The ELISA method was made possible because of scientific advances in a number of related fields. Technology enabling the
production of antigen-specific monoclonal antibodies by Kohler and Milstein (1975) led to their use as probes for detecting
individual molecules in complex protein mixtures or tissue samples. Initially, detection was achieved by radioimmunoassay
using antibodies labeled with radioisotopes, but because of health risks alternatives were sought. Avramais (1966, 1969) and
Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable
signal with solutions containing appropriate substrates. With the development of fluorescence technology, signal generation
using fluorophore-labeled antibodies has also become prevalent, especially in multiplex arrays.