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

1985 ◽  
Vol 31 (7) ◽  
pp. 1198-1200 ◽  
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.

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

1993 ◽  
Vol 39 (7) ◽  
pp. 1404-1407 ◽  
Author(s):  
M Mattiazzo ◽  
I Ramasamy
Keyword(s):  
Alkaline Phosphatase ◽  
Wheat Germ ◽  
Serum Alkaline Phosphatase ◽  
Single Step ◽  
Agarose Gels ◽  
Lectin Affinity ◽  
Affinity Electrophoresis ◽  
Alkaline Phosphatase Isoenzymes ◽  
Electrophoresis Method ◽  
Wheat Germ Lectin

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.

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

1986 ◽  
Vol 32 (8) ◽  
pp. 1570-1573 ◽  
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.

Cellulose Acetate Electrophoresis of Alkaline Phosphatase Isoenzymes in Human Serum and Tissue

1972 ◽  
Vol 18 (5) ◽  
pp. 417-421 ◽  
Author(s):  
H A Fritsche ◽  
H R Adams-Park
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Cellulose Acetate ◽  
High Sensitivity ◽  
Heat Inactivation ◽  
Cellulose Acetate Electrophoresis ◽  
Electrophoretic Method ◽  
Large Numbers ◽  
Alkaline Phosphatase Isoenzymes ◽  
Isoenzyme Patterns

Abstract We describe a new electrophoretic method for the characterization of human serum and tissue alkaline phosphatases on cellulose acetate plates. Enzymes are localized fluorometrically with the substrate α-naphthol AS-MX phosphate or colorimetrically by coupling the reaction product with Fast Blue RR. Both localization techniques are sensitive enough to demonstrate isoenzyme patterns in micro-scale samples of normal sera. Our electrophoretic studies indicate that sera of children and adults normally contain isoenzymes originating from both liver and bone. The high sensitivity of the method allows the use of normal sera as markers rather than tissue extracts, and isoenzyme patterns may be visually assessed after heat inactivation and chemical inhibition. The method is suitable for the electrophoretic fractionation of alkaline phosphatase in large numbers of sera, with equipment and technique familiar to many laboratories.

Quantification of bone alkaline phosphatase in serum by precipitation with wheat-germ lectin: a simplified method and its clinical plausibility.

1986 ◽  
Vol 32 (10) ◽  
pp. 1960-1966 ◽  
Author(s):  
W Behr ◽  
J Barnert
Keyword(s):  
Alkaline Phosphatase ◽  
Wheat Germ ◽  
Biliary Tree ◽  
Bone Alkaline Phosphatase ◽  
Original Method ◽  
Bone Diseases ◽  
Skeletal Involvement ◽  
Number Of Patients ◽  
Comparison Groups ◽  
Wheat Germ Lectin

Abstract We report an easy, rapid method for quantifying bone isoenzyme of alkaline phosphatase (EC 3.1.3.1., ALP) in serum. The original method described by Rosalki and Ying Foo (Clin Chem 1984;30:1182-6) was somewhat simplified. In contrast to their results, we found that bone ALP is precipitated quantitatively by wheat-germ lectin. To check the clinical plausibility of the method, we used samples from several comparison groups (blood donors, children, pregnant women, patients with neoplasms but without skeletal involvement) and a large number of patients suffering from bone diseases and diseases of the liver and biliary tree. Measured activities of bone ALP nearly always correlated with the clinical diagnosis. Only patients with hepatitis often had pathological bone activities not in accord with the other findings. Possible reasons for this observation are discussed.

Quantitative alkaline phosphatase isoenzyme determination by electrophoresis on cellulose acetate membranes.

1977 ◽  
Vol 23 (1) ◽  
pp. 28-34 ◽  
Author(s):  
W H Siede ◽  
U B Seiffert
Keyword(s):  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Clinical Laboratory ◽  
Kinetic Assay ◽  
Specific Staining ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Elution Method ◽  
Routine Clinical Laboratory ◽  
Alkaline Phosphatase Isoenzyme

Abstract We present a new method for quantitative determination of alkaline phosphatase isoenzymes. This method consists of electrophoretic separation on cellulose acetate membranes, special fixation technique to avoid elution and diffusion of enzyme protein during incubation, specific staining, and quantitative evaluation by densitometric measurement. We highly recommend the precedure for routine clinical laboratory use. In all normal individuals we observe two isoenzymes of hepatic origin and one isoenzyme each of osseous, intestinal, and biliary origin. Quantitative normal values are presented. Precision of the method is calculated, the CV being less than 10%. The exactness of densitometric quantification is proved by comparison with kinetic assay of alkaline phosphatase isoenzymes by use of an elution method. Clinical implications of alkaline phosphatase isoenzymograms are reported and discussed in detail.

Reference standards for quantification of skeletal alkaline phosphatase activity in serum by heat inactivation and lectin precipitation

1993 ◽  
Vol 39 (9) ◽  
pp. 1878-1884 ◽  
Author(s):  
J R Farley ◽  
S L Hall ◽  
S Herring ◽  
C Libanati ◽  
J E Wergedal
Keyword(s):  
Alkaline Phosphatase ◽  
Alkaline Phosphatase Activity ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Bone Cells ◽  
Heat Inactivation ◽  
Osteosarcoma Cells ◽  
Con A ◽  
Alp Activity ◽  
Skeletal Alkaline Phosphatase

Abstract Putative standards of skeletal alkaline phosphatase (ALP) (from bone, bone cells, osteosarcoma cells, and Pagetic serum) and hepatic ALP (from cholestatic serum and bile) were used to compare three methods for quantifying skeletal ALP activity in serum: heat inactivation, precipitation with wheat germ agglutinin (WGA), and precipitation with concanavalin A (Con A). All the skeletal ALP standards were similarly sensitive to heat inactivation, as were the hepatic ALP standards. Heat inactivation separated skeletal from hepatic ALP by a 50% difference in remaining ALP activities (e.g., 23% and 74% remaining skeletal and hepatic ALP activities after 30 min at 52 degrees C). Differential precipitations with WGA and with Con A were less efficient at separating skeletal from hepatic ALP (maximum differences of < 30% remaining ALP activity). Although both types of hepatic ALP standard (cholestatic serum and bile) were precipitated with similar efficiencies by WGA and Con A, the skeletal ALP standards were not (e.g., at 2.7 g/L, WGA precipitated 78-86% of the ALP activity in Pagetic serum, but only 49% of the ALP activity in extracts of human bone). These data suggest that heat inactivation is preferable to precipitation with WGA or Con A for quantifying skeletal ALP activity in serum: it better separates skeletal from hepatic ALP activity and is not sensitive to glycosyl heterogeneity.

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