Solid phase system for ligand assay

In an analytical method for conducting a solid phase fluoresence immunoassay of a fluid sample by immunological differentiation of complex molecules in such fluid by contacting such fluid, containing an unknown level of analyte, and a fluorophore-labeled indicator, with an immobilized binding material within a reaction zone of a porous medium under conditions favoring immunochemical interaction of said binding material with the analyte and the labeled indicator, whereby the analyte of interest of said fluid and the labeled indicator compete for available sites on said binding material, said labeled indicator comprising a fluorophore conjugated to a ligand, applying a wash fluid to said porous medium to remove unreacted labeled indicator from said medium, and measuring the amount of labeled indicator immunochemically bound to the binding material within the porous medium, the improvement comprising:

• a. applying, to substantially the center of a reaction zone within an inert porous medium, a stream of eluting solvent, the quantity of such eluting olvent applied to said reaction zone being sufficient to effect radial separation, within said inert porous medium, of unbound fluorophore-labeled indicator from bound fluorophore-labeled indicator within said reaction zone;
• and
• b. observing the extent to which the bound fluorophore-labeled indicator is present within a delimited area of said reaction zone by measurement of the level of fluoresence, within a delimited area of the reaction zone, said delimited area of said reaction zone being essentially free of unbound indicator.

A sandwich method for conducting a fluoresence immunoassay of a fluid sample within the interstices of a solid, inert porous medium, said fluid sample containing an unknown level of analyte, the method comprising:

• a. providing a binding material immobilized within a finite zone of a solid, inert porous medium, said binding material being capable of immunological reaction with said analyte from among the constituents of the fluid sample;
• b. applying, under binding conditions, to substantially the center of said finite zone containing said immobilized binding material, a fluid sample containing the analyte for which the said binding material is specific, said analyte being applied as a solution so as to permit diffusion thereof within a reaction zone of the solid, porous medium containing the immobilized binding material, whereby substantially all of said analyte is bound to the binding material;
• c. appying a fluorophore-labeled indicator to substantially the center of said reaction zone, under conditions which allow said labeled indicator to become immunochemically bound to said analyte in a manner which can be correlated to the amount of analyte in the reaction zone, said indicator comprising a fluorophore conjugated to a ligand;
• d. applying, to substantially the center of said reaction zone, a stream of eluting solvent said solvent being applied in a quantity sufficient to effect radial separation, within said porous medium, of unbound labeled indicator from the indicator which is bound within said reaction zone;
• and
• e. observing the extent to which the bound fluorophore labeled indicator is present within a delimited area of said reaction zone, said delimited area of said reaction zone being essentially free of unbound indicator.

In an analytical method for a solid phase fluoresence immunoassay of a fluid sample by immunological differentiation of complex molecules in such fluid by contacting such fluid, containing an unknown level of analyte, with an immobilized binding material within a reaction zone of a porous medium under conditions favoring immunochemical interaction of said binding material with the analyte, whereby substantially all of the analyte of interest of said fluid is immunochemically bound to the binding material, further contacting the analyte with an fluorophore labeled indicator under conditions favoring immunochemical interaction of said indicator with the analyte, said indicator comprising a fluorophore conjugated to a ligand, applying a wash fluid to said porous medium to remove unreacted labeled indicator from said medium, and measuring the amount of labeled indicator which is immunochemically bound to the analyte, the improvement comprising:

• a. applying, to substantially the center of a reaction zone within an inert, porous medium, a stream of eluting solvent, the quantity of solvent applied to said reaction zone being sufficient to effect radial separation within said porous medium, of unbound fluorophore labeled indicator from bound fluorophore labeled indicator within said reaction zone;
• and
• b. observing the extent to which the bound fluorophore labeled indicator is present within a delimited area of said reaction zone by measurement of the level of fluoresence within a delimited area of said zone, said delimited area of said reaction zone being essentially free of unbound labeled indicator.