scholarly journals Characterization of quail intestinal mucin as a ligand for endogenous quail lectin

1993 ◽  
Vol 293 (3) ◽  
pp. 867-872 ◽  
Author(s):  
R Fang ◽  
M Mantle ◽  
H Ceri
Keyword(s):  
Goblet Cells ◽  
Cross Reactivity ◽  
Mucosal Surface ◽  
Immunoperoxidase Technique ◽  
Western Blots ◽  
Intestinal Mucin ◽  
Sds Page ◽  
Endogenous Lectins ◽  
Antibody Preparations ◽  
Intestinal Mucins

The S-type lectins have been shown to be components of mucosal scrapings, and in avian systems these lectins have been localized immunohistochemically to the mucosal surface and goblet cells of the intestine. The interaction of lectin specifically with purified mucin has not, however, been established. Quail intestinal mucin was purified by two subsequent isopycnic density-gradient centrifugations in CsCl and chromatography on Sepharose Cl-2B. Purified mucin, obtained from the void volume of the Sepharose column, was characterized by SDS/PAGE, amino acid and carbohydrate analyses, sensitivity to thiol reduction, and cross-reactivity with antibody preparations to rat and human intestinal mucins on Western blots. Antibody raised against purified quail mucin partially cross-reacts with purified rat, rabbit and human intestinal mucins, and specifically labels the mucosal surface and goblet cells of quail intestine by the immunoperoxidase technique. Protein eluted by lactose from an affinity matrix composed of quail intestinal mucin possessed the same molecular mass on SDS/PAGE as intestinal lectin and reacted on Western blots with a lectin-specific antibody. The data clearly demonstrate the co-localization of lectin and mucin in the quail intestine and also the ability of the lectin to specifically interact with the purified mucin, raising the question of the role of endogenous lectins in secretions.

scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Genetic and immunological characterization of the microsporidianSeptata intestinalisCali, Kotler and Orenstein, 1993: reclassification toEncephalitozoon intestinalis

Parasitology ◽  
1995 ◽  
Vol 110 (3) ◽  
pp. 277-285 ◽  
Author(s):  
R. A. Hartskeerl ◽  
T. Van Gool ◽  
A. R. J. Schuitema ◽  
E. S. Didier ◽  
W. J. Terpstra
Keyword(s):  
Protein Composition ◽  
Rflp Analysis ◽  
Small Subunit ◽  
Cross Reactivity ◽  
Antigenic Structure ◽  
Phenotypic Characterization ◽  
Western Blots ◽  
Microsporidian Species ◽  
Sequence Identity ◽  
Sds Page

SUMMARYTh relationships between theEncephalitozoon-likeSeptata intestinalisand other microsporidia that occur in humans, notablyEncephalitozoon cuniculiandEncephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects ofS. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of theS. intestinalis rrsgene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplifiedrrsgenes and establishment of the degree of sequence identity betweenrrsgenes of various microsporidian species. The unique sequence ofrrsofS. intestinalisas well as the distinct RFLP and SDS-PAGE profiles indicate thatS. intestinalisis clearly different from other human microsporidian species. However, itsrrsgene shared about 90% sequence identity withrrsof bothEncephalitozoonspp.,E. cuniculiandE. hellem. This is remarkably higher than the about 70% identity observed betweenrrsof microsporidian species which belong to different genera and thus suggests that S.intestinalisshould be regarded as a species of the genusEncephalitozoon. Western blots revealed a marked cross-reactivity betweenS. intestinalisand both species ofEncephalitozoonwhich also stresses the close relationship between these organisms. It is concluded thatS. intestinalisis so closely related toE. cuniculi, the type species ofEncephalitozoon, that it should be reclassified asEncephalitozoon intestinalis.

scholarly journals Secretion of endogenous lectin by chicken intestinal goblet cells.

1982 ◽  
Vol 92 (1) ◽  
pp. 28-33 ◽  
Author(s):  
E C Beyer ◽  
S H Barondes
Keyword(s):  
Goblet Cells ◽  
Mucosal Surface ◽  
Immunoperoxidase Staining ◽  
Secretory Vesicles ◽  
Intestinal Epithelial ◽  
Epithelial Surface ◽  
Adult Chicken ◽  
Intestinal Mucin ◽  
Cholinergic Agent ◽  
Staining Methods

The two lactose-binding lectins found in adult chicken intestine, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11), were localized within the vesicles of the mucin-secreting goblet cells by indirect immunofluorescence and immunoperoxidase staining methods. Attention was concentrated on CLL-11 which is 200 time more abundant than CLL-1 in adult intestine. The localization of CLL-11 in secretory vesicles, combined with its demonstration on the intestinal epithelial surface by immune staining methods and by specific elution with lactose, suggested that at least a portion of the CLL-11 in the vesicles was secreted by the goblet cells and then became associated with the mucosal surface. In support of this, treatment of isolated intestinal strips with a cholinergic agent, bethanechol (10(-7 M) produced a small but significant increase in the amount of CLL-11 that could be eluted from their surface with lactose. Secretion of lectin may occur in conjunction with mucin because both are localized in the secretory vesicles and CLL-1 and CLL-11 apparently bind to purified chicken intestinal mucin, which is a potent inhibitor of their hemagglutination activities. The mucin is six orders of magnitude more potent than lactose as a hemagglutination inhibitor of CLL-1 or CLL-11 on a molar basis, and three orders of magnitude more potent when expressed per mole of hexose. These results suggest that CLL-11, and perhaps CLL-1, are secreted from the goblet cells along with mucin. They may function in the organization of mucin for secretion and/or in its association with the intestinal mucosal surface.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

2008 ◽  
Vol 43 (10) ◽  
pp. 1405-1411 ◽  
Author(s):  
Paula Radaelli ◽  
Thor Vinícius Martins Fajardo ◽  
Osmar Nickel ◽  
Marcelo Eiras ◽  
Gilvan Pio-Ribeiro
Keyword(s):  
Virus Disease ◽  
Disease Diagnosis ◽  
Coat Proteins ◽  
Grapevine Virus ◽  
Western Blots ◽  
E Coli ◽  
Sds Page ◽  
Grapevine Virus B ◽  
Polyclonal Antisera ◽  
Infected Grapevine

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

Cross-Reactivity between IgE-Binding Proteins from Anisakis Simplex and Dermatophagoides Pteronyssinus

2005 ◽  
Vol 18 (4) ◽  
pp. 671-675 ◽  
Author(s):  
R. Bernardini ◽  
G. Mistrello ◽  
E. Novembre ◽  
D. Roncarolo ◽  
S. Zanotta ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Specific Ige ◽  
Dermatophagoides Pteronyssinus ◽  
Cross Reactivity ◽  
Anisakis Simplex ◽  
Cross Reaction ◽  
Ige Binding ◽  
Sds Page ◽  
The Cross

An association was found between Anisakis simplex (As) and Dermatophagoides pteronyssinus (Dp) sensitization. One recent study shows a cross-reactivity between As and Dp and tropomyosin (tr) is suspected as being one of the proteins responsible of this cross-reaction. The aim of our study was: 1) to confirm the cross-reactivity between Dp and As; 2) to determine the importance of tr in this cross reaction. SDS-PAGE analysis of Dp and As (metabolic and somatic) extracts was carried out. Then an IgE immunoblotting test using serum from a patient who had specific IgE only to Dp and As and immunoblotting inhibition experiments using Dp extract and tr as inhibitors were performed. We found that patient's serum reacted: 1) against larval As antigens with a molecular weight (mw) of 25 kilodalton (kD) and a mw > 100 kD, 2) against various metabolic As antigens with a mw > 100 kD, a mw ranging approximately from 35 to 50 kD, and a mw around 20 kD, and 3) against Dp proteins with mw between 35 and 55 kD. Preincubation of patient's serum with Dp extract caused the disappearance of reactivity against antigens with a mw > 100 kD in both larval and metabolic As extracts and against proteins with mw ranging approximately from 35 to 50 kD in the metabolic As extract. Preincubation of patient's serum with As extract caused the disappearance of reactivity against antigens with mw between 35 and 55 kD in the Dp extract. Pre-incubation of patient's serum with tr did not induce any change in the immunoblotting profile. The results show that 1) cross-reactive components between Dp and As are some proteins with a mw ranging approximately from 35 to 50 kD and with a mw > 100 kD, and 2) tr is not involved in cross-reactivity between As and Dp.

scholarly journals Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA

2019 ◽  
Vol 25 (3) ◽  
pp. 310-319
Author(s):  
Yuan Dong ◽  
Hanjin Hou ◽  
An Chen ◽  
Wei Ma ◽  
Moli Yin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Enzyme Hydrolysis ◽  
Clinical Samples ◽  
Sds Page ◽  
Thrombotic Diseases ◽  
D Dimer

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

Distribution of meprin in kidneys from mice with high- and low-meprin activity

1987 ◽  
Vol 253 (4) ◽  
pp. C535-C540 ◽  
Author(s):  
S. S. Craig ◽  
J. F. Reckelhoff ◽  
J. S. Bond
Keyword(s):  
Electron Microscopy ◽  
Brush Border ◽  
Inbred Mouse ◽  
Light And Electron Microscopy ◽  
Mouse Strains ◽  
Immunoperoxidase Technique ◽  
Inbred Mouse Strains ◽  
Western Blots ◽  
Reactive Material ◽  
Low Activity

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.

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