Novel immunochemical method for assaying stable glycosylated hemoglobin

Claim 1

A method of assaying the amount of stable glucohemoglobin in a glucohemoglobin-containing sample comprising the steps of: (a) admixing said sample with a phthalic acid solution having a pH value of about 3 to about 6, at a ratio of at least about 1.5 milIimoles of said acid per milligram of hemoglobin to form an acid reaction admixture; (b) maintaining said acid reaction admixture for a predetermined time period at a temperature above about 0 degrees C. to about 37 degrees C. sufficient to remove the labile glucohemoglobin present, while maintaining the stable glucohemoglobin present in the original sample; (c) admixing said acid reaction admixture with a water-compatible borohydride reductant at a ratio of at least about 0.015 moles of borohydride per milligram of hemoglobin to form a reduction reaction admixture; (d) maintaining said reduction reaction admixture for a predetermined time period at a temperature above about 0 degrees C. to about 37 degrees C. sufficient to form glucitollysine-hemoglobin; (e) separating said glucitolylsine-hemoglobin from any unreacted borohydride to form isolated glucitollysine-hemoglobin; (f) admixing said isolated glucitollysine-hemoglobin with glucitollysine-specific antibody molecules or antibody combining site-containing portions thereof to form an immunoreaction admixture; (g) maintaining said immunoreaction admixture under biological assay conditions for a time period sufficient for the antibody molecules or antibody combining site-containing portions thereto to immunologically bind to said glucitollysine-hemoglobin to form an immunoreactant; and (h) determining the amount of immunoreactant that formed in the maintained immunoreaction admixture and thereby the amount of stable glucohemoglobin in said sample.

Claim 2

A method of assaying the amount of stable glucohemoglobin in a glucohemoglobin-containing sample comprising the steps of: (a) admixing said sample with a phthalic acid solution having a pH value of about 3 to about 6, at a ratio of at least about 1.5 milIimoles of said acid per milligram of hemoglobin to form an acid reaction admixture; (b) maintaining said acid reaction admixture for a predetermined time period at a temperature above about 0 degrees C. to about 37 degrees C. sufficient to remove the labile glucohemoglobin present, while maintaining the stable glucohemoglobin present in the original sample; (c) admixing said acid reaction admixture with a water-compatible borohydride reductant at a ratio of at least about 0.015 moles of borohydride per milligram of hemoglobin to form a reduction reaction admixture; (d) maintaining said reduction reaction admixture for a predetermined time period at a temperature above about 0 degrees C. to about 37 degrees C. sufficient to form glucitollysine-hemoglobin; (e) separating said glucitolylsine-hemoglobin from any unreacted borohydride to form isolated glucitollysine-hemoglobin; (f) admixing said isolated glucitollysine-hemoglobin with glucitollysine-specific antibody molecules or antibody combining site-containing portions thereof to form an immunoreaction admixture; (g) maintaining said immunoreaction admixture under biological assay conditions for a time period sufficient for the antibody molecules or antibody combining site-containing portions thereto to immunologically bind to said glucitollysine-hemoglobin to form an immunoreactant; and (h) determining the amount of immunoreactant that formed in the maintained immunoreaction admixture and thereby the amount of stable glucohemoglobin in said sample.

Claim 11

A method of assaying the amount of stable glucohemoglobin in a glucohemoglobin-containing sample comprising the steps of: (a) providing a predetermined volume of a packaged red blood cell pellet; (b) admixing said predetermined volume of pellet with aqueous 0.05M phthalic acid at a pH value of about 4 to about 5 at a ratio of about 1 volume pellet to about 62.5 volumes of aqueous acid to form an acid reaction admixture; (c) maintaining said reaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C, to about 37 degrees C. to form a hemolysate; (d) admixing in a microtiter plate well a predetermined volume of said hemolysate with an equal volume of an aqueous solution having a sodium borohydride or potassium borohydride concentration of about 400 mM to form a reduction reaction admixture containing a ratio of at least about 0.15 millimoles of borohydride per milligram of hemoglobin; (e) maintain said reduction reaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C. to about 37 degrees C. to form microtiter plate well-bound glucitollysine-hemoglobin; (f) separating said well-bound glucitollylsine-hemoglobin from said borohydride to form isolated well-bound glucitollysine-hemoglobin; (g) admixing said isolated well-bound glucitollysine-hemoglobin with a predetermined about of enzyme-labeled antibody molecules secreted by the hybridomas having ATCC accession numbers HB 8356 or HB 8358, or antibody combining site-containing portions thereof, to form an immunoreaction admixture; (h) maintaining said immunoreaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C. to about 37 degrees C., said time period being sufficient for said antibody molecules or antibody combining side-containing portions thereof to immunologically bind said glucitolysine-hemoglobin and form a microtiter plate well-bound immunoreactant; and (i) determining the amount of microtiter plate well-bound immunoreactant that formed in step (h) and thereby the amount of stable glucohemoglobin in said sample.

Claim 12

A method of assaying the amount of stable glucohemoglobin in a glucohemoglobin-containing sample comprising the steps of: (a) providing a predetermined volume of a packaged red blood cell pellet; (b) admixing said predetermined volume of pellet with aqueous 0.05M phthalic acid at a pH value of about 4 to about 5 at a ratio of about 1 volume pellet to about 62.5 volumes of aqueous acid to form an acid reaction admixture; (c) maintaining said reaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C, to about 37 degrees C. to form a hemolysate; (d) admixing in a microtiter plate well a predetermined volume of said hemolysate with an equal volume of an aqueous solution having a sodium borohydride or potassium borohydride concentration of about 400 mM to form a reduction reaction admixture containing a ratio of at least about 0.15 millimoles of borohydride per milligram of hemoglobin; (e) maintain said reduction reaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C. to about 37 degrees C. to form microtiter plate well-bound glucitollysine-hemoglobin; (f) separating said well-bound glucitollylsine-hemoglobin from said borohydride to form isolated well-bound glucitollysine-hemoglobin; (g) admixing said isolated well-bound glucitollysine-hemoglobin with a predetermined about of enzyme-labeled antibody molecules secreted by the hybridomas having ATCC accession numbers HB 8356 or HB 8358, or antibody combining site-containing portions thereof, to form an immunoreaction admixture; (h) maintaining said immunoreaction admixture for a time period of about 15 minutes to about 30 minutes at a temperature of about 20 degrees C. to about 37 degrees C., said time period being sufficient for said antibody molecules or antibody combining side-containing portions thereof to immunologically bind said glucitolysine-hemoglobin and form a microtiter plate well-bound immunoreactant; and (i) determining the amount of microtiter plate well-bound immunoreactant that formed in step (h) and thereby the amount of stable glucohemoglobin in said sample.