Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase with commercially available agarose gels

Abstract We have modified a commercially available procedure involving precast agarose gels (Paragon Isopal System) to measure alkaline phosphatase (EC 3.1.3.1) isoenzymes. Including wheat germ lectin in the equilibration buffer improved the resolution of the bone and liver isoenzymes. Unlike previously described wheat germ lectin affinity electrophoresis methods, the procedure measures bone, liver, and biliary isoenzymes in a single step. There was good correlation between the affinity electrophoresis and the neuraminidase preincubation methods for the measurement of bone (r = 0.958) and liver (r = 0.962) alkaline phosphatase isoenzymes. However, the affinity electrophoresis method also quantified minor isoenzyme fractions that were poorly resolved by the neuraminidase method. The method is technically simple, reproducible, and capable of rapid handling of large workloads.

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Reference limits of bone and liver alkaline phosphatase isoenzymes in the serum of healthy subjects according to age and sex as determined by wheat germ lectin affinity electrophoresis

Clinica Chimica Acta ◽

10.1016/0009-8981(88)90014-9 ◽

1988 ◽

Vol 173 (3) ◽

pp. 273-280 ◽

Cited By ~ 14

Author(s):

Tomomi Kuwana ◽

Osamu Sugita ◽

Minoru Yakata

Keyword(s):

Alkaline Phosphatase ◽

Healthy Subjects ◽

Wheat Germ ◽

Lectin Affinity ◽

Affinity Electrophoresis ◽

Reference Limits ◽

Alkaline Phosphatase Isoenzymes ◽

Wheat Germ Lectin ◽

Age And Sex

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Alkaline phosphatase isoenzymes resolved by electrophoresis on lectin-containing agarose gel.

Clinical Chemistry ◽

10.1093/clinchem/32.8.1570 ◽

1986 ◽

Vol 32 (8) ◽

pp. 1570-1573 ◽

Cited By ~ 14

Author(s):

W E Schreiber ◽

L Whitta

Keyword(s):

Heat Treatment ◽

Alkaline Phosphatase ◽

Cellulose Acetate ◽

Wheat Germ ◽

Agarose Gel ◽

Electrophoretic Method ◽

Activity Staining ◽

Agarose Gels ◽

Alkaline Phosphatase Isoenzymes ◽

Wheat Germ Lectin

Abstract With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.

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Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma.

Clinical Chemistry ◽

10.1093/clinchem/31.7.1198 ◽

1985 ◽

Vol 31 (7) ◽

pp. 1198-1200 ◽

Cited By ~ 15

Author(s):

S B Rosalki ◽

A Y Foo

Keyword(s):

Alkaline Phosphatase ◽

Cellulose Acetate ◽

Wheat Germ ◽

Bone Alkaline Phosphatase ◽

Heat Inactivation ◽

Cellulose Acetate Membrane ◽

Affinity Electrophoresis ◽

Alkaline Phosphatase Isoenzymes ◽

Wheat Germ Lectin ◽

Tissue Origin

Abstract Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.

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Alkaline phosphatase isoenzymes of liver and bone origin are incompletely resolved by wheat-germ-lectin affinity chromatography.

Clinical Chemistry ◽

10.1093/clinchem/35.1.29 ◽

1989 ◽

Vol 35 (1) ◽

pp. 29-32 ◽

Cited By ~ 10

Author(s):

D G Gonchoroff ◽

E L Branum ◽

J F O'Brien

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Affinity Chromatography ◽

Wheat Germ ◽

Solid Phase ◽

Lectin Affinity Chromatography ◽

Serum Samples ◽

Lectin Affinity ◽

Alp Activity ◽

Wheat Germ Lectin

Abstract We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.

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