The invention relates to a detection method and kit for the identification and quantification of analytes in biological specimens by non-captive substrate liquid phase immunoassay techniques.
Observations of colloidal gold or silver concentrations have been used in immunoassays in conjunction with solid phase diffusion assays. For example, European Patent No. 207,152 discloses a solid phase diffusion assay utilizing a porous sheet having ligands or receptors prebound to the sheet prior to the application of an analyte and a colloidal gold or silver labeled ligand or receptor. However, assays performed on ligand or antiligand coated porous films must be specifically tailored for a particular analyte and, in view of the limited surface area and reaction time with antigens, this process may be ineffective if the affinity of the substrate bound antiligand for the labeled analyte is low. Furthermore, specific areas of the porous sheet substrate may become overloaded with analyte due to restricted radial diffusion, thereby allowing colloidal metals already bound to analytes to pass through the porous substrate without binding to the surface of the porous substrate for subsequent visual detection.
U.S. Pat. No. 4,853,335 to Olsen et al., discloses a sandwich immunoassay method by premixing a biological specimen with: (1) a colloidal gold labeled ligand or antiligand and (2) solid phase captive particles coated with a ligand or antiligand and applying the subsequent mixture onto the surface of a porous film. By requiring solid phase captive particles this process may distort the visual detection measurements because uncoupled captive particles may block the pores of the substrate and prevent rapid passage of uncoupled colloidal gold.
In both of the foregoing patents a ligand, antiligand or receptor is necessarily prebound to a captive substrate in the form of an insoluble porous sheet or a solid captive particle such as a latex particle. It is desirable to develop a method for quantitative and qualitative detection of an analyte, such as C-reactive protein, using a non-captive substrate in order to accelerate passage of non-complexed immunoassay constituents through the pores of the test support. The use of a non-captive substrate technique presents the most simplistic and flexible approach to the rapid and efficient identification and quantification of ligands and analytes and diagnosis of a disease.
By eliminating the need for prebinding ligands to solid supports or substrates as a part of the assay system, non-captive substrate techniques promote accurate detection of analytes and cost savings in the production and subsequent use of diagnostic test kits.
The object of the present invention is to overcome the deficiencies of prior art solid phase captive immunoassay techniques through the development of a non-captive substrate immunoassay that is easily conducted and accurate in detection and measurement.
The present invention is an immunoassay performed on a liquid biological specimen, e.g. serum, plasma, whole blood, etc., for a specific analyte by forming a test sample by contacting the liquid biological specimen with: (a) a binding substance of a ligand, antiligand or receptor capable of binding the analyte and (b) a detector substance of a colloidal metal labeled ligand or antiligand to form a test sample. Both the binding substance and the detector substance are in liquid phases. The test sample is then applied onto a defined zone of an insoluble porous support film having a pore size impassable to a complex formed between the analyte, if present, with the binding substance and the detector substance, but passable to the binding substance and detector substance while remaining uncomplexed in the absence of the desired analyte.
If the analyte is present in the test specimen, the analyte binds with both the detector substance and the binding substance to form a visually discernable precipitable complex on the surface of the porous support film. After application of the test sample to the porous support, the surface of the porous support is visually inspected for color to determine the presence and approximate quantity or the absence of the analyte being assayed.