Interligand metal transfer assay

Claim 2

In a method of chemical analysis for determining an analyte in a test material comprising carrying out a biospecific reaction with the analyte and thereafter detecting the occurrence of the biospecific reaction and employing a reporter mechanism to indicate the occurrence of the biospecific reaction the improvement comprising employing as the reporter mechanism an interligand metal ion transfer in which a metal ion is directly transferred from a first chelate complex with a first ligand to a second ligand thereby forming a second chelate complex which is separate from and detectably different from the first chelate complex.

Claim 3

The method of analysis of claim 1 wherein the analyte is isolated from the test material prior to the interligand metal ion transfer.

Claim 4

The method of claim 2 wherein the biospecific reaction is a competitive reaction in which the analyte competes with an added chelate-labeled competitor of the analyte.

Claim 5

The method of claim 3 comprising the steps of a. biospecifically reacting in the presence of the test material two species capable of biospecific reaction with one another, the second of which carries a first metal ion chelate complex and competes with the analyte, if present, for a reaction-limiting amount of one or more biospecific binding sites on the first species thereby forming a biospecific reaction product, b. separating the biospecific reaction product from unreacted second species and analyte, c. contacting the separated biospecific reaction product with an excess of a ligand which directly removes metal ion from the first metal ion chelate and forms a second metal ion chelate which is separate from and detectably different from the first metal ion chelate, d. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and e. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 6

The method of claim 4 wherein the first species is attached to a solid support.

Claim 7

The method of claim 3 comprising the steps of a. biospecifically reacting in the presence of the test material first and second biospecific species, said first species carrying a first metal ion chelate complex and capable of biospecific reaction with the analyte and said second species being capable of reaction with said first species in competition with said analyte if present, for a reaction-limiting amount of biospecific binding sites on the first species thereby forming a biospecific reaction product, b. separating the biospecific reaction product from unreacted second species and analyte, c. contacting the separated biospecific reaction product with an excess of a ligand which directly removes metal ion from the first metal ion chelate complex and forms a second metal ion chelate complex which is separate from and detectably different from the first metal ion chelate complex, d. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and e. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 8

The method of claim 6 wherein the second species is attached to a solid support.

Claim 9

The method of claim 2 comprising the steps of a. contacting the test material with an excess of a biospecific partner for a first site on the analyte, thereby biospecifically coupling any analyte present in the test sample to form a first biospecific pair, b. contacting the first biospecific pair with an excess of a second biospecific partner for a second site on the analyte, which second biospecific partner carries a first metal ion chelate complex thereby forming a first metal ion chelate-labeled biospecific trio, c. separating the biospecific trio from unreacted second biospecific partner, d. contacting the separated biospecific trio with an excess of a ligand which directly removes metal ion from the first metal ion chelate complex and forms a second metal ion chelate complex which is separate from and detectably different from the first metal ion chelate and the metal ion-containing biospecific trio, e. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and f. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 10

The method of analysis of claim 8 wherein the biospecific partner for the first site on the analyte is attached to a solid support.

Claim 11

The method of analysis of claim 1 wherein the analyte is not isolated from the test material prior to the interligand metal ion transfer.

Claim 12

The method of claim 10 wherein the biospecific reaction is a competitive reaction in which the analyte competes with an added chelate-labeled competitor of the analyte.

Claim 13

The method of claim 10 comprising the steps of a. biospecifically reacting in the presence of the test material two species capable of biospecific reaction with one another, the first of which carries a first metal ion chelate complex and the second of which carries a second metal ion chelate-forming ligand, said second species competing with the analyte, if present, for a reaction-limiting amount of one or more biospecific binding sites on the first species thereby forming a biospecific reaction product comprising the couple of the first and second species and the couple of the first species and the analyte, the ligand on the second species being positioned in such proximity to its biospecific group that as a result of the biospecific reaction the ligand can contact and react with the first metal ion chelate complex carried by the first species, b. incubating the product of the biospecific reaction for a period sufficient to effect direct interligand transfer of metal ion from the first metal ion chelate complex to the second metal ion chelate-forming ligand thereby forming a second metal ion chelate complex, c. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and d. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 14

The method of claim 11 comprising the steps of a. biospecifically reacting in the presence of the test material two species capable of biospecific reaction with one another, the second of which carries a first metal ion chelate complex and the first of which carries a second metal ion chelate-forming ligand, said second species competing with the analyte, if present, for a reaction-limiting amount of one or more biospecific binding sites on the first species thereby forming a biospecific reaction product comprising the couple of the first and second species and the couple of the first species and the analyte, the ligand on the first species being positioned in such proximity to its biospecific group that as a result of the biospecific reaction the ligand can contact and react with the first metal ion chelate complex carried by the second species, b. incubating the product of the biospecific reaction for a period sufficient to effect direct interligand transfer of metal ion from the first metal ion chelate complex to the second metal ion chelate-forming ligand thereby forming a second metal ion chelate complex, c. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and d. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 15

The method of claim 10 comprising the steps of a. biospecifically reacting in the presence of the test material two biospecific species, one of which carries a first metal ion chelate complex and the other of which carries a second metal ion chelate-forming ligand, each of these biospecific species being present in excess and each biospecifically reacting with the analyte if present but with different sites thereon, the chelate on the first species and the ligand on the second species each being positioned in such proximity that as a result of the biospecific reactions the ligand can contact and react with the first metal ion chelate complex, b. incubating the product of the biospecific reaction for a period sufficient to effect direct interligand transfer of metal ion from the first metal ion chelate complex to the second metal ion chelate-forming ligand thereby forming a second metal ion chelate complex, c. detecting the presence of the second metal ion chelate complex and generating a signal related thereto, and d. quantitatively comparing the generated signal with signals from known standards to determine the amount of the analyte in the test material.

Claim 16

The method of claim 1 wherein the first chelate complex is fluorescent and the detectable difference between the first and second chelate complexes is a difference in fluorescence.

Claim 17

The method of claim 15 wherein the fluorescence is measured using a fluorescent background rejection method in which the difference in fluorescence is measured after background fluorescence has decayed.

Claim 18

The method of claim 1 wherein the second chelate complex is fluorescent and the detectable difference between the first and second chelate complexes is a difference in fluorescence.