Heterogeneous immunoassay for digoxin using ouabain as a separation means

Claim 2

A noncompetitive immunoassay for the measurement of digoxin in a test sample, said assay comprising the steps of: (a) forming a reaction mixture by contacting a molar excess of labeled monovalent or divalent anti-digoxin antibody with the test sample, whereby a fraction of said antibody forms a complex with the digoxin and a fraction remains free; and (b) contacting the reaction mixture with a solid phase having ouabain immobilized thereon, the ouabain being present in an amount capable of binding all of the free antibody, whereby the free antibody is bound to the solid phase; and (c) measuring the amount of complex which elutes from the solid phase by measuring the activity of the label.

Claim 3

The immunoassay of claim 1 wherein the label is an enzyme, radioisotope, chromophore or fluorophore.

Claim 4

The immunoassay of claim 1 wherein the monovalent antibody is an Fab fragment or Fab' fragment.

Claim 5

The immunoassay of claim 1 wherein the divalent antibody is an F(ab') 2 fragment.

Claim 6

The immunoassay of claim 1 wherein the solid phase is beaded agarose, beaded dextran, polyacrylamide or glass.

Claim 7

The immunoassay of claim 5 wherein the solid phase is packed in a column.

Claim 8

The immunoassay of claim 1 wherein the solid phase is beaded dextran, the monovalent antibody is an Fab' fragment and the label is β-galactosidase.

Claim 9

The immunoassay of claim 1 wherein the solid phase is beaded dextran, the divalent antibody is an F(ab') 2 fragment and the label is β-galactosidase.

Claim 10

The immunoassay of claim 7 wherein the measurement step (c) is performed by contacting the eluted β-galactosidase labeled, Fab' anti-digoxin antibody/digoxin complex with o-nitrophenyl-β-D-galactopyranoside.

Claim 11

The immunoassay of claim 8 wherein the measurement step (c) is performed by contacting the eluted β-galactosidase labeled, F(ab') 2 anti-digoxin antibody/digoxin complex with o-nitrophenyl-β-D-galactopyranoside.

Claim 12

A competitive immunoassay for the measurement of digoxin in a test sample, said assay comprising the sequential steps of: (a) forming a reaction mixture by contacting a sample suspected of containing digoxin with a molar excess of ouabain immobilized on a solid phase; (b) contacting the reaction mixture with a labeled, monovalent or divalent anti-digoxin antibody, said antibody being in immunochemical excess over digoxin, but in immunochemical deficiency relative to ouabain; (c) allowing a reaction to take place whereby a fraction of the antibody forms a first complex with the digoxin and a second fraction forms a complex with the immobilized ouabain; (d) separating the first fraction from the second fraction; and (e) measuring the amount of label present in either the first or the second fraction.

Claim 13

A competitive immunoassay for the measurement of digoxin in a test sample, said assay comprising the sequential steps of: (a) forming a reaction mixture by contacting a sample suspected of containing digoxin with a molar excess of ouabain immobilized on a solid phase; (b) contacting the reaction mixture with a labeled, monovalent or divalent anti-digoxin antibody, said antibody being in immunochemical excess over digoxin, but in immunochemical deficiency relative to ouabain; (c) allowing a reaction to take place whereby a fraction of the antibody forms a first complex with the digoxin and a second fraction forms a complex with the immobilized ouabain; (d) separating the first fraction from the second fraction; and (e) measuring the amount of label present in either the first or the second fraction.

Claim 14

The immunoassay of claim 11 wherein the label is an enzyme, radioisotope, chromophore or fluorophore.

Claim 15

The immunoassay of claim 11 wherein the monovalent antibody is an Fab fragment or Fab' fragment.

Claim 16

The immunoassay of claim 11 wherein the divalent antibody is an F(ab') 2 fragment.

Claim 17

The immunoassay of claim 11 wherein the solid phase is beaded agarose, beaded dextran, polyacrylamide or glass.

Claim 18

The immunoassay of claim 11 wherein the solid phase is beaded dextran, the monovalent antibody is an Fab' fragment and the label is β-galactosidase.

Claim 19

The immunoassay of claim 11 wherein the solid phase is beaded dextran, the monovalent antibody is an F(ab') 2 fragment and the label is β-galactosidase.

Claim 20

The immunoassay of claim 16 wherein the measurement step (e) is performed by contacting the fraction to be measured with o-nitrophenyl-β-D-galactopyranoside.

Claim 21

The immunoassay of claim 17 wherein the measurement step (e) is performed by contacting the fraction to be measured with o-nitrophenyl-β-D-galactopyranoside.

Claim 22

A test kit for measuring the amount of digoxin in a liquid sample, comprising: (a) a labeled anti-digoxin antibody; and (b) a solid phase having ouabain bound thereto; and (c) directions for the use of said kit.

Claim 23

A test kit for measuring the amount of digoxin in a liquid sample, comprising: (a) a labeled anti-digoxin antibody; and (b) a solid phase having ouabain bound thereto; and (c) directions for the use of said kit.