o Enzyme linked immunosorbent assays (ELISA)

Label for immunoassay (labels can detect substances present in concentrations as low as

picograms/mL) is enzyme

Label is attached to Ab or Ag to allow visualization or quantification of Ab-Ag reaction

Enzymes: alkaline phosphatase, horseradish peroxidase – enzyme causes conversion of

added substrate to colored product that is either sol or insoluble

Commonly used in clinical and exp labs or sold OTC

EMIT (enzyme-multiplied immunoassay technique) – ELISA variation that doesn’t

require separating free from bound ligand; competitive binding assay in solution: free

ligand competes with enzyme-labeled ligand for binding an Ab this binding

inactivates enzyme prevents cleavage of substrate and release of colored product; if

free ligand present binding of enzyme-labeled ligand to Ab more inhib more

colored product formed when substrate added

Color change proportional to concentration of ligand in test sample: more free ligand

more color

Pregnancy tests – ligand to be detected is captured by solid phase Ab and then detected

with labeled Ab

o Radioimmunoassay (RIA)

Label is radioactive isotope

Iodine-125, sulfer-35, carbon-14, tritium

Labels detected by autoradiography (x-ray exposed to radioactive surface to generate

image)

Can also detect label by counters that count number of disintegrations per minute

Can use phosphorescence plate imager – expose sample to plate image scanned and

generated

Quantitative but require special instruments and facilities; therefore have been replaced

by ELISA