Multicenter evaluation of a specific pancreatic isoamylase assay based on a double monoclonal-antibody technique.

1988 ◽  
Vol 34 (10) ◽  
pp. 2096-2102 ◽  
Author(s):  
N W Tietz ◽  
A Burlina ◽  
W Gerhardt ◽  
W Junge ◽  
P Malfertheiner ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Wheat Germ ◽  
Reference Intervals ◽  
Antibody Technique ◽  
Clinical Sensitivity ◽  
Activity Concentrations ◽  
Interlaboratory Studies ◽  
Inhibitor Method ◽  
Pancreatic Isoamylase ◽  
High Concentrations

Abstract Eleven evaluators from nine laboratories in five countries evaluated a new immunoinhibition method for pancreatic isoamylase determination that is as simple to perform as that for total amylase. The precision at low and intermediate activity concentrations was superior, and at high concentrations it equalled that of the wheat-germ inhibitor method. The test was linear to approximately 2000 U/L, depending on the instrumentation used. The percentage salivary isoamylase activities remaining in specimens after reaction with two monoclonal antibodies ranged from 2 to 4.4%. Comparative studies showed good correlation with the wheat-germ inhibitor (r greater than 0.978) and electrophoresis methods (r = 0.920). Hemolysis, lipemia, and bilirubinemia have no effect on results. Interlaboratory studies demonstrated excellent transferability of the method, if instruments are calibrated with the same calibrator. Reference intervals for pancreatic isoamylase are 13 to 64 U/L (25 degrees C), 13 to 83 U/L (30 degrees C), and 17 to 115 U/L (37 degrees C). A clinical evaluation of patients with acute pancreatitis showed that pancreatic isoamylase has a greater clinical sensitivity than total amylase.

Pancreatic amylase measured in serum by use of a monoclonal antibody immunochemically immobilized to a solid phase.

1989 ◽  
Vol 35 (1) ◽  
pp. 110-114 ◽  
Author(s):  
T E Mifflin ◽  
M Hamilton ◽  
E Hubbard ◽  
M J Kline ◽  
D E Bruns
Keyword(s):  
Monoclonal Antibody ◽  
Polyvinylidene Fluoride ◽  
Solid Phase ◽  
Amylase Activity ◽  
Reference Intervals ◽  
New Method ◽  
Pancreatic Amylase ◽  
Salivary Amylase ◽  
Electrophoretic Method ◽  
Activity Concentrations

Abstract We studied a method for measuring the pancreatic isoenzyme of amylase (EC 3.2.1.1) by use of a mouse monoclonal antibody against human salivary-type amylase (Clin Chem 1985;31:1283) coupled indirectly to particles of polyvinylidene fluoride via polyclonal goat anti-mouse immunoglobulin. These particles, in 200 microL of a suspension, could remove salivary amylase (activity 2200 U/L) from an equal volume of serum in 5 min. Measurement of amylase activity in the supernatant fluids from treated sera thus provided an assay of pancreatic amylase. Precision studies at three activity concentrations yielded within-run CVs of 1.6% to 1.7% (n = 25) and total CVs of 2.2% to 5.1% (20 days). Salivary amylase added to each of 10 sera was completely (99.8%, SD 1.6%) removed. The new method (y) showed the following regression statistics when compared with an electrophoretic method (x): slope = 0.989 (SD 0.019), intercept = -0.220% (SD 1.48%), SEE 4.0%, n = 51. Similar respective regression values were found for urine samples: slope = 0.934 (SD 0.053), intercept = 2.3 U/L (SD 3.2), SEE 8.4 U/L, n = 26. The following respective values were found when the new method (y) was compared with the previously described immunoprecipitation assay (x): slope = 1.02 (SD 0.02), intercept = 2.2% (SD 1.4%), SEE 3.3%, n = 23 sera. Reference intervals for pancreatic amylase activity in serum were established for three different substrates: maltotetraose, maltopentaose, and p-nitrophenylheptaoside.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of beta-spectrin in filaments quantitatively resembling those of erythrocytes

1995 ◽  
Vol 108 (9) ◽  
pp. 3145-3154 ◽  
Author(s):  
D.W. Pumplin
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Cytoplasmic Membrane ◽  
Membrane Surface ◽  
Membrane Skeleton ◽  
Immunogold Labeling ◽  
Platinum Coating ◽  
High Concentrations ◽  
Receptor Clusters

I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of beta-spectrin colocalizing with acetylcholine receptors. The filaments are 32 +/- 11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of beta-spectrin.

Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall.

1987 ◽  
Vol 33 (7) ◽  
pp. 1235-1236 ◽  
Author(s):  
S Hiroishi ◽  
S Matsuyama ◽  
S Kurooka ◽  
N Sunahara ◽  
S Inoue ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Bacterial Cell ◽  
Present Method ◽  
Bacterial Cell Wall ◽  
Clinical Tests ◽  
Salivary Amylase ◽  
Inhibitor Method ◽  
Serum And Urine ◽  
Serum And Urine Samples

Abstract We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.

scholarly journals Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1094-1102 ◽  
Author(s):  
Y Ozaki ◽  
J Iwata ◽  
T Ohashi
Keyword(s):  
Membrane Proteins ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Binding Property ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Cell Extracts ◽  
High Concentrations

Abstract Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

scholarly journals Thrombospondin stimulates motility of human neutrophils.

1990 ◽  
Vol 111 (6) ◽  
pp. 3077-3086 ◽  
Author(s):  
P J Mansfield ◽  
L A Boxer ◽  
S J Suchard
Keyword(s):  
Monoclonal Antibody ◽  
Cell Types ◽  
Human Neutrophils ◽  
Heparin Binding ◽  
Directed Movement ◽  
Surface Receptors ◽  
Chemotactic Peptides ◽  
Heparin Binding Domain ◽  
High Concentrations ◽  
Priming Activity

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)-mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20-70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.

Role of endotoxin in intestinal reperfusion-induced expression of E-selectin

1999 ◽  
Vol 276 (2) ◽  
pp. G479-G484 ◽  
Author(s):  
Philippe Bauer ◽  
Janice M. Russell ◽  
D. Neil Granger
Keyword(s):  
Monoclonal Antibody ◽  
Cell Adhesion Molecule ◽  
Inflammatory Responses ◽  
Enteric Bacteria ◽  
Ischemia And Reperfusion ◽  
Induced Expression ◽  
Antibody Technique ◽  
Dose Dependent ◽  
Endothelial Cell Adhesion

Products of enteric bacteria, including endotoxin [lipopolysaccharide (LPS)], have been implicated in the acute inflammatory responses elicited by ischemia and reperfusion (I/R) of the small intestine. The objective of this study was to assess the contribution of LPS to the increased E-selectin expression observed in the intestinal vasculature after I/R. The dual radiolabeled monoclonal antibody technique was used in LPS-sensitive (C3HeB/FeJ) and LPS-insensitive (C3H/HeJ) mice that were exposed to either exogenous LPS or to gut I/R (45 min ischemia, 5 h reperfusion). LPS elicited a dose-dependent (0.5–50 μg LPS/animal) increase in E-selectin expression (at 3 h) in LPS-sensitive mice, whereas LPS-insensitive mice were largely unresponsive. E-selectin expression was increased fivefold by I/R in the small bowel of both LPS-sensitive and -insensitive mice. These results indicate that, although exogenous LPS is capable of eliciting profound dose-dependent increases in E-selectin expression, endogenous LPS does not contribute significantly to I/R-induced expression of this endothelial cell adhesion molecule.

Surface carbohydrate changes onOnchocerca lienalislarvae as they develop from microfilariae to the infective third-stage inSimulium ornatum

1988 ◽  
Vol 62 (3) ◽  
pp. 195-205 ◽  
Author(s):  
P. J. Ham ◽  
A. J. Smail ◽  
B. K. Groeger
Keyword(s):  
Wheat Germ ◽  
Helix Pomatia ◽  
Kidney Bean ◽  
Con A ◽  
The Third ◽  
Lentil Lectin ◽  
Natural Vector ◽  
Third Stage ◽  
High Concentrations ◽  
Surface Carbohydrates

ABSTRACTUse was made of seven FITC labelled lectins as tools to investigate the surface ofOnchocerca lienalislarvae as they develop through to the infective third-stage in a natural vector,Simulium ornatum. The lectins were derived fromCanavalia ensiformis(Con A),Lens culinaris(lentil),Triticum vulgaris(wheat germ),Arachis hypogaea(peanut),Helix pomatia, Phaseolus vulgaris(kidney bean) andTetragonolobus purpureus(asparagus pea). Between 70 and 100 living parasites were examined for each developmental stage; i.e. skin microfilariae, late first-stages, second-stages, preinfective third-stages and infective third-stages isolated from the mouth parts of the flies. None of the lectins used bound to the surface of the microfilariae. However, progressive binding to the cuticle of the first- and second-stages was observed using Con. A, lentil lectin and wheat germ agglutinin (WGA). Following moulting to the third-stage, binding of these three lectins declined. Furthermore, as these lectins decreased, peanut andHelix pomatialectins progressively increased in their binding, despite the fact that they showed little or no binding to the first- and second-stages; stages at which Con A, lentil and WGA were at their maximum. Asparagus pea and kidney bean lectins failed completely to bind to any of the larvae examined. Carbohydrate inhibition tests showed that the lectin was indeed binding specifically to glycoconjugates on the parasite surface. WGA binding was not inhibited by prior incubation with N-acetyl-D-glucosamine, even at high concentrations, but neuraminic acid did completely inhibit its binding. Judging from the patterns of binding on the nematodes themselves, the carbohydrates may not be vector in origin, but derive from the worms. The lectin specificities indicate that initially mannose/glucose type derivatives are present on the surface. Following moulting to the third-stage these are progressively replaced, or overlaid with galactosamine type derivatives, also present on the infective third-stage as it enters the bovine host. The availability of these surface glycoconjugates to attack mediated by natural insect lectins may be of importance in the parasite regulatory mechanisms of the blackfly. Variability in these surface carbohydrates, and in the response to them could well be a contributing factor in the cytospecific variation inS. damnosumsusceptibility to geographical variants ofO. volvulus.

Total Amylase and Pancreatic Isoamylase in Serum and Urine: Considerations from Data on Biological Variation

1989 ◽  
Vol 26 (4) ◽  
pp. 335-340 ◽  
Author(s):  
Steven T Cummings ◽  
Callum G Fraser
Keyword(s):  
Clinical Decision Making ◽  
Amylase Activity ◽  
Clinical Decision ◽  
Population Based ◽  
Reference Intervals ◽  
Similar Data ◽  
Creatinine Ratio ◽  
Additional Information ◽  
Pancreatic Isoamylase ◽  
Urine Amylase

The analytical, within-subject and between-subject components of variation were estimated for amylase activity and pancreatic isoamylase activity in serum measured using newer analytical methods. Desirable analytical imprecisions based on within-subject variation were CV ≤ 4·4% and CV ≤ 7·0%, respectively. Conventional population-based reference intervals were not useful because of marked individuality; clinical decision-making points should be derived from the desired sensitivity and specificity. Serial results must change more than about 30% and 40% respectively before significance (P ≤ 0·05) can be claimed. Similar data on total amylase and pancreatic isoamylase activities in random and first morning urines showed that the use of conventional reference intervals was appropriate. Very large changes (> 100%) were required before a difference in serial results was significant. Calculation of the urine amylase/creatinine ratio appeared to confer no advantage. Derivation of the ratio of pancreatic isoamylase to total amylase activity in serum or urine was unlikely to provide additional information of value in either diagnosis or monitoring.

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