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.

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.

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.

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.

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.

Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum.

1988 ◽  
Vol 34 (9) ◽  
pp. 1857-1862 ◽  
Author(s):  
V O Van Hoof ◽  
L G Lepoutre ◽  
M F Hoylaerts ◽  
R Chevigné ◽  
M E De Broe
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
High Molecular Mass ◽  
High Reproducibility ◽  
Neuraminidase Treatment ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Before And After ◽  
Alkaline Phosphatase Isoenzymes

Abstract A modified agarose electrophoretic system for the separation of alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes is described. Bone, liver, high-molecular-mass, and intestinal ALP are separated with high reproducibility. The sensitivity of the agarose system is superior to cellulose acetate in detecting high-Mr ALP. Correlation is good between bone ALP fractions scanned before and after treatment with neuraminidase. Immunoglobulin-bound ALPs, the ALP-lipoprotein-X complex, and the additional ALP fraction observed in transient hyperphosphatasemia in children are detected by their peculiar electrophoretic mobility in the proposed system. Approximately 25% of the samples contained an additional fraction of intestinal-type ALP, as evidenced by neuraminidase treatment and use of polyclonal and monoclonal antibodies. Because the electrophoretic mobilities of this "intestinal variant" and of some immunoglobulin-bound ALP fractions are identical to those of bone and intestinal ALP, respectively, treatment of the samples with a polyclonal antibody that reacts with intestinal ALP is advised.

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.

Precipitation of serum proteins by the lectin from wheat-germ.

1987 ◽  
Vol 33 (1) ◽  
pp. 185-186 ◽  
Author(s):  
W E Schreiber ◽  
L Whitta
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Protein ◽  
Wheat Germ ◽  
Serum Proteins ◽  
Total Serum Protein ◽  
Total Serum ◽  
Wheat Germ Lectin

Abstract We investigated the composition of the precipitate that forms when wheat-germ lectin derived from Triticum vulgaris is added to serum. A number of serum proteins are precipitated, representing about 2.5% of the total serum protein. This study demonstrates that the interaction of this lectin with the bone isoenzyme of alkaline phosphatase is not specific.

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

1989 ◽  
Vol 35 (1) ◽  
pp. 29-32 ◽  
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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