Syva Company

Moshe Schwarzberg

Edwin F Ullman

A method is provided for preparing immunoassay reagents involving labeled members of specific binding pairs substantially enriched relative to contaminating labeled materials. The method involves conjugating a member of a specific binding pair to a support by a covalent bond which is cleavable under mild conditions to provide a binding pair member-support conjugate. Combining the binding pair member-support conjugate with a labeled composition containing the reciprocal member of the binding pair, so that the labeled reciprocal member becomes bound to the support through the binding of the specific binding pair. Separating the support to which is bound the labeled member from the remaining labeled material and then cleaving the bond joining the labeled specific binding pair to the support to provide labeled reagent for immunoassays. In particular, an antibody is linked to a support by disulfide linkage and a composition containing the reciprocal antigen to the antibody is labeled with a chromophore, particularly fluorescer. The support is freed of labeled material other than the desired labeled antigen and the disulfide link cleaved to provide labeled reagent for immunoassays.

Purification of reagents by disulfide immobilization

Immunoassays are provided employing antibodies and fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.

Fluorescence quenching with immunological pairs in immunoassays

Methods and compositions are provided for improved protein binding assays by preparing compositions having indirectly labeled ligands substantially free of label conjugated to materials other than the indirectly labeled ligand. The method for preparing the compositions involves employing a monovalent receptor to which the label is conjugated and combining the monovalent receptor labeled conjugate to the ligand, either in pure or impure form. The mixture is then segregated according to molecular weight and the ligand conjugated to the labeled receptor isolated. This conjugate may then be directly used as a reagent in a protein binding assay, where the assay mixture is substantially free of label other than labeled receptor bound to ligand. The labels will be for the most part of relatively low molecular weight, while the receptor is preferably a Fab fragment. The ratio of receptor to ligand will be chosen so as to provide reasonable molecular weight distinctions between unbound ligand, unbound labeled receptor, ligand bound to labeled receptor, and other materials which may be in the mixture. Various techniques may be used for the separation. The assays are performed in accordance with known methods employing a second receptor composition, which may be labeled or unlabeled.

Assay employing a labeled Fab-fragment ligand complex

Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, or whether antibodies are to be assayed, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand or antibody present in the assay medium. In carrying out the assay for ligands, the unknown and antiody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antiobodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The fluorescar is electronically excited, for example by irradiation with light at a wavelength, absorbed by the fluorescing molecule and the amount of emitted light determined. By employing appropriate standards, the presence and amount of the ligand can be determined. The technique for the determination of antibodies in a competitive mode is substantially the same except that ligand is added as a reagent.

Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.

Immunoassays employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.

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Palo Alto, CA, United States of America

Moshe Schwarzberg