Bio-rad Laboratories, Inc.

Nathan Lewin

Judith I Blakemore

Vito J Mangiardi

Henry J Jeong

James F Monthony

Michael W Burgett

Paul F Wegfahrt

James E Fries

Emmy Tong-In Wong

C Steven Richards

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.

Single-incubation two-site immunoassay

Improved competitive binding serum folate assay in which serum folate bound by folate-selective protein is separated from free serum folate by removal with an anion exchanger, preferably intermediate base anion exchange resin. Preferable are resins characterized by having an aliphatic lattice, particularly, a polyalkyleneamine lattice having tertiary and quaternary amine groups. The resin is advantageously added for the separation function in tablet form.

Folate assay with ion exchange resin bound-free separation

The method of this invention is characterized by the use of hydrolyzed cross-linked polyacrylamide particles to which have been bonded, by means of covalent bonds, antibiodies against the thyroid hormone to be determined. The particles selected are of a size which forms a stable hydrophyllic suspension. In the preferred embodiment a measured quantity of unextracted human serum is mixed together with a blocking agent in an amount sufficient to displace the thyroid hormone to be measured from thyroxine binding globulin (TBG), a radioactively labeled thyroid hormone of the type to be measured, and the antibody-polyacrylamide complex. The hormone to be measured is displaced by the blocking agent followed by the competitive binding of the labeled and unlabeled hormone to the antibody-polyacrylamide particles. The particles are readily separated from the sample liquid and the radioactivity of the particle material and/or in the liquid is determined. Particles containing Alcian yellow or blue dye facilitate practice of the method.

Radioimmunoassay for thyroid hormone

This invention provides a method for the radioimmunoassay of thyroid-stimulating hormone which utilizes a rapid and convenient version of a double antibody procedure. Highly purified second antibody is bound, by means of covalent bonds, to hydrolyzed polyacrylamide particles to produce a two-phase system. The solid phase comprises immobilized second antibody bound to the reaction product of labeled and unlabeled thyroid-stimulating hormone with the first antibody (first antibody-antigen complex) and the liquid phase comprises free (unbound) labeled and unlabeled thyroid-stimulating hormone. The two phases are separated and the radioactivity of either phase is measured.

Radioimmunoassay for thyroid-stimulating hormone (TSH)

Serum folate assay procedure by competitive binding in which serum folate is stabilized while being freed from serum folate proteins in the presence of a dithiopolyol, preferably a 1,4-dithiotetritol such as dithiothreitol. Subsequently in the procedure, free folate is separated from competitively bound folate by the addition of dry dextran coated charcoal in a tablet form.

Assay method for serum folate

Improved competitive binding thyroid hormone assay in which thyroid hormone bound by thyroid binding protein is separated from free thyroid hormone by removal with an intermediate base anion exchange resin. Typical are resins characterized by having an aliphatic lattice, particularly, a polyalkyleneamine lattice having tertiary and quaternary amine groups. The resin is advantageously added for the separation function in tablet form.

Competitive binding thyroid assay with improved bound-free separation

Radioassay for vitamin B-12 using the unknown quantity of non-radioactive vitamin B-12 released from serum mixed with the radioactivity of a known quantity of radioactive vitamin B-12 tracer. A solution of intrinsic factor having a binding capacity less than the quantity of serum vitamin B-12 and radioactive vitamin B-12 is used to bind a portion of the vitamin B-12 mixture. The vitamin B-12 not bound to intrinsic factor is removed by addition of a bentonite-containing tablet. The quantity of radioactive vitamin B-12 bound to intrinsic factor is compared with standard values and the unknown serum vitamin B-12 obtained. In the steps of the procedure the acid assay medium is pre-combined with the radioactive tracer so that the radioactive vitamin B-12 tracer receives the same treatment as serum vitamin B-12. Certain of the other reagent solutions are pre-combined and the concentration of the components adjusted so that the volume used of each of these other reagent solutions is the same in different assay steps. Thus, fewer pipetting steps are necessary.

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Corte Madera, CA, United States of America

Nathan Lewin