Method for determining analyte level

A method of determining a level of an analyte on a solid surface comprising the steps of:

• providing a source of hydrogen peroxide;
• providing a source of an affinity-enzymatic compound of formula A-C-B, wherein A is a detecting agent having affinity for said analyte, B is an enzymatic visualizing agent consisting of peroxidase and C is a binding agent linking said detecting agent and said visualizing agent to one another;
• providing a multilayer analytical element comprising a solid support and a hydrophilic layer thereon, said hydrophilic layer being formed of gelatin, polyvinyl alcohol or a mixture thereof and containing a developing agent and a hydrophobic color-producing agent in a total amount of 5 to 30 weight % based on the weight of said hydrophilic layer, said developing agent and said color-producing agent being present in a weight ratio of 1:0.3 to 1:3.0;
• applying a predetermined amount of said affinity-enzymatic compound onto said surface in a selected area thereof and allowing said compound to stand thereon for a period of time sufficient to cause binding of said detecting agent to said analyte on said surface;
• removing any affinity-enzymatic compound with unbound detecting agent from said surface;
• applying a predetermined amount of hydrogen peroxide onto said surface in said selected area;
• and
• pressing said multilayer analytical element against said surface in said selected area;
• wherein said developing agent undergoes oxidation by the hydrogen peroxide in the presence of said peroxidase, bound via said binding and detecting agents to said analyte, and the developing agent in oxidized form reacts with said color-producing agent to form a colored product having a color indicative of the level of said analyte.