Clinically significant concentrations of digoxin can be determined by reacting an unknown amount of digoxin, a known amount of labelled digoxin, and a composite consisting of anti-digoxin antibodies coupled through an intermediate silane to magnetically responsive particles; magnetically separating the immunochemical complexes formed thereby, counting the radioactivity of either the separated products or remaining solution, and relating the count to a standard curve.
Method and apparatus suitable for automatic assay of fluid samples, particularly immunoassays, in which a reaction product of the assay is bound to a magnetically attractable particle and separated from the reaction mixture by use of a magnetic trap. The method and apparatus are of especial use in radioimmunoassays carried out in an automated continuous-flow manner.
A mixture of two different kinds of particles having distinctive, different properties is employed for determining the presence of a select protein in, or the absence of a select protein from, a solution. The first kind of particle provides a property facilitating separation, while the second kind of particle provides a property facilitating detection. The particles are coated with the same protein, a protein able to interact specifically with the select protein.
The concentration of thyroxine binding globulin (TBG) in a fluid sample can be determined via radioassay technique with an immunochemical composite comprising anti-TBG antibodies immobilized on a water-insoluble carrier. The composite is incubated with the sample in a quantity sufficient to assure complexation of substantially all TBG in the sample to the antibodies of the composite. The quantity of TBG complexed is determined with radiolabeled thyroxine (T.sub.4) which complexes with at least a portion of the complexed TBG. By separating the labeled composite from the remaining labeled T.sub.4, and counting the bound (or unbound) T.sub.4, the concentration of TBG can be determined from a standard curve.
A secondary antibody capable of stabilizing the binding of a small molecule to its binding protein is described which secondary antibody is capable of binding said binding protein in the presence of an in the absence of the small molecule but is not capable of binding said small molecule in the absence of binding protein. Such antibodies may be obtained by forming a complex between a small molecule and its binding protein, using the complex to raise antibodies and selecting the antibodies. The antibodies may be used in competitive assays in which it is desired to improve the binding of a small molecule or labelled small molecule to its binding protein.
Immunoassays for digoxin and its congeners are provided by pre-treating the serum sample with dilute aqueous alkaline solutions under mild conditions and for short periods of time, preferably in combination with a chelating agent, prior to carrying out the immunoassay. The method finds particular application with radioimmunoassays and homogeneous enzyme immunoassays.
