CLS 1113Introduction to Clinical

Laboratory Practices

Unit 5

Labeled Immunoassays

Chapter 10

Labeled Immunoassays

• Designed for Ags and Abs that DO NOT react in precipitation or agglutination tests due to their small size or low concentrations.

• Indirect method of detection:

• Competitive vs. Non-Competitive– Test Antigen or Antibody competes for binding

sites

Elements of Labeled Immunoassays

• Ligands

• Antibodies

• Standards or Calibrators

• Separation Methods

• Detection of Label

Radioimmunoassay (RIA)

• Competitive binding assay

• Uses a radioactive substance as a label– 3H - Tritiated hydrogen– 125I - Iodine 125

Radioimmunoassay

• Two Types– Number 1

Radioimmunoassay

Number 2

Enzyme Immunoassay

• Immunoassay labels

• Enzymes– Cheap– Readily available– Long shelf life– Easily adapted to automation

ELISA, Figure 10-4, page 149

Enzyme Immunoassay

• Enzymes are naturally occurring molecules that catalyze specific biochemical reactions.

• They react with suitable substances to produce products that are chromogenic (color), fluorogenic, or luminescent.

ELISA: Sandwich method

Figure 10-5, page 149

Fluorescent Immunoassay

• Similar to ELISA but a fluorochrome is used rather than an enzyme.

• Fluorochromes have the ability to absorb energy from light an emit it at a longer wavelength.

Fluorescent Immunoassay: Direct and Indirect

Chemiluminescent Immunoassays

• Chemiluminescence is the production of light energy due to a chemical reaction.

• Certain substances when oxidized can give off short or long bursts of light energy.