Macroamylases: Differences in Activity against Various-Sized Substrates

1992 ◽  
Vol 38 (9) ◽  
pp. 1725-1729
Author(s):  
J L Rosenblum ◽  
G L Hortin ◽  
C H Smith ◽  
G E Pashos ◽  
M Landt
Keyword(s):  
Acute Pancreatitis ◽  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
Amylase Activity ◽  
High Molecular Mass ◽  
Pancreatic Amylase ◽  
Serum Samples ◽  
Automated Methods

Abstract Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-mass (high-M(r)) substrates rather than oligosaccharide substrates. We hypothesized that high-M(r) substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83), but we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding antihuman pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and to purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-M(r) substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparing activities determined from automated methods that use different substrates.

Macroamylases: Differences in Activity Against Various-Size Substrates

1992 ◽  
Vol 38 (8) ◽  
pp. 1454-1458
Author(s):  
J L Rosenblum ◽  
G L Hortin ◽  
C H Smith ◽  
G E Pashos ◽  
M Landt
Keyword(s):  
Molecular Weight ◽  
Acute Pancreatitis ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
Amylase Activity ◽  
Pancreatic Amylase ◽  
Serum Samples ◽  
Automated Methods

Abstract Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-weight rather than oligosaccharide substrates. We hypothesized that high-molecular-weight substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83); we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding anti-human pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-molecular-weight substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparison of activities determined from automated methods that use different substrates.

scholarly journals Immunochemical characterization of mucins. Polypeptide (M1) and polysaccharide (A and Leb) antigens

1988 ◽  
Vol 254 (1) ◽  
pp. 185-193 ◽  
Author(s):  
J Bara ◽  
R Gautier ◽  
J Le Pendu ◽  
R Oriol
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Density Gradient Ultracentrifugation ◽  
Blood Group Antigens ◽  
Lewis Blood Group ◽  
Progressive Loss ◽  
Cscl Density Gradient ◽  
Mucinous Cyst

Seven monoclonal antibodies (MAbs) reacting with high-molecular-mass components (greater than 20,000 kDa) isolated from an ovarian mucinous cyst of an A Le(a-b+) patient are described. By the use of immunoradiometric methods, these MAbs characterized seven different epitopes associated with components having a density of 1.45 g/ml by CsCl-density-gradient ultracentrifugation, like mucins. Two MAbs reacted with A and Lewis blood-group antigens respectively (polysaccharide epitopes). The five other MAbs characterized five M1 epitopes (called a, b, c, d and e), mainly associated with components of more than 20,000 kDa and 2000 kDa. They were completely destroyed by papain and 2-mercaptoethanol treatment (polypeptide epitopes). Moreover, timed trypsin digestion of native mucin resulted in a progressive loss of M1 activity and degraded these mucins into smaller M1-positive fragments. The a and c epitopes were partially degraded from relatively high-molecular-mass fragments (2000 kDa to 500 kDa) into a 100 kDa fragment. The b and d epitopes were completely degraded into smaller fragments ranging from 100 kDa to 40 kDa. The e epitope was completely destroyed by trypsin. These different pathways of M1 antigen degradation suggest the occurrence of different epitopes located in separate regions of the mucin molecules.

Determination of amylase activity in serum by using a wheat germ inhibitor with the Du Pont aca.

1986 ◽  
Vol 32 (8) ◽  
pp. 1539-1541 ◽  
Author(s):  
D A Lacher ◽  
M B Harize
Keyword(s):  
Acute Pancreatitis ◽  
Wheat Germ ◽  
Serum Amylase ◽  
Amylase Activity ◽  
Reference Interval ◽  
Pancreatic Amylase ◽  
Wheat Germ Extract ◽  
Rapid Procedure ◽  
Assay Conditions

Abstract A rapid procedure for determining salivary- and pancreatic-type amylase (EC 3.2.1.1) in serum by incorporating a wheat germ inhibitor (from Triticum aestivum) was developed for the Du Pont aca IV analyzer. Under optimal assay conditions, activities of salivary and pancreatic amylase were inhibited by 93% and 19%, respectively. The 95% central reference interval for the percentage of inhibition of serum amylase was 38-84%. Patients with acute pancreatitis showed less than 26% inhibition of amylase after addition of the wheat germ extract, reflecting the prevalence of pancreatic-type amylase in this disorder.

Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum.

1986 ◽  
Vol 32 (8) ◽  
pp. 1503-1505 ◽  
Author(s):  
E Sykes ◽  
F L Kiechle ◽  
E Epstein
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Cereus ◽  
Molecular Mass ◽  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Alkaline Phosphatase Activity ◽  
Plasma Membranes ◽  
High Molecular Mass ◽  
Serum Samples ◽  
Liver Membrane

Abstract Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin. This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes. Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band. Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.

scholarly journals Total and Pancreatic Amylase Measured with 2-Chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside

2000 ◽  
Vol 46 (7) ◽  
pp. 928-933 ◽  
Author(s):  
Yoshitaka Morishita ◽  
Yoshitsugu Iinuma ◽  
Nobuo Nakashima ◽  
Keiichi Majima ◽  
Katsuhiko Mizuguchi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amylase Activity ◽  
Biological Fluids ◽  
Transfer Reactions ◽  
Pancreatic Amylase ◽  
Salivary Amylase ◽  
Specific Determination ◽  
Routine Procedures

Abstract Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex. Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-d-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm. Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-d-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4-O-β-d-galactopyranosyl-β-maltotetraoside method or the modified IFCC method. Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.

A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase

1997 ◽  
Vol 110 (24) ◽  
pp. 3031-3041
Author(s):  
K. Shimada ◽  
M. Harata ◽  
S. Mizuno
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
Nuclear Matrix ◽  
Protein S ◽  
S Phase ◽  
High Molecular Mass ◽  
Nuclear Antigen ◽  
Double Positive ◽  
Embryonic Fibroblasts ◽  
Triton X

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.

scholarly journals Presence of free haptoglobin alpha 1S-subunit in acute porcine reproductive and respiratory syndrome virus infection

2008 ◽  
Vol 89 (11) ◽  
pp. 2746-2753 ◽  
Author(s):  
Josephine S. Gnanandarajah ◽  
Cheryl M. T. Dvorak ◽  
Craig R. Johnson ◽  
Michael P. Murtaugh
Keyword(s):  
Molecular Mass ◽  
Clinical Signs ◽  
High Molecular Mass ◽  
Respiratory Syndrome Virus ◽  
Tandem Ms ◽  
Laser Desorption Ionization ◽  
Serum Samples ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Significant Disease

The biochemical events triggered by viral infection are critical to the outcome of a host immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) causes the most significant disease of swine worldwide. Onset of infection is insidious and subclinical. Clinical signs may not appear for days and antibodies cannot be detected for a week or more. To understand better the early pathophysiological response of swine to PRRSV infection and its inapparent onset, we examined serum samples in the first days of infection for evidence of early biochemical changes. Sera from pigs infected with various isolates of PRRSV were extracted to remove high molecular mass proteins, desalted and analysed by matrix assisted laser desorption/ionization–time of flight mass spectrometry (MS). Comparative analysis of low molecular mass serum protein profiles revealed that one protein, with an m/z value of 9244±2, appeared within 1 day of infection. The 9244±2 peak was identified as the alpha 1S (α1S)-subunit of porcine haptoglobin (Hp) by tandem MS sequencing and confirmed by immunoblotting with anti-porcine Hp antibody. Hp is an acute phase haem-binding protein consisting of α–β heterodimers that is secreted from the liver in response to stresses, including infection. However, the presence of free α1S-subunit in response to infection is novel and may provide new insights into biochemical processing of Hp and its role in disease pathogenesis, including PRRS.

scholarly journals Radioimmunoassay of Human Pancreatic Amylase with Monoclonal Antibody

1989 ◽  
Vol 26 (5) ◽  
pp. 416-421 ◽  
Author(s):  
Chiyo Fujita ◽  
Chihiro Kasai ◽  
Hiromi Kosuge ◽  
Kenji Ogata ◽  
Ichiyo Oshima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Acute Pancreatitis ◽  
Normal Subjects ◽  
Enzymic Activity ◽  
Pancreatic Amylase ◽  
Normal Range ◽  
Salivary Amylase ◽  
Serum Samples ◽  
The Mean ◽  
Mean Concentration

A monoclonal antibody (E-21) was obtained that specifically binds to human pancreatic amylase and shows negligible cross-reaction with human salivary amylase. Using this antibody a radioimmunoassay was developed for pancreatic amylase in human serum. The assay was shown to be sensitive (detectable up to 7 mg/L), reproducible, and specific for pancreatic amylase. In normal subjects, the mean concentration of serum pancreatic amylase determined by this method was 36·3 mg/L with a 95% confidence range of 16·5 to 79·2 mg/L. A good correlation was observed between the concentrations of immunoreactive pancreatic amylase (IR-PA) and enzymatic activities in 20 serum samples ( r = 0·97). The concentration of serum IR-PA was below the detectable limit in pancreatectomised patients, and was greatly increased in patients with acute pancreatitis; the latter was accompanied by parallel changes in total enzymic activity. In patients with mumps, the serum IR-PA level was within the normal range whereas the total enzymic activity was elevated.

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