Ion exchange chromatographic isoenzyme separation and isolation

Claim 2

A process of separating and isolating fractions containing isoenzymes from a sample of tissue extract or blood serum comprising ion-exchange column chromatographically separating said sample containing said isoenzymes into a first fraction containing the hepatic isoenzyme of lactic acid dehydrogenase (LDH 5 ) and a second fraction containing the myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), the myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phophatase by ion-exchange column chromatographing said sample on a weakly-basic anion-exchange chromatographic resin and sequentially eluting and collecting said first fraction and said second fraction, said chromatographing, eluting and collecting being conducted in accordance with the following sequence:

Claim 3

a. passing said sample through the anion-exchange resin in a pre-swelled form in a chromatographic column and collecting a first eluate passing through the column; b. passing a first aqueous buffered salt solution through the anion-exchange resin in the chromatographic column and collecting a second eluate with said first eluate to obtain said first fraction containing the hepatic isoenzyme of lactic acid dehydrogenase (LDH 5 ), said first aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to passing said sample, having a concentration in terms of the anion of about 0.01 to 0.15 equivalent/liter and being passed in an amount such that up to about 3 volume equivalents, based on said sample, of said first aqueous buffered salt solution are passed; c. sequentially passing a second aqueous buffered salt solution and a third aqueous buffered salt solution through the anion-exchange resin in the chromatographic column to obtain a third eluate, said second aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.01 to 0.15 equivalent/liter and being passed in an amount such that at least about 4 volume equivalents, based on said sample, of said second aqueous buffered salt solution are passed, and said third aqueous buffered salt solution having a pH of about 7.5 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.15 to 0.25 equivalent/liter and being passed in an amount such that about 4 or less volume equivalents, based on said sample, of said third aqueous buffered salt solution are passed; and d. passing a fourth aqueous buffered salt solution through the anion-exchange resin in the chromatographic column and collecting a fourth eluate to obtain said second fraction containing the myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), the myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phosphatase, said fourth aqueous buffered salt solution having a pH of about 6.5 to 7.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.4 to 0.6 equivalent/liter and being passed in an amount such that about 1 to 6 volume equivalents, based on said sample, of said fourth aqueous buffered salt solution are passed.

Claim 4

a. passing said sample through the anion-exchange resin in a pre-swelled form in a chromatographic column and collecting a first eluate passing through the column; b. passing a first aqueous buffered salt solution through the anion-exchange resin in the chromatographic column and collecting a second eluate with said first eluate to obtain said first fraction containing the hepatic isoenzyme of lactic acid dehydrogenase (LDH 5 ), said first aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to passing said sample, having a concentration in terms of the anion of about 0.01 to 0.15 equivalent/liter and being passed in an amount such that up to about 3 volume equivalents, based on said sample, of said first aqueous buffered salt solution are passed; c. sequentially passing a second aqueous buffered salt solution and a third aqueous buffered salt solution through the anion-exchange resin in the chromatographic column to obtain a third eluate, said second aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.01 to 0.15 equivalent/liter and being passed in an amount such that at least about 4 volume equivalents, based on said sample, of said second aqueous buffered salt solution are passed, and said third aqueous buffered salt solution having a pH of about 7.5 to 8.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.15 to 0.25 equivalent/liter and being passed in an amount such that about 4 or less volume equivalents, based on said sample, of said third aqueous buffered salt solution are passed; and d. passing a fourth aqueous buffered salt solution through the anion-exchange resin in the chromatographic column and collecting a fourth eluate to obtain said second fraction containing the myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), the myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phosphatase, said fourth aqueous buffered salt solution having a pH of about 6.5 to 7.5, containing a salt with an anion corresponding to the anion of the anion-exchange resin prior to the passing of said sample, having a concentration in terms of the anion of about 0.4 to 0.6 equivalent/liter and being passed in an amount such that about 1 to 6 volume equivalents, based on said sample, of said fourth aqueous buffered salt solution are passed.

Claim 5

The process of claim 1, wherein said sample is a sample of blood serum.

Claim 6

The process of claim 1, wherein said sample is a tissue extract.

Claim 7

The process of claim 1, wherein said anion exchange resin is a diethylaminoethyl dextran anion-exchange resin.

Claim 8

The process of claim 1, wherein said anion exchange resin is a diethylaminoethyl cellulose anion-exchange resin.

Claim 9

The process of claim 1, wherein said anion exchange resin is a diethylaminoethyl agarose anion-exchange resin.

Claim 10

The process of claim 1, wherein said anion exchange resin is a deithylaminoethyl acrylamide anion-exchange resin.

Claim 11

The process of claim 1, wherein said process further includes concentrating said first fraction.

Claim 12

The process of claim 1, wherein said process further includes concentrating said second fraction.

Claim 13

The process of claim 1, wherein said process further includes concentrating said first fraction and said second fraction.

Claim 14

The process of claim 5, wherein said first aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8, having a concentration in terms of the chloride of about 0.1 equivalent/liter and being passed in an amount such that about 1 volume equivalent, based on said sample, of said first aqueous buffered salt solution is passed; said second aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8, having a concentration in terms of the chloride of about 0.1 equivalent/liter and being passed in an amount such that about 7 volume equivalents, based on said sample of said second aqueous buffered salt solution are passed; said third aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8, having a concentration in terms of the chloride of about 0.2 equivalent/liter and being passed in an amount such that about 2 volume equivalents, based on said sample, of said third aqueous buffered salt solution, are passed; and said fourth aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 7, having a concentration in terms of the chloride of about 0.5 equivalent/liter and being passed in an amount such that about 2 volume equivalents of said fourth aqueous buffered salt solution, based on said sample, are passed.

Claim 15

The process of claim 11, wherein said alkali metal chloride is sodium chloride.

Claim 16

A process for isolating a fraction containing isoenzymes of diagnostic importance in diagnosing heart disorders from a sample of tissue extract or blood serum comprising ion-exchange column chromatographically separating said sample containing said enzymes into a fraction containing the myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), the myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phosphatase by ion-exchange column chromatographing said sample on a weakly-basic anion-exchange chromatographic resin and sequentially eluting and collecting to obtain said fraction containing said myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), said myocardial isoenzyme of creatine phosphokinase (CPK-MB) and said prostatic acid phosphatase, and said chromatographing, eluting and collecting being conducted in accordance with the following sequence: a. passing said sample through said anion-exchange resin in a pre-swelled form in a chromatographic column and discarding the eluate obtained therefrom; b. sequentially passing (i) a combination of a first aqueous buffered salt solution and a second aqueous buffered salt solution, and (ii) a third aqueous buffered salt solution through said anion-exchange resin in said chromatographic column to obtain a second eluate and discarding said eluate; said first aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to passing said sample and having a concentration in terms of said anion of about 0.01 to 0.15 equivalent/liter; said second aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample and having a concentration in terms of said anion of about 0.01 to 0.15 equivalent/liter; and the combination being passed in an amount such that at least about 4 volume equivalents based on said sample, of said combination, are passed; and said third aqueous buffered salt solution having a pH of about 7.5 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample, having a concentration in terms of said anion of about 0.15 to 0.25 equivalent/liter and being passed in an amount such that about 4 or less volume equivalents, based on said sample, of said third aqueous buffered salt solution, are passed; and c. passing a fourth aqueous buffered salt solution through said anion-exchange resin in said chromatographic column and collecting the eluate to obtain said fraction containing said isoenzymes; said fourth aqueous buffered salt solution having a pH of about 6.5 to 7.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample, having a concentration in terms of said anion of about 0.4 to 0.6 equivalent/liter and being passed in an amount such that about 1 to 6 volume equivalents, based on said sample, of said fourth aqueous buffered salt solution are passed.

Claim 17

A process for isolating a fraction containing isoenzymes of diagnostic importance in diagnosing heart disorders from a sample of tissue extract or blood serum comprising ion-exchange column chromatographically separating said sample containing said enzymes into a fraction containing the myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), the myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phosphatase by ion-exchange column chromatographing said sample on a weakly-basic anion-exchange chromatographic resin and sequentially eluting and collecting to obtain said fraction containing said myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), said myocardial isoenzyme of creatine phosphokinase (CPK-MB) and said prostatic acid phosphatase, and said chromatographing, eluting and collecting being conducted in accordance with the following sequence: a. passing said sample through said anion-exchange resin in a pre-swelled form in a chromatographic column and discarding the eluate obtained therefrom; b. sequentially passing (i) a combination of a first aqueous buffered salt solution and a second aqueous buffered salt solution, and (ii) a third aqueous buffered salt solution through said anion-exchange resin in said chromatographic column to obtain a second eluate and discarding said eluate; said first aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to passing said sample and having a concentration in terms of said anion of about 0.01 to 0.15 equivalent/liter; said second aqueous buffered salt solution having a pH of about 7.0 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample and having a concentration in terms of said anion of about 0.01 to 0.15 equivalent/liter; and the combination being passed in an amount such that at least about 4 volume equivalents based on said sample, of said combination, are passed; and said third aqueous buffered salt solution having a pH of about 7.5 to 8.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample, having a concentration in terms of said anion of about 0.15 to 0.25 equivalent/liter and being passed in an amount such that about 4 or less volume equivalents, based on said sample, of said third aqueous buffered salt solution, are passed; and c. passing a fourth aqueous buffered salt solution through said anion-exchange resin in said chromatographic column and collecting the eluate to obtain said fraction containing said isoenzymes; said fourth aqueous buffered salt solution having a pH of about 6.5 to 7.5, containing a salt with an anion corresponding to the anion of said anion-exchange resin prior to said passing of said sample, having a concentration in terms of said anion of about 0.4 to 0.6 equivalent/liter and being passed in an amount such that about 1 to 6 volume equivalents, based on said sample, of said fourth aqueous buffered salt solution are passed.

Claim 18

The process of claim 13, wherein said sample is a sample of blood serum.

Claim 19

The process of claim 13, wherein said sample is a tissue extract.

Claim 20

The process of claim 13, wherein said anion-exchange resin is a diethylaminoethyl dextran anion-exchange resin.

Claim 21

The process of claim 13, wherein said anion-exchange resin is a diethylaminoethyl cellulose anion-exchange resin.

Claim 22

The process of claim 13, wherein said anion-exchange resin is a diethylaminoethyl agarose anion-exchange resin.

Claim 23

The process of claim 13, wherein said anion-exchange resin is a diethylaminoethyl acrylamide anion-exchange resin.

Claim 24

The process of claim 13, wherein said process further includes concentrating said fraction containing said myocardial isoenzymes of lactic acid dehydrogenase (LDH 1 ,2), said myocardial isoenzyme of creatine phosphokinase (CPK-MB) and prostatic acid phosphatase.

Claim 25

The process of claim 16, wherein said first aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8 and having a concentration in terms of the chloride of about 0.1 equivalent/liter; said second aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8, and having a concentration in terms of the chloride of about 0.1 equivalents/liter; and the combination is passed in an amount such that about 8 volume equivalents, based on said sample, of said combination are passed; said third aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 8, having a concentration in terms of the chloride of about 0.2 equivalent/liter and being passed in an amount such that about 2 volume equivalents, based on said sample, of said third aqueous buffered salt solution are passed; and said fourth aqueous buffered salt solution is an aqueous buffered alkali metal chloride solution having a pH of about 7, having a concentration in terms of the chloride of about 0.5 equivalent/liter and being passed in an amount such that about 2 volume equivalents of said fourth aqueous buffered salt solution, based on said sample, are passed.

Claim 26

The process of claim 21, wherein said alkali metal chloride is sodium chloride.