for Therapeutic Drug Monitoring ( TDM ) 15 Fluorescence Polarization Immunoassay 16 Lumininescence/Chemiluminescence • The process of exciting molecules by chemical means and measuring the light emitted as the molecules return to their ground/unexcited state. • Competitive & heterogeneous • Applications include quantitation of drugs, steroid and peptide hormones, etc. 17 Labeled Immunoassay • Heterogeneous or homogeneous • Heterogeneous assays called separation assays – Require multiple steps – Careful washing of surface to remove unbound reagents and samples. • Homogeneous assays do NOT require a separation step. – Mix reagents and patient sample. – Measure the labeled product. Competitive Immunoassays • Labeled known and patient unknown are added to reaction and “compete” for the target. – For example, looking for an antibody. – Add labeled reagent antibody of known specificity, patient sample and known antigen. – Patient antibody competes with reagent antibody for the target antigen. – Concentration is inversely proportional to results. • High concentrations of patient antigen means that more of the antibody-antigen complexes are unlabeled • Low concentrations of patient antigen means that more of the antibody-antigen complexes are labeled 19 Competitive Labeled Immunoassays 20 Non-Competitive Labeled Immunoassays “Sandwich” • A labeled reagent antibody is used to detect an antigen • Direct relationship between the concentration of the antigen and the bound antibody. 21 References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson . 22