Method of detection of analyte active forms and determination of the ability of substances to bind into analyte active sites

A method for detection of active form of analytes in a sample and/or for determination of ability of tested substances to bind to the active site of these analytes, characterized in that it comprises the following steps:

• a) analyte or group of analytes from the sample is immobilized on the surface of a solid carrier either by non-specific non-covalent adsorption or by covalent binding of surface functional groups of the analyte and corresponding functional groups of the solid carrier, or preferably via a binding molecule which is bound to the surface of the solid carrier before immobilization of the analyte or group of analytes and is capable of selectively binding the analyte or group of analytes contained in the sample during incubation of the solid carrier with the sample;
• b) analyte or group of analytes is incubated with a detection probe which binds selectively to the analyte or group of analytes via a compound for selective binding to the analyte active site;
• whereas the probe consists of
• a low molecular compound having molecular weight of up to 2500 Da for selective binding to the analyte active site;
• an oligonucleotide tag, optionally with a covalently attached fluorophore, biotin or a chemical group, and
• a chemical linker covalently linking the compound for selective binding to the analyte active site and the oligonucleotide tag;
• and
• optionally, the incubation is carried out in the presence of a tested substance whose ability to bind to the active site is to be tested, or a mixture of such substances;
• c) then the solid carrier is washed to remove unbound detection probe;
• d) subsequently, the amount of bound detection probe is determined, preferably directly on the solid carrier or after releasing, whereas this amount is proportional to the amount of the analyte or group of analytes in the sample.