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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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Title: CLINICAL CHEMISTRY CHAPTER 5 Author: KIRK NEWPORT Last modified by: kbrophym Created Date: 9/12/2002 6:30:52 PM Document presentation format – PowerPoint PPT presentation

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Title: MLAB 2401: Clinical Chemistry Keri Brophy-Martinez


1
MLAB 2401 Clinical ChemistryKeri Brophy-Martinez
  • Immunoassays

2
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

3
General Considerations
  • Meet the antibody
  • Protein
  • IgG important in chemistry
  • Produced in response to foreign invaders
  • Humoral
  • Acquired

4
Antibodies
  • Monoclonal
  • Arise from one cell line
  • Provide high specificity
  • Polyclonal
  • Arise from many cell lines

5
General Considerations
  • the antigen
  • Elicits an antibody response
  • Has multiple sites (epitopes)to bind antibodies

6
General Considerations
  • An antigen-antibody reaction
  • Requires affinity and avidity
  • Determined by Law of Mass Action

Antigen Antibody Antigen-Antibody
Complex
7
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

8
Avidity
  • Overall strength of antigen/antibody bond formed
  • Property of the antibody
  • The greater the avidity- the less likely to have
    cross-reactivity

9
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.

10
Zone of EquivalenceAntigen-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

11
Prozone
  • Antibody excess
  • No precipitation

12
Postzone
  • Antigen excess
  • No precipitation

13
General Principles of Immunoassays
  • Immunoassay Labels
  • Competitive Immunoassays
  • Noncompetitive Sandwich Immunoassays

14
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

15
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 )

16
Fluorescence Polarization Immunoassay
17
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.

18
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.

19
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

20
Competitive Labeled Immunoassays
21
Non-Competitive Labeled ImmunoassaysSandwich
  • A labeled reagent antibody is used to detect an
    antigen
  • Direct relationship between the concentration of
    the antigen and the bound antibody.

22
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
    .
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