[Detection and quantification of weak concentrations of antigens using a sensitive and direct method of demonstrating...

Using agarose gel coated on GelBond film sheets, and using Coomassie blue stain followed by silver stain, a sensitive microassay has been developed for detecting small amounts of antigen-antibody precipitates, and for quantitating low concentrations of antigen. In order to obtain a high sensitivity, antigen-antibody ratios were adjusted imperatively close to the equivalence in double-diffusion, and for quantitative estimation, single radial immunodiffusion was performed, according to Mancini, by measuring circles at the end point. The use of a double staining procedure allows to detect as little as 8 micrograms/ml antigen by Ouchterlony and 200 ng/ml by Mancini technique. The sensitivity of the method is 100 times greater than classical techniques and other advantages such as the need for minimal amounts of unconcentrated samples, the absence of radioactive labelling, and the absence of interference due to a second or a third antibody coat, make this assay useful for analyzing and quantitating monoclonal antibodies obtained by hybridoma or B-cell immortalization.