Radioimmunoassay for isoenzymes of creative kinase having a B-subunit and reagents therefor

Claim 2

A competitive displacement radioimmunoassay method for quantitative determination of the concentration of a B subunit-containing isoenzyme of human creatine kinase in a sample, said method comprising: (1) incubating said sample with a pre-determined stoichiometric excess quantity of antibodies to purified MB creatine kinase to form a first reaction mixture comprising (a) the product of antigen-antibody association of B subunit-containing isoenzymes in the sample with antibodies to purified MB creatine kinase, and (b) unreacted antibodies to purified MB creatine kinase; (2) incubating the reaction mixture of step (1) with a stoichiometric excess of radioisotopically labelled, purified B subunit-containing isoenzymes of creatine kinase to form a second reaction mixture comprising the association product of step (1) and the product of antigen-antibody association of said labelled isoenzymes with said unreacted antibodies in said first reaction mixture; and (3) removing unreacted labelled isoenzymes from said second reaction mixture and ascertaining the concentration of B subunit-containing isoenzymes in said sample on the basis of the extent of antigen-antibody association between said antibodies and said labelled isoenzyme.

Claim 3

The method of claim 1 wherein said antibody to purified MB creatine kinase is isolated from an animal immunized with purified MB creatine kinase.

Claim 4

A competitive displacement radioimmunoassay method for quantitative determination of the concentration of a B subunit-containing isoenzyme of human creatine kinase in a sample, said method comprising: (1) incubating said sample in a medium having reducing agent activity with a pre-determined stoichiometric excess quantity of antibodies to purified MB creatine kinase to form a first reaction mixture comprising (a) the product of antigen-antibody association of B subunit-containing isoenzymes in the sample with antibodies to purified MB creatine kinase, and (b) unreacted antibodies to purified MB creatine kinase; (2) incubating the reaction mixture of step (1) in a medium having reducing agent activity with a stoichiometric excess of radioisotopically labelled, purified B subunit-containing isoenzymes of creatine kinase to form a second reaction mixture comprising the association product of step (1) and the product of antigen-antibody association of said labelled isoenzymes with said unreacted antibodies in said first reaction mixture, said labelled isoenzymes prepared by attaching a suitable radioisotopically labelled carrier molecule to the isoenzyme thus avoiding exposure of said isoenzyme to oxidative damage during labelling; and (3) removing unreacted labelled isoenzymes from said second reaction mixture and ascertaining the concentration of B subunit-containing isoenzymes in said sample on the basis of the extent of antigen-antibody association between said antibodies and said labelled isoenzyme.

Claim 5

A competitive displacement radioimmunoassay method for quantitative determination of the concentration of a B subunit-containing isoenzyme of human creatine kinase in a sample, said method comprising: (1) incubating said sample in a medium having reducing agent activity with a pre-determined stoichiometric excess quantity of antibodies to purified MB creatine kinase to form a first reaction mixture comprising (a) the product of antigen-antibody association of B subunit-containing isoenzymes in the sample with antibodies to purified MB creatine kinase, and (b) unreacted antibodies to purified MB creatine kinase; (2) incubating the reaction mixture of step (1) in a medium having reducing agent activity with a stoichiometric excess of radioisotopically labelled, purified B subunit-containing isoenzymes of creatine kinase to form a second reaction mixture comprising the association product of step (1) and the product of antigen-antibody association of said labelled isoenzymes with said unreacted antibodies in said first reaction mixture, said labelled isoenzymes prepared by attaching a suitable radioisotopically labelled carrier molecule to the isoenzyme thus avoiding exposure of said isoenzyme to oxidative damage during labelling; and (3) removing unreacted labelled isoenzymes from said second reaction mixture and ascertaining the concentration of B subunit-containing isoenzymes in said sample on the basis of the extent of antigen-antibody association between said antibodies and said labelled isoenzyme.

Claim 6

The method of claim 3 wherein the medium wherein said incubation steps are carried out retards multiple reactions between antigen and antibody moieties and retards dissociation of the reacted moieties.

Claim 7

The method of claim 3 wherein said incubation steps are carried out in an aqueous buffer medium comprising Tris and having a pH of from 7.0 to about 8.0.

Claim 8

The method of claim 5 wherein the pH of said buffer is about 7.4.

Claim 9

The method of claim 6 wherein said reducing agent is mercaptoethanol.

Claim 10

The method of claim 3 wherein the radioisotopically labelled, purified B subunit-containing isoenzyme is an acylation product of the reaction of purified isoenzyme with iodinated N-succinimidyl-3-(4-hydroxyphenyl propionate).

Claim 11

The method of claim 8 wherein the medium wherein said incubation steps are carried out is an aqueous buffer having a pH of about 7.4 and comprising Tris, gamma globulin, serum albumin and mercaptoethanol.

Claim 12

A combination of reagents for use in the quantitative detection, by radioimmunoassay of the concentration of a B subunit-containing isoenzyme of creatine kinase in a sample, said combination of reagents comprising (1) a pre-determined quantity of an antibody to purified MB creatine kinase, reactive with B subunit-containing isoenzymes of creatine kinase to form an antigen-antibody association upon contact therewith and being substantially unreative with MM creatine kinase; and (2) a pre-determined quantity of a radioisotopically labelled, purified B subunit-containing isoenzyme of creatine kinase, capable of antigen-antibody association with said antibody upon contact therewith, said labelled isoenzymes prepared by attaching a suitable radioisotopically labelled carrier molecule to the isoenzymes thus avoiding exposure of said isoenzyme to oxidative damage.

Claim 13

A combination of reagents for use in the quantitative detection, by radioimmunoassay of the concentration of a B subunit-containing isoenzyme of creatine kinase in a sample, said combination of reagents comprising (1) a pre-determined quantity of an antibody to purified MB creatine kinase, reactive with B subunit-containing isoenzymes of creatine kinase to form an antigen-antibody association upon contact therewith and being substantially unreative with MM creatine kinase; and (2) a pre-determined quantity of a radioisotopically labelled, purified B subunit-containing isoenzyme of creatine kinase, capable of antigen-antibody association with said antibody upon contact therewith, said labelled isoenzymes prepared by attaching a suitable radioisotopically labelled carrier molecule to the isoenzymes thus avoiding exposure of said isoenzyme to oxidative damage.

Claim 14

A combination of reagents in claim 10 further including a medium which has reducing agent activity and which retards multiple reactions between the antigen and antibody moieties and which retards dissociation of the reacted moieties.

Claim 15

A combination of reagents as set forth in claim 11 wherein the medium is an aqueous buffer solution having a pH of about 7.4 comprising Tris, gamma globulin, serum albumin and mercaptoethanol.

Claim 16

A combination of reagents as set forth in claim 11 wherein said radioisotopically labelled, purified isoenzyme is an acylation product of the reaction of purified isoenzyme with iodinated N-succinimidyl-3-(4-hydroxphenyl propionate).

Claim 17

A combination of reagents as set forth in claim 13 wherein said radioisotopically labelled, purified isoenzyme is MB isoenzyme.