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
  .