scholarly journals LM6-M: a high avidity rat monoclonal antibody to pectic α-1,5-L-arabinan

2017 ◽  
Author(s):  
Valérie Cornuault ◽  
Fanny Buffetto ◽  
Susan E Marcus ◽  
Marie-Jeanne Crépeau ◽  
Fabienne Guillon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
High Sensitivity ◽  
In Vitro Assays ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Avidity ◽  
Plant Cell Walls ◽  
Rhamnogalacturonan I ◽  
Phosphate Buffered Saline

Abstract1,5-arabinan is an abundant structural feature of side chains of pectic rhamnogalacturonan-I which is a matrix constituent of plant cell walls. The study of arabinan in cells and tissues is driven by putative roles for this polysaccharide in the generation of cell wall and organ mechanical properties. The biological function(s) of arabinan is still uncertain and high quality molecular tools are required to detect its occurrence and monitor its dynamics. Here we report a new rat monoclonal antibody, LM6-M, similar in specificity to the published rat monoclonal antibody LM6 (Willats et al. (1998) Carbohydrate Research 308: 149-152). LM6-M is of the IgM immunoglobulin class and has a higher avidity for α-1-5-L-arabinan than LM6. LM6-M displays high sensitivity in its detection of arabinan in in-vitro assays such as ELISA and epitope detection chromatography and in in-situ analyses.AbbreviationsAraArabinoseBSABovine Serum AlbuminGalGalactoseGalAGalacturonic acidEDCEpitope detection chromatographyELISAEnzyme-Linked Immunosorbent AssaymAbMonoclonal antibodyPBSPhosphate-buffered salineRhaRhamnoseRG-IRhamnogalacturonan-I

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Immunomagnetic Separation and Coagglutination of Vibrio parahaemolyticus with Anti-Flagellar Protein Monoclonal Antibody

2008 ◽  
Vol 15 (10) ◽  
pp. 1541-1546 ◽  
Author(s):  
S. Datta ◽  
M. E. Janes ◽  
J. G. Simonson
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Cell Suspension ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Immunomagnetic Separation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Isolation ◽  
Flagellar Protein ◽  
Phosphate Buffered Saline

ABSTRACT Mice were immunized by injection of Vibrio parahaemolyticus ATCC 17802 polar flagellin in order to produce monoclonal antibodies (mAbs). mAbs were analyzed by anti-H enzyme-linked immunosorbent assay using V. parahaemolyticus polar flagellar cores. The mAb exhibiting the highest anti-H titer was coated onto Cowan I Staphylococcus aureus cells at a concentration of 75 μg/ml cell suspension and used for slide coagglutination. Of 41 isolates identified genetically as V. parahaemolyticus, 100% coagglutinated with the anti-H mAb within 30 s, and the mAb did not react with 30 isolates identified as Vibrio vulnificus. A strong coagglutination reaction with V. parahaemolyticus ATCC 17802 was still observed when the S. aureus cells were armed with as little as 15 μg of mAb/ml S. aureus cell suspension. At this concentration, the mAb cross-reacted with three other Vibrio species, suggesting that they share an identical H antigen or antigens. The anti-H mAb was then used to optimize an immunomagnetic separation protocol which exhibited from 35% to about 45% binding of 102 to 103 V. parahaemolyticus cells in phosphate-buffered saline. The mAb would be useful for the rapid and selective isolation, concentration, and detection of V. parahaemolyticus cells from environmental sources.

scholarly journals Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

2006 ◽  
Vol 13 (3) ◽  
pp. 420-422 ◽  
Author(s):  
S. E. Burastero ◽  
C. Paolucci ◽  
D. Breda ◽  
G. Monasterolo ◽  
R. E. Rossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Positive Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

Expression of the SMP antigen by oligodendrocytes in the developing avian central nervous system

Development ◽  
1989 ◽  
Vol 107 (4) ◽  
pp. 825-833
Author(s):  
P. Cameron-Curry ◽  
C. Dulac ◽  
N.M. Le Douarin
Keyword(s):  
Monoclonal Antibody ◽  
Schwann Cell ◽  
Basic Protein ◽  
Culture Conditions ◽  
Cell Marker ◽  
Cellular Expression ◽  
Early Expression ◽  
Different Parts

Expression of the avian antigen SMP (Schwann cell Myelin Protein, Mr 75-80000), first characterized in the PNS with a monoclonal antibody as an early and strictly specific Schwann cell marker, was further studied in the CNS. Comparing SMP immunoreactive areas in the different parts of the CNS with those expressing the Myelin Basic Protein (MBP), we showed a strict colocalisation of both phenotypes. In vitro, MBP+ oligodendrocytes express the surface antigen SMP as well. SMP cellular expression was followed in situ and in culture using nervous tissues from embryos at different stages. We were thus able to detect an early expression of this marker by oligodendroblasts before the first appearance of MBP immunoreactivity. We have also identified a subpopulation of SMP+/MBP- and SMP+/GC- cells, which persists under our culture conditions as precursors remaining in an immature state.

scholarly journals Immunoassay of muscle-specific creatine kinase with a monoclonal antibody and application to myogenesis and muscular dystrophy

1983 ◽  
Vol 213 (2) ◽  
pp. 417-425 ◽  
Author(s):  
G E Morris ◽  
L P Head
Keyword(s):  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Enzyme Activity ◽  
Total Protein ◽  
Plasma Concentrations ◽  
Thigh Muscle ◽  
Cell Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Activity Measurements

A competition e.l.i.s.a. (enzyme-linked immunosorbent assay) is described that enables direct measurement of the muscle-specific polypeptide of chick creatine kinase (M-CK) in extracts of differentiating muscle-cell cultures and in blood plasma samples, even in the presence of embryonic, or brain-type, creatine kinase. The characteristics of the assay can be considerably improved by the use of a monoclonal antibody, CK-ART, instead of rabbit antisera, and we offer an explanation for this in terms of heterogeneity of antibody affinities in polyclonal antisera. In addition to native enzyme, the assay will measure creatine kinase unfolded and inactivated by 8 M-urea treatment. During chick muscle differentiation in vitro, M-CK increased from 7.5% of the total creatine kinase at 24h to 76.0% at 143h, in good agreement with isoenzyme separation data. As a percentage of the total cell protein, M-CK increased by 156-340-fold over the same period and constituted 0.38-0.56% of the total protein in late cultures. E.l.i.s.a. measurements on 17-20-day embryonic thigh-muscle extracts, which contain almost exclusively M-CK, agree well with enzyme activity and radioimmunoassay. M-CK constituted 0.7-1.6% of the total protein in 17-19-day embryonic thigh muscle. Plasma M-CK concentrations in normal 2-8-week-old chickens were found to be in the range 0.5-0.9 micrograms/ml. Plasma concentrations of 32-56 micrograms/ml were found in 8-week-old dystrophic chickens by both e.l.i.s.a. and enzyme-activity measurements. The results suggest that inactive or unfolded forms of M-CK do not normally exist, in any significant amounts, in cell and tissue extracts or in freshly prepared samples of plasma.

scholarly journals Modulation of the Lung Inflammatory Response to Serotype 8 Pneumococcal Infection by a Human Immunoglobulin M Monoclonal Antibody to Serotype 8 Capsular Polysaccharide

2005 ◽  
Vol 73 (8) ◽  
pp. 4530-4538 ◽  
Author(s):  
Tamika Burns ◽  
Maria Abadi ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibody ◽  
Inflammatory Response ◽  
Capsular Polysaccharide ◽  
Human Monoclonal Antibody ◽  
Pneumococcal Infection ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Monocyte Chemoattractant Protein 1

ABSTRACT The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(κ)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >107 CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.

In vitro degradability of feed proteins in the rumen: use of non-rumen proteases

2005 ◽  
Vol 56 (8) ◽  
pp. 797 ◽  
Author(s):  
M. Aslam Mirza ◽  
E. L. Miller
Keyword(s):  
Correlation Coefficient ◽  
Soybean Meal ◽  
Streptomyces Griseus ◽  
Correlation Coefficients ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Bacterial Protease ◽  
Ruminal Protein Degradability

Various feed proteins were incubated independently with bacterial protease from Streptomyces griseus (SGP), papain (Corica papaya), and ficin (Ficus glabrata) in a simple laboratory assay to predict ruminal protein degradability. The estimates obtained from in vitro assays were compared with those obtained from an in situ analysis using synthetic fibre bags. The rate and extent of degradation in vitro using proteases from non-rumen sources differed among substrates used. A high correlation coefficient (r2 = 0.99) was observed between N-degradability from the in vitro method using SGP and in situ estimates when soybean meal was the substrate. Soybean meal nitrogen (N) was almost completely hydrolysed (0.99) in vitro. The correlation coefficients were low and variable with assays using other enzymes. The correlation coefficient was also high (r2 = 0.77–0.84) with in vitro methods using either SGP, papain, or ficin when incubated with fish meal. The N disappearance from barley in vitro was slow to moderate. The ‘b’ estimate of barley obtained with the in vitro assay was significantly (P < 0.01) lower than that observed in situ. Slower proteolysis observed in barley may possibly be linked to poor accessibility of structural proteins rather than the degradability of N per se. None of the enzymes could rank barley in the same order as the in situ method.

Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model

1996 ◽  
Vol 80 (6) ◽  
pp. 1984-1992 ◽  
Author(s):  
J. M. Graf ◽  
C. W. Smith ◽  
M. M. Mariscalco
Keyword(s):  
Monoclonal Antibody ◽  
Polymorphonuclear Leukocytes ◽  
Rabbit Model ◽  
Transendothelial Migration ◽  
Peritoneal Exudate ◽  
Adult Rabbit ◽  
Primary Mechanism ◽  
Phosphate Buffered Saline

Human neonatal polymorphonuclear leukocytes (PMNs) exhibit decreased mobility, adherence, and transendothelial migration in vitro compared with adult PMNs. These deficits, in part, are due to functional and quantitative defects in neonatal Mac-1 (CD11b/CD18), whereas LFA-1 (CD11a/CD18) function is similar to that found in adults (D.C.Anderson, O.Abbassi, T.K.Kishimoto, J.M.Koenig, L.V.McIntire, and C.W.Smith, J.Immunol. 146: 3372-3379, 1991; C. W. Smith, T. K. Kishimoto, O. Abbassi, B.J.Hughes, R.Rothlein, L.V.McIntire, E.Butcher, and D.C. Anderson, J. Clin. Invest. 87: 609-618, 1991). We tested the hypothesis that the primary mechanism for the neonatal PMN CD18-dependent emigration in vivo is due to LFA-1. Neutrophils from 1-day-old rabbit pups had 32 and 60% of adult rabbit levels of CD11a and CD11b, respectively. Rabbit pups or adult rabbits received the monoclonal antibody (MAb) R7.1 (anti-CD11a) or R15.7 (anti-CD18) or the vehicle phosphate-buffered saline (PBS) before the instillation of intraperitoneal thioglycollate. Six hours later peritoneal exudate was collected. The administration of MAbs R7.1 and R15.7 in adult animals resulted in 60 and 83% inhibition of leukocyte emigration, respectively, compared with PBS-treated animals (P < 0.01). In neonatal animals, R7.1 and R15.7 inhibited leukocyte peritoneal accumulation to the same extent (50 and 60%, respectively) compared with PBS-treated animals (P < 0.01). Adult animals were also treated with the anti-CD11b MAb 198. MAb 198 decreased emigration by 25%, although this was not significant compared with PBS-treated animals. We conclude that although neonatal animals have significantly less neutrophil CD11a, the diminished levels did not contribute to a reduced ability to emigrate to the peritoneum and, like adult animals, neonatal animals primarily utilize LFA-1 for accumulation in this model. The contribution of Mac-1 to neonatal leukocyte emigration remains uncertain.

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