Immobilization of active anti-cortisol antibodies onto negatively charged solid phase surfaces results in a shift of the antibody pH optimum to an acidic range thereby permitting effective yet minimal use of deblocking agents and an extraction-free immunoassay for cortisol.

This invention provides a two-site immunoassay method for a ligand utilizing a single incubation. The multivalent ligand, labeled receptor for the ligand and unlabeled receptor for the ligand covalently bound to a solid-phase support are incubated as a substantially stable suspension to produce a solid and liquid phase. The solid and liquid phases are separated from each other and the labeled receptor in either phase is quantitated. The method may be used to assay for human thyroid stimulating hormone using purified, radioactively labeled antibodies and unlabeled antibodies covalently bound to hydrolyzed polyacrylamide particles.

A method and an implementation are disclosed for measuring the contents of a certain component (e.g. a hormone) in a biological fluid. The basic principle of the method is that a complex is formed starting from the antagonist of the component sought for with the product of conjugation of the latter with a tracer (e.g. a radioisotope), said complex is introduced in the biological fluid to be tested and the quantity of the tracer which has been set free is measured with a measuring or dosing technique which is appropriate for the tracer which has been selected.

A reagent is disclosed for determining the concentration of a ligand in blood serum. The reagent includes a protenaceous, three-dimensional, serum-insoluble complex which is readily collected by centrifugation, the complex comprising non-specific immunoglobulins, anti-ligand immunoglobulins, immunoglobulins which are antibodies to the previously mentioned immunoglobulins and which are complexed therewith, and a labeled ligand complexed with the anti-ligand immunoglobulins. The reagent is particularly useful in competitive binding analyses such as radioimmunoassays.

The present invention provides methods for the simultaneous measurement of triiodothyronine (T.sub.3) and thyroxine (T.sub.4) in biological fluids such as serum by direct equilibrium dialysis and immunoassay. Specifically, the method comprises dialyzing the serum sample to equilibrium in a physiological buffer system so that the free T.sub.3 and the free T.sub.4 are separated from T.sub.3 and T.sub.4 bound to serum proteins. The method further comprises combining a measured quantity of the dialyzed serum sample having free T.sub.3 and free T.sub.4 with reagents comprising a measured quantity of T.sub.3 labelled with a detectable marker and a measured quantity of T.sub.4 labelled with a detectable marker; an anti-T.sub.3 antibody of sufficient specificity and in sufficient quantity to bind a measurable quantity of the free T.sub.3, and an anti-T.sub.4 antibody of sufficient specificity and in sufficient quantity to bind a measurable quantity of the free T.sub.4. The method then comprises allowing reaction of the free T.sub.3 and the free T.sub.4 and the labelled T.sub.3 and the labelled T.sub.4, with the anti-T.sub.3 antibody and the anti-T.sub.4 antibody to proceed substantially to equilibrium to thereby produce antibody bound labelled T.sub.3 and antibody bound labelled T.sub.4. Finally, the method comprises separating the unbound labelled T.sub.3 from the antibody bound labelled T.sub.3 and the unbound labelled T.sub.4 from the antibody bound labelled T.sub.4 ; and determining the levels of T.sub.3 and T.sub.4 in the sample by comparing relative amounts of antibody bound labelled T.sub.3 and T.sub.4 and unbound labelled T.sub.3 and T.sub.4. In one embodiment of this method, the assay is a radioimmunoassay.