Non-captive substrate liquid phase immunoassay

A method for the quantitative or qualitative detection of a C-reactive protein in a biological specimen of whole blood, serum, or plasma, comprising the steps of:

• (a) adding to a biological specimen a reagent solution consisting essentially of (i) a binding substance in the liquid phase comprising an unlabeled antibody to C-reactive protein, and (ii) a detector substance comprising an antibody to C-reactive protein said antibody labeled with colloidal gold, the reagent solution forming a precipitable complex with the C-reactive protein if the C-reactive protein is present and forming no such complex in the absence of the C-reactive protein in the biological specimen, the reagent solution free of solid phase particles;
• (b) applying the biological specimen and the added reagent solution to a defined zone on a porous support, the support having a maximum effective pore size smaller than a complex formed between the C-reactive protein, the binding substance and the detector substance and having minimal effective pore size larger than each of the substances contained within the reagent solution if a precipitable complex is not formed with the C-reactive protein;
• and
• (c) assessing the defined zone for color development and intensity caused by the formation of the complex formed by the C-reactive protein, the binding substance and the detector substance, the color development correlating with the presence of the C-reactive protein in the biological specimen.

A kit for the quantitative, semi-quantitative and qualitative determination of an analyte in a biological sample consisting essentially of:

• (a) an insoluble porous support, said porous support being covered by a layer of water-impermeable material having at least one hole therein, the porous support having an maximum effective pore size smaller than a complex formed between the analyte, the binding substance and the detector substance and having minimal effective pore size larger than each of the analyte, the binding substance and the detector substance if a precipitable complex with an analyte is not formed, so that the analyte, the binding substance and the detector all can pass through the support;
• (b) a solution comprising:
• (i) a binding substance in liquid phase selected from the group consisting of a ligand and an antiligand which specifically binds to an analyte, and (ii) a detector substance selected from the group consisting of a colloidal gold labeled ligand, a colloidal silver labeled ligand, and colloidal gold labeled antiligand and a colloidal silver labeled antiligand, wherein the antiligand or the ligand in the detector substance binds to the analyte, all components of the solution being small enough to pass through the effective pore size of the pores in the porous support if binding of the binding substance to the analyte does not occur the solution free of solid phase particles.