IMMUNOASSAY FOR NATIVE ENZYME QUANTIFICATION IN BIOLOGICAL SAMPLES

In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was develope d since currently available methods could not be used because of eithe r their lack of sensitivity or the conditions prevailing in our sample s: turbidity of the medium, presence of redox systems other than gluco se oxidase, and high concentration of proteins. The principle of the m ethod is to coat a polystyrene surface with a fragment Fc-specific ant i-IgG, then with an antibody directed against the looked-for enzyme, w hich is simultaneously the antigen and the enzyme activity required fo r immunoenzymatic detection. We applied this concept to biological sam ples after glucose oxidase administration to mice. This method achieve s specificity and sensitivity (20 ng/mL or 1 ng) with samples of biolo gical origin. No marker is needed since the antigen itself possesses a n enzyme activity. This method, which requires a small sample volume ( 50 mu L, 20 mu L, if necessary), can be extended easily to the many en zymes currently used as markers. It could also be applied to the nativ e enzymes of medical interest for which antibodies and a colorimetric reaction are available.