Immunoassays
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What are immunoassays?
Utilize antibodies or antigens as reagents to measure analytes Based on antigen:antibody interactions Ag + Ab ↔ Ag:Ab These reactions are reversible
What is the advantage of immunoassays?
Very sensitive and reliable.
What can immunoassays be used for?
Can use immunoassays to measure proteins, hormones, drugs (therapeutic and abuse), metabolites, tumour markers, etc.
What are the five primary classes of immunoglobulins? and their chains?
Antibodies are produced in response to antigen exposure Five primary classes: IgA, IgD, IgE, IgG, and IgM Two identical heavy chains (α,δ,ε,γ, and μ) and two identical light chains (κ and λ).
What is the type of bonds between the chains?
Disulfide bonds between chains.
What molecule that the immune system recognizes as non-self can be used as a reagent in an immunoassay?
Antigens
What is affinity?
Strength of binding between an antibody and epitope of an antigen Antibody attaches via the antigen-binding site of the Fab region Determines sensitivity of method
What is avidity?
Binding strength between a number of binding sites Likelihood of separation is inversely related to the binding strength of the antibody and epitopes
What is specificity?
Specificity allows detection of one epitope in the complex matrix of the sample. Specific antibodies will react with one epitope and not others.
When does cross-reactivity occur?
Occurs when antibodies bind with structurally similar epitopes. Dependent on chemical composition, physical forces, and molecular structure.
What is a monoclonal antibody?
Arising from one cell line Antibody made by cloning Each antibody is identical
How are monoclonal antibodies produced?
Produced by fusing mice cells (sensitized lymphocytes from spleen) with myeloma cells (cancerous cells) to produce one clonal line of antibodies
What are monoclonal antibody reagents specific for?
Monoclonal antibody reagents will have one antibody specificity
What are polyclonal antibodies? How are they produced?
• Arising from many cell lines • Immunizing animals to produce antibodies –> Many B cells producing multiple antibodies • Polyclonal antibody reagents will have a mixture of antibody specificities
Define ligand.
Analyte being measured (antibody, hormone, drug, etc.)
What is a conjugate?
Labeled reagent. Antigen or antibody covalently attached to label.
What is a substrate?
A substrate creates the reaction to be measured.
Given an example of a substrate.
Example of the substrate peroxidase: 1. Reagent antibody labeled with horseradish peroxidase (Ab-HRP) + Peroxidase → O2 2. O2 + chromagen → coloured product read by spec
What are labeled immunoassays?
• Tagged with a component that allows detection or visualization of immune complexes • Measure concentration or activity of label • Not part of ag:ab reaction • Ag or ab can be labeled
What are some examples of types of labels used?
Labels may be: radioactive, enzymes, fluorescent, or luminescent.
What are common enzymes used with labeled immunoassays?
Common enzymes: alkaline phosphatase, horseradish peroxidase, glucose-7-P-dehydrogenase
How do you measure unlabeled immunoassays?
You can measure Ag:Ab complexes using nephelometry (scattered light). Note: No colour change, complexes become insoluble. Exists as particles in a solution when bound, soluble on own.
What may be added to unlabeled immunoassays to enhance the reaction?
Latex beads may be added to enlarge complex and make the reaction more visible.
What is a competitive immunoassay? How do you relate the signal to concentration?
Patient antigen and labeled reagent antigen with the same specificity compete for the same binding site on the antibody Inversely proportional More signal = less analyte of interest Getting signal from labeled reagent antigen