Characterization of a Panel of Monoclonal Antibodies to Human Coagulation Factor XI and Detection of Factor XI in Hep G2 Cell Conditioned Medium

1990 ◽  
Vol 63 (03) ◽  
pp. 417-423 ◽  
Author(s):  
Y Ohkubo ◽  
D P O’Brien ◽  
T Kanehiro ◽  
H Fukui ◽  
E G D Tuddenham
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Coagulation Factor ◽  
High Yield ◽  
Functional Assay ◽  
Activity Levels ◽  
Hep G2 ◽  
Factor Xi

SummaryWe have produced a panel of ten monoclonal antibodies specific to coagulation factor XI. Western blot analysis demonstrates that 9 of these antibodies react with the heavy chain of factor XI and one with the light chain. Seven of these antibodies inhibit factor XI and factor XIa activity.We have used immobilised monoclonal antibody for the production of factor XI deficient plasma and to purify factor XI to homogeneity with high yield in a simple two-step procedure. These monoclonal antibodies were used to develop highly sensitive immunoassays capable of detecting less than 0.01 u factor XI antigen ml−1. A strong correlation was found between antigen and activity levels in 11 patients with hereditary deficiency indicating that none was cross-reacting material positive. Cultured Hep G2 cells were found to synthesize small amounts of factor XI antigen and this could also be detected by functional assay and by western blot analysis.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

scholarly journals A comparison of antigenic peptides in muscle larvae of severalTrichinellaspecies by two-dimensional western-blot analysis with monoclonal antibodies

Parasite ◽  
2001 ◽  
Vol 8 ◽  
pp. S117-S119 ◽  
Author(s):  
M.A. Dea-Ayuela ◽  
F.M. Ubeira ◽  
A. Pitarch ◽  
C. Gil ◽  
A.R. Martínez-Fernández ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Muscle Larvae

scholarly journals Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2439
Author(s):  
Song Hee Lee ◽  
Tae-Kyun Oh ◽  
Sung Oh ◽  
Seongdae Kim ◽  
Han Byul Noh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Rapid Detection ◽  
Western Blot Analysis ◽  
Apis Cerana ◽  
Korean Isolate ◽  
Sacbrood Virus ◽  
Positive Reactivity

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

Abstract P071: Transglutaminases Are Active In Perivascular Adipose Tissue

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Alexis Orr ◽  
Janice Thompson ◽  
Janae Lyttle ◽  
Stephanie W Watts
Keyword(s):  
Adipose Tissue ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Coagulation Factor ◽  
Sprague Dawley ◽  
Perivascular Adipose Tissue ◽  
Tissue Sections ◽  
Mesenteric Resistance Artery ◽  
Immunohistochemical Analyses

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model in which to test interactions of amines and TGs. We hypothesized that TG2 and FXIII are active in PVAT. Sprague-Dawley rat aortic, superior mesenteric (SMA), and mesenteric resistance artery (MR) PVAT express TG2 and blood coagulation factor XIII (FXIII) mRNA (Figure 1A). Consistent with this, immunohistochemical analyses support that PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs and a catalyzing calcium solution, visualized with TRITC fluorescence (Figure 1B,C). Both TG2 and FXIII are active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT by 6.14% of total normalized signal (p<0.0001, N=7). Further Western blot analysis proved that experimentally added 5-HT competitively inhibits incorporation of experimentally added BAP into MRPVAT adipocytes, reducing total normalized signal by 10.75% (p=0.001, N=4). Further studies to determine what proteins TGs are amidating will give insight into how these enzymes contribute to the development of hypertension.

scholarly journals Monoclonal Antibodies Detecting Regulatory Polypepttdes of the Insect Neuronal Acetylcholine Receptor

1989 ◽  
Vol 147 (1) ◽  
pp. 329-342
Author(s):  
D. BENKE ◽  
I. STAHMER ◽  
H. BREER
Keyword(s):  
Monoclonal Antibodies ◽  
Ligand Binding ◽  
Western Blot ◽  
Acetylcholine Receptor ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Receptor Protein ◽  
Neuronal Membranes ◽  
Neuronal Acetylcholine Receptor

Please address reprint requests to Professor H. Breer, Universität Stuttgart, Hohenheim Farbenstrasse 30, 7000 Stuttgart 70 1. Monoclonal antibodies affecting the ligand binding of the neuronal acetylcholine receptor were prepared. 2. A clone was detected which produced antibodies that increased [125I]-αbungarotoxin binding, but decreased [3H]acetylcholine binding. 3. Western blot analysis established that these antibodies did not recognize the receptor protein, but labelled a 20xl03Mr polypeptide. 4. This putative regulatory polypeptide was purified and was found to inhibit [125I]-α-bungarotoxin binding to pretreated neuronal membranes.

Development and regulation of (1→3,1→4)-β-glucan endohydrolases in germinating wheat (Triticum aestivum)

1993 ◽  
Vol 3 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Doreen M. L. Lai ◽  
Amanda M. Slade ◽  
Geoffrey B. Fincher
Keyword(s):  
Monoclonal Antibody ◽  
Triticum Aestivum ◽  
Monoclonal Antibodies ◽  
Gibberellic Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Apparent Effect ◽  
Glucanase Activity

AbstractThe development of (1→3,1→4)-β-glucan 4-glucanohydrolase (EC 3.2.1.73) has been examined in germinating wheat (Triticum aestivum cv. Millewa) grain, and in isolated aleurone layers and scutella. Activity is first detected in extracts of intact grain 2–3 days after the initiation of germination and thereafter increases until 6 days. In isolated aleurone layers, (1→3,1→4)-β-glucanase activity is secreted for up to 4 days. Treatment of aleurone layers with gibberellic acid (GA3) results in a 2-fold enhancement of secreted enzyme for the first 2 days, but activity decreases after 2 days. (1→3,1→4)-β-Glucanase secretion from GA3-treated aleurone layers clearly precedes the secretion of amylase and (1→4)-β-xylanase. Small but significant levels of cellulase are secreted from aleurone layers after GA3 treatment. Isolated scutella also secrete (1→3,1→4)-β-glucanase but GA3 has little apparent effect on secretion patterns from this tissue. Proteins secreted from excised aleurone layers and scutella, together with those in homogenates of intact, germinated grain, have been examined by Western blot analysis, using monoclonal antibodies against barley (1→3,1→4)-β-glucanases as probes. In each case the wheat (1→3,1→4)-β-glucanase is recognised only by the monoclonal antibody that is specific for barley isoenzyme El.

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