Assay of isoenzymes by ion exchange chromatography

A method of determining the presence of isoenzymes of different net electrical charges derived from lactate dehydrogenase in a sample of blood serum or tissue extracts, comprising the steps of

• a. equilibrating an ion exchange agent of the amine type based on a polysaccharide matrix with an aqueous buffered alkali metal chloride having about 0.1 molar alkali chloride at about pH 8.0 to 9.5 so that the net electrical charge characteristics of the ion exchange agent are similar to those of the blood serum sample in undialyzed, unaltered ionic form or the tissue extract sample in undialyzed, unaltered ionic form;
• b. passing said sample of blood serum in undialyzed, unaltered ionic form or tissue extract in undialyzed, unaltered form through said equilibrated ion exchange agent and collecting the effluent, then
• c. passing through said ion exchange agent successive separate volumes of an aqueous buffered alkali metal chloride solution of a first molar ion concentration of about 0.1 and a pH of about 8.0 to 9.5, collecting the effluent separate from the effluent of step (b), then passing through the same column of the ion exchange agent an aqueous buffered alkali metal chloride solution of a second molar ion concentration of about 0.2 and a pH of about 8.0 to 9.5 and collecting the effluent separately from the effluents of step (b) and the first effluent of step (c);
• (d) assaying each of said separate effluents to determine whether the isoenzymes of lactate dehydrogenase are present therein.

A method of determining the presence of isoenzymes of different net electrical charges derived from an enzyme selected from the group consisting of acid phosphatase, aspartate transaminase, gamma glutamyl transpeptidase, amylase and aldolase in a sample of blood serum tissue extracts, comprising the steps of

• a. equilibrating an ion exchange agent of the amine type based on a polysaccharide matrix with an aqueous buffered alkali metal chloride having about 0.1 molar alkali metal chloride at about pH 8.0 to 9.5 so that the net electrical charge characteristics of the ion exchange agent are similar to those of the blood serum sample in undialyzed, unaltered ionic form or the tissue extracts sample in undialyzed, unaltered ionic form;
• b. passing said sample of blood serum in undialyzed, unaltered ionic form or tissue extracts in undialyzed, unaltered form through said equilibrated ion exchange agent and collecting the effluent, then
• c. passing through said ion exchange agent successive separate volumes of an aqueous buffered alkali metal chloride solution of a first molar ion concentration of about 0.1 and a pH of about 8.0 to 9.5, collecting the effluent separate from the effluent of step (b), then passing through the same column of the ion exchange agent an aqueous buffered alkali metal chloride solution of a second molar ion concentration of about 0.2 and a pH of about 8.0 to 9.5, and collecting the effluent separately from the effluents of step (b) and the first effluent of step (c);
• d. assaying each of said separate effluents to determine whether the isoenzymes of said enzymes are present therein.