Immunochemical assay for creatine kinase-MB isoenzyme

Claim 2

A process for determining the amount of creatine kinase-MB isoenzyme in a biological fluid sample containing creatine kinase having subunit B or M or both, the process comprising: (a) incubating the sample with (i) a first antibody raised in a first animal species, the first antibody capable of immuno-reactively binding selectively one of the B or M subunits of the creatine kinase in the sample; (ii) a second antibody raised in a second animal species, the second antibody capable of immunoreactively binding selectively as an immunoprecipitin with the first antibody; and (iii) a third antibody raised in an animal species other than the first animal species and labeled with a measurable label, the third antibody capable of immunoreactively binding selectively with the other of said M or B subunits to provide as a precipitate a mixture of immuno-complexes containing labeled third antibody-creatine kinase-MB isoenzyme-first antibody-second antibody and creatine kinase isoenzyme-first antibody-second antibody; (b) isolating the precipitate containing the immunocomplexes; (c) measuring the amount of label in the precipitate; and (d) comparing the amount of measured label to a standard curve thereby determining the amount of creatine kinase-MB isoenzyme in the biological fluid sample.

Claim 3

A process according to claim 1 wherein the third antibody has a radioactive label.

Claim 4

A process according to claim 2 wherein the radioactive label is iodine-125.

Claim 5

A process according to claim 1 wherein the biological fluid sample is blood serum.

Claim 6

A process according to claim 4 wherein the blood serum is derived from a patient suspected of a myocardial infarct.

Claim 7

A process according to claim 1 wherein the first, second, and third antibodies are each obtained from different animal species.

Claim 8

A process according to claim 6 wherein the first antibody is rabbit anti-(CK-MM), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-BB).

Claim 9

A process according to claim 6 wherein the first anti-body is rabbit anti-(CK-BB), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-MM).

Claim 10

A process according to claim 1 wherein the second antibody is attached to an insoluble solid support material.

Claim 11

A process for determining the amount of creatine kinase-MB isoenzyme in a biological fluid sample containing creatine kinase having subunits B or M or both, the process comprising: (a) incubating the sample with (i) a first antibody raised in a first animal species, the first antibody capable of immunoreactively binding selectively the B subunit of the creatine kinase in the sample, and (ii) a second antibody raised in a second animal species, the second antibody capable of immunoreactively binding selectively as an immunoprecipitin with the first antibody to provide as an immunoprecipitin a mixture containing creatine kinase-MB isoenzyme-first antibody-second antibody and creatine kinase-BB isoenzyme-first antibody-second antibody; (b) isolating the immunoprecipitins from the mixture, (c) incubating the isolated immunoprecipitins with a third antibody raised in an animal species other than the first animal species and labeled with a measurable label, the third antibody capable of immunoreactively binding selectively with the M subunit of the creatine-kinase-MB isoenzyme to provide as a precipitate a mixture of immunocomplexes containing labeled third antibody creatine kinase-MB isoenzyme-first antibody-second antibody and creatine kinase-BB isoenzyme-first antibody-second antibody; (d) isolating the precipitate containing the immunocomplexes; (e) measuring the amount of label in the precipitate; and (f) comparing the amount of measured label to a standard curve thereby determining the amount of creatine kinase-MB isoenzyme in the biological fluid sample.

Claim 12

A process for determining the amount of creatine kinase-MB isoenzyme in a biological fluid sample containing creatine kinase having subunits B or M or both, the process comprising: (a) incubating the sample with (i) a first antibody raised in a first animal species, the first antibody capable of immunoreactively binding selectively the B subunit of the creatine kinase in the sample, and (ii) a second antibody raised in a second animal species, the second antibody capable of immunoreactively binding selectively as an immunoprecipitin with the first antibody to provide as an immunoprecipitin a mixture containing creatine kinase-MB isoenzyme-first antibody-second antibody and creatine kinase-BB isoenzyme-first antibody-second antibody; (b) isolating the immunoprecipitins from the mixture, (c) incubating the isolated immunoprecipitins with a third antibody raised in an animal species other than the first animal species and labeled with a measurable label, the third antibody capable of immunoreactively binding selectively with the M subunit of the creatine-kinase-MB isoenzyme to provide as a precipitate a mixture of immunocomplexes containing labeled third antibody creatine kinase-MB isoenzyme-first antibody-second antibody and creatine kinase-BB isoenzyme-first antibody-second antibody; (d) isolating the precipitate containing the immunocomplexes; (e) measuring the amount of label in the precipitate; and (f) comparing the amount of measured label to a standard curve thereby determining the amount of creatine kinase-MB isoenzyme in the biological fluid sample.

Claim 13

A process according to claim 10 wherein the third antibody has a radioactive label.

Claim 14

A process according to claim 11 wherein the radioactive label is iodine-125.

Claim 15

A process according to claim 10 wherein the biological fluid sample is blood serum.

Claim 16

A process according to claim 13 wherein the blood serum is derived from a patient suspected of a myocardial infarct.

Claim 17

A process according to claim 10 wherein the first, second, and third antibodies are each obtained from different animal species.

Claim 18

A process according to claim 15 wherein the first antibody is rabbit anti-(CK-BB), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-MM).

Claim 19

A process according to claim 10 wherein the second antibody is attached to an insoluble solid support material.

Claim 20

A process for determining the amount of creatine kinase-MB isoenzyme in a biological fluid sample containing creatine kinase having subunits B or M or both, the process comprising: (a) incubating the sample with (i) a first antibody raised in a first animal species, the first antibody capable of immunoreactively binding selectively with the M subunit of the creatine kinase in the sample, and (ii) a third antibody raised in an animal species other than the first animal species and labeled with a measurable label, the third antibody capable of immunoreactively binding selectively with the B subunit of the creatine kinase in the sample to provide a mixture of immunocomplexes containing labeled third antibody-creatine kinase-MB isoenzyme first antibody; labeled third antibody-creatine kinase-BB isoenzyme; and creatine kinase-MM isoenzymefirst antibody; (b) incubating the mixture with a second antibody raised in a second animal species, the second antibody capable of immunoreactively binding selectively as an immunoprecipitin with the first antibody to provide as a precipitate a mixture of immunocomplexes containing labeled third antibody-creatine kinase-MB isoenzymefirst antibody-second antibody and creatine kinase-MM isoenzyme-first antibody-second antibody; (c) isolating the precipitate containing immunocomplexes; (d) measuring the amount of label in the precipitate; and (e) comparing the amount of measured label to a standard curve thereby determining the amount of creatine kinase-MB isoenzyme in the biological fluid sample.

Claim 21

A process for determining the amount of creatine kinase-MB isoenzyme in a biological fluid sample containing creatine kinase having subunits B or M or both, the process comprising: (a) incubating the sample with (i) a first antibody raised in a first animal species, the first antibody capable of immunoreactively binding selectively with the M subunit of the creatine kinase in the sample, and (ii) a third antibody raised in an animal species other than the first animal species and labeled with a measurable label, the third antibody capable of immunoreactively binding selectively with the B subunit of the creatine kinase in the sample to provide a mixture of immunocomplexes containing labeled third antibody-creatine kinase-MB isoenzyme first antibody; labeled third antibody-creatine kinase-BB isoenzyme; and creatine kinase-MM isoenzymefirst antibody; (b) incubating the mixture with a second antibody raised in a second animal species, the second antibody capable of immunoreactively binding selectively as an immunoprecipitin with the first antibody to provide as a precipitate a mixture of immunocomplexes containing labeled third antibody-creatine kinase-MB isoenzymefirst antibody-second antibody and creatine kinase-MM isoenzyme-first antibody-second antibody; (c) isolating the precipitate containing immunocomplexes; (d) measuring the amount of label in the precipitate; and (e) comparing the amount of measured label to a standard curve thereby determining the amount of creatine kinase-MB isoenzyme in the biological fluid sample.

Claim 22

A process according to claim 18 wherein the third antibody has a radioactive label.

Claim 23

A process according to claim 19 wherein the radioactive label is iodine-125.

Claim 24

A process according to claim 18 wherein the biological fluid sample is blood serum.

Claim 25

A process according to claim 21 wherein the blood serum is derived from a patient suspected of a myocardial infarct.

Claim 26

A process according to claim 18 wherein the first, second, and third antibodies are each obtained from different animal species.

Claim 27

A process according to claim 23 wherein the first antibody is rabbit anti-(CK-MM), the second antibody is goat antirabbit IgG, and the third antibody is sheep anti-(CK-BB).

Claim 28

A process according to claim 18 wherein the second antibody is attached to an insoluble solid support material.

Claim 29

A composition for measuring the amount of creatine kinase-MB isoenzyme in biological fluid sample containing creatine kinase having subunits B or M or both, the composition comprising a first antibody raised in a first animal species and capable of immunoreactively binding selectively one of the B or M subunits of creatine kinase in the sample; a second antibody raised in a second animal species and capable of immunoreactively binding selectively as an immunoprecipitin the first antibody, and a third antibody raised in an animal species other than the first animal species, labeled with a measurable label and capable of immunoreactively binding selectively the other of said M or B subunits.

Claim 30

A composition for measuring the amount of creatine kinase-MB isoenzyme in biological fluid sample containing creatine kinase having subunits B or M or both, the composition comprising a first antibody raised in a first animal species and capable of immunoreactively binding selectively one of the B or M subunits of creatine kinase in the sample; a second antibody raised in a second animal species and capable of immunoreactively binding selectively as an immunoprecipitin the first antibody, and a third antibody raised in an animal species other than the first animal species, labeled with a measurable label and capable of immunoreactively binding selectively the other of said M or B subunits.

Claim 31

A composition according to claim 26 wherein the third antibody has a radioactive label.

Claim 32

A composition according to claim 27 wherein the radioactive label is iodine-125.

Claim 33

A composition according to claim 26 wherein the biological fluid sample is blood serum.

Claim 34

A composition according to claim 26 wherein the first, second and third antibodies are obtained from different animal species.

Claim 35

A composition according to claim 30 wherein the first antibody is rabbit anti-(CK-BB), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-MM).

Claim 36

A composition according to claim 30 wherein the first antibody is rabbit anti-(CK-MM), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-BB).

Claim 37

A composition according to claim 26 wherein the second antibody is attached to an insoluble support material.

Claim 38

A diagnostic test kit system for determining the amount of creatine kinase-MB isoenzyme in an immunoassay, the test kit system comprising in combination: (a) a container of a first antibody raised in a first animal species and capable of immunoreactively binding selectively one of the B or M subunits of creatine kinase, (b) a container of a second antibody raised in a second animal species and capable of immunoreactively binding selectively as an immunoprecipitin the first antibody, and (c) a container of a third antibody raised in an animal species other than the first animal species, labeled with a measurable label and capable of immunoreactively binding selectively the other of said M or B subunits bound by the first antibody, all of said antibodies being immunoreactive binders and present in relative amounts sufficient to provide an immunocomplex containing creatine kinase-MB isoenzyme selectively bound to all three of said antibodies.

Claim 39

A diagnostic test kit system for determining the amount of creatine kinase-MB isoenzyme in an immunoassay, the test kit system comprising in combination: (a) a container of a first antibody raised in a first animal species and capable of immunoreactively binding selectively one of the B or M subunits of creatine kinase, (b) a container of a second antibody raised in a second animal species and capable of immunoreactively binding selectively as an immunoprecipitin the first antibody, and (c) a container of a third antibody raised in an animal species other than the first animal species, labeled with a measurable label and capable of immunoreactively binding selectively the other of said M or B subunits bound by the first antibody, all of said antibodies being immunoreactive binders and present in relative amounts sufficient to provide an immunocomplex containing creatine kinase-MB isoenzyme selectively bound to all three of said antibodies.

Claim 40

A test kit system according to claim 34 wherein the third antibody has a radioactive label.

Claim 41

A test kit system according to claim 35 wherein the radioactive label is iodine-125.

Claim 42

A test kit system according to claim 34 wherein the biological fluid sample is blood serum.

Claim 43

A test kit system according to claim 34 wherein the first, second, and third antibodies are each obtained from different animal species.

Claim 44

A test kit system according to claim 38 wherein the first antibody is rabbit anti-(CK-MM), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-BB).

Claim 45

A test kit system according to claim 38 wherein the first antibody is rabbit anti-(CK-BB), the second antibody is goat anti-rabbit IgG, and the third antibody is sheep anti-(CK-MM).

Claim 46

A test kit system according to claim 34 wherein the second antibody is attached to an insoluble solid support material.

Claim 47

A test kit system according to claim 34 wherein the first antibody and the third antibody are in the same container.

Claim 48

A test kit system according to claim 34 further comprising standards for establishing a standard curve.