A high throughput screening assay to quantify, visualize, and standardize biological activities: Enzyme-Linked Immuno-Culture Assay (ELICA)

A high throughput screening assay, termed enzyme-linked immuno-culture assay (ELICA), was originally developed to identify, visualize, and purify biological activities of unknown compounds, e.g., human recombinant proteins and morphogenetic proteins. The ELICA was based on concepts inherent to an ELISA for the identification and characterization of antibodies binding to antigens. The ELISA is performed by absorbing either antigens or antibodies to a nylon membrane, probing with either antibody or antigen with an attached HRP, then incubating with HRP substrate. The ELICA was based on a 96-well plate format. Cultured cells (or unknown compounds) are fixed to the plate surface using the ELICA fixative. The wells are then incubated in succession with an antibody to a phenotypic expression marker (PEM), the primary probe; followed by antibody against species of primary probe with an attached biotin, the secondary probe; followed by avidin with attached horseradish peroxidase enzyme, the tertiary probe. Then the wells are incubated with a soluble HRP substrate. The soluble HRP substrate is removed and scanned in a spectrophotometer to quantify amount of phenotypic expression marker, based on a titration curve. The wells are then incubated with an insoluble HRP which precipitates at sites of antibody binding, allowing visualization of labeled cells/material. The wells are photographed and then processed for DNA quantification. Each of the above procedures were performed in each well of 96-well plates. Results are reported as quantity of PEM standardized to quantity of DNA.