MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Immunoassays

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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

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Antibodies

• Monoclonal– Arise from one cell

line– Provide high

specificity

• Polyclonal– Arise from many cell

lines

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General Considerations

• the antigen– Elicits an antibody

response– Has multiple sites

(epitopes)to bind antibodies

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General Considerations

• An antigen-antibody reaction– Requires affinity and

avidity– Determined by Law of

Mass Action

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Antigen + Antibody Antigen-Antibody Complex

Affinity

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• 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

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

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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

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Prozone– Antibody excess– No precipitation

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Postzone

• Antigen excess– No precipitation

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General Principles of Immunoassays

• Immunoassay Labels• Competitive Immunoassays• Noncompetitive Sandwich Immunoassays

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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 moleculesLuminescent labels

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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 )

Fluorescence Polarization Immunoassay

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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.

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

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Competitive Labeled Immunoassays

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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.

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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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