Immunochemical potentiometric method for HIgG and anti-HIgG determination, using a NH3 probe and immunoprecipitation as preconcentration procedure

With the aim of improving the LOD value of a previous method, able to determine HIgG using a classic immunosensor, a pretreatment involving immunoprecipitation of the urease enzyme marked immunocomplex was introduced. By new method we determined HIgG using anti-HIgG conjugated with urease for immunocomplex formation, and then protein G-agarose was added for the immunoprecipitation process, which was able to concentrate the immunocomplex (and thus also the HIgG) up to 10,000-fold. In this way the marked immunocomplex was separated from the marked anti-HIgG excess, subsequently detected after urea addition in solution, by a gas diffusion potentiometric electrode for NH3. The formation of the marked antigen-antibody complex was the first step of the assay, the immunoprecipitation of the complex; the second and third step consisted first in removal of the nonspecific binding protein and then in eluting the precipitated immunocomplex from the microcolumn. The last step was the enzymatic measurement as checked by NH3 sensor. In addition, using the same procedure, namely, using the same conjugated urease and the same sensor for the NH3, it was possible to determine also the anti-HIgG, in any case preceding the process of preconcentration by immunoprecipitation by means of a classical competitive process in homogeneous solution.