Process and composition for determining the activity of creatinekinase-MB

Claim 3

The method of claim 1, wherein said enzymatic activity is determined photometrically in step (b).

Claim 4

The method of claim 1, wherein the biological sample is blood serum and the blood serum and antibodies are incubated in the presence of a CK substrate.

Claim 5

The method of claim 1, further comprising a preliminary step, prior to incubation of said sample with said antibodies, of determining total CK activity in said sample.

Claim 6

The method of claim 4, wherein said complete inhibition of said enzymatic activity of said M subunit leaves less than 3 U./l. of said enzymatic activity intact and said non-inhibition of said enzymatic activity of said B subunit is inhibition of less than 5 U./l.

Claim 7

The method of claim 5, wherein said antibodies are anti-human skeletal muscle CK-MM, anti-Rhesus monkey skeletal muscle CK-MM, anti-pig skeletal muscle CK-MM, or anti-cattle skeletal muscle CK-MM.

Claim 8

The method of claim 5, wherein said antibodies are anti-human skeletal muscle CK-MM from goats.

Claim 9

The method of claim 1, wherein said antibodies are able to inhibit completely up to 2,500 U./l. of M subunit of creatinekinases MM and MB in said sample.

Claim 10

The method of claim 1, wherein said biological sample is blood serum and the blood serum and antibodies are incubated in the presence of a CK substrate; including a preliminary step, prior to incubation of said sample with said antibodies, of determining total CK activity in said sample; complete inhibition of said enzymatic activity of said M subunit leaves less than 3 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is inhibition of less than 5 U./l.; said antibodies are anti skeletal muscle CK-MM from goats; and said antibodies are able to inhibit completely up to 2,500 U./l. of M subunit of creatinekinases MM and MB in said sample.

Claim 11

A method according to claim 1, wherein said biological liquid sample is derived from a patient suspected of myocardial infraction.

Claim 12

A method according to claim 4, wherein said biological liquid sample is derived from a patient suspected of myocardial infraction.

Claim 13

A method according to claim 9, wherein said blood serum is derived from a patient suspected of a myocardial infraction.

Claim 14

A method according to claim 1, wherein said antibodies are substantially free of CK-BB isoenzyme activity and have a molecular weight of 130,000 - 210,000 and a sedimentation constant between 6 S and 8 S; and said antibodies are able to effect complete inhibition of said enzymatic activity of said M subunit in CK-MM and CK-MB without inhibiting said enzymatic activity of said B subunit, even in the presence of CK substrates.

Claim 15

A method according to claim 13, wherein the molecular weight is about 160,000 and the sedimentation constant is about 7 S.

Claim 16

A composition for determining the activity of creatinekinase-MB in a liquid biological sample comprising antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and an enzyme-coenzyme and substrate reagent for determining CK activity.

Claim 17

A composition for determining the activity of creatinekinase-MB in a liquid biological sample comprising antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and an enzyme-coenzyme and substrate reagent for determining CK activity.

Claim 18

A composition for determining the activity of creatinekinase-MB in a liquid biological sample comprising a. antiskeletal muscle CK-MM antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and b. an enzyme-coenzyme and substrate mixture containing hexokinase, glucose-6-phosphate-dehydrogenase, adenosine diphosphate, nicotinamide-adenine-dinucleotidephosphate, creatine phosphate and glucose.

Claim 19

A composition for determining the activity of creatinekinase-MB in a liquid biological sample comprising a. antiskeletal muscle CK-MM antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and b. an enzyme-coenzyme and substrate mixture containing hexokinase, glucose-6-phosphate-dehydrogenase, adenosine diphosphate, nicotinamide-adenine-dinucleotidephosphate, creatine phosphate and glucose.

Claim 20

The composition of claim 15, wherein said antibodies are able to effect complete inhibition of said enzymatic activity of said M subunit in CK-MM and CK-MB without inhibiting said enzymatic activity of said B subunit, even in the presence of CK substrates.

Claim 21

The composition of claim 15, wherein said antibodies are anti-skeletal muscle CK-MM obtained by immunization of goats.

Claim 22

The composition of claim 15, wherein said antibodies are substantially free of CK-BB isoenzyme activity and have a molecular weight of 130,000 - 210,000 and a sedimentation constant between 6 S and 8 S; and said antibodies are able to effect complete inhibition of said enzymatic activity of said M subunit in CK-MM and CK-MB without inhibiting said enzymatic activity of said B subunit, even in the presence of CK substrates.

Claim 23

The composition of claim 19, wherein the molecular weight is about 160,000 and the sedimentation constant is about 7 S.

Claim 24

A test kit for the determination of the activity of creatinekinase-MB in a biological sample, which comprises: a. antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and b. an enzyme-coenzyme and substrate reagent for determining CK activity.

Claim 25

A test kit for the determination of the activity of creatinekinase-MB in a biological sample, which comprises: a. antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l., and b. an enzyme-coenzyme and substrate reagent for determining CK activity.

Claim 26

A test kit for the determination of the activity of creatinekinase-MB in a biological sample, which comprises: a. antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l.; b. an enzyme-coenzyme and substrate mixture comprising disodium creatine phosphate hexahydrate, reduced glutathione or N-acetylcysteine, disodium adenosine diphosphate hexahydrate, disodium nicotinamideadeninedinucleotide phosphate, disodium adenosine monophosphate, hexokinase, glucose-6-phosphatdehydrogenase, glucose and magnesium acetate; and c. a buffer solution of triethanolamine or imidazole acetate.

Claim 27

A test kit for the determination of the activity of creatinekinase-MB in a biological sample, which comprises: a. antibodies being substantially free of CK-BB isoenzyme activity and being able to inhibit completely the enzymatic activity of the M subunit of creatinekinases MM and MB in solution without precipitation and without inactivating the enzymatic activity of the B subunit of any creatinekinase-MB in the sample, wherein complete inhibition of said M subunit leaves less than 5 U./l. of said enzymatic activity intact and non-inhibition of said enzymatic activity of said B subunit is less than 10 U./l.; b. an enzyme-coenzyme and substrate mixture comprising disodium creatine phosphate hexahydrate, reduced glutathione or N-acetylcysteine, disodium adenosine diphosphate hexahydrate, disodium nicotinamideadeninedinucleotide phosphate, disodium adenosine monophosphate, hexokinase, glucose-6-phosphatdehydrogenase, glucose and magnesium acetate; and c. a buffer solution of triethanolamine or imidazole acetate.

Claim 28

A kit according to claim 21, wherein said reagent contains hexokinase, glucose-6-phosphate-dehydrogenase, adenosine diphosphate, nicotinamide-adenine-dinucleotidephosphate, creatine phosphate and glucose.