Adaptation of commercial immunoassays to analysis of complex biological substances

An Intracellular Adhesion Molecule I (ICAM-1) immunoassay from R and D Systems, and a Melanoma Inhibitory Activity (MIA) immunoassay from Roche Diagnostics were tested for accurate quantitation within complex biological substances such as cell lysates. Prior to assay, lysates of melanoma cells were treated with detergents to obtain soluble antigens. Maximum ICAM-1 and maximum MIA were detected after treatment using 0.8% Triton X-100. Two key aspects of assay accuracy were: 1) determining the dilutions of test sample that provided accurate quantitation (sample range), and 2) performing spiking experiments at these dilutions to determine absence or presence of a "matrix" effect due to biological complexity of the sample. A high degree of accuracy was found by diluting this particular cellular extract 50-fold prior to ICAM-1 assay, or only 5-fold prior to MIA assay. In addition, the bicinchoninic acid protein assay was analyzed to test the accuracy of protein quantitation of cellular lysates. Precision, limits of detection, and quantitation, robustness, linearity, and specificity also were tested for the immunoassays.