Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms

2011 ◽  
Vol 106 (07) ◽  
pp. 90-101 ◽  
Author(s):  
Niraj Mishra ◽  
Ellen Vercauteren ◽  
Jan Develter ◽  
Riet Bammens ◽  
Paul J. Declerck ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Lysis Time ◽  
Human Thrombin ◽  
Fibrinolysis Inhibitor ◽  
Dose Dependent ◽  
Clot Lysis Time

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFITI- wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eightfold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MATCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MATCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.

scholarly journals Characterization of Monoclonal Antibodies against σA Protein and Cross-Reactive Epitope Identification and Application for Detection of Duck and Chicken Reovirus Infections

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 140
Author(s):  
Xueming Chen ◽  
Tongtong Li ◽  
Xiaodan Chen ◽  
Chenxi Li ◽  
Weiwei Lin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Core Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Samples ◽  
Independent Manner ◽  
Competitive Binding Assay ◽  
Diagnostic Potential ◽  
Peptide Phage Display

Although σA is an important major core protein of duck reovirus (DRV), the B-cell epitopes of this protein remain unknown to reseacrhers. Six monoclonal antibodies (MAbs) (1A7, 3F4, 5D2, 4E2, 3C7, and 2B7) were developed by using prokaryotic-expressed recombinant His-σA protein. Five of six MAbs (1A7, 3F4, 4E2, 3C7, and 2B7) reacted with His-σA protein in a conformation-independent manner, while 5D2 reacted with σA in a conformation-dependent manner. Immunofluorescence assays showed that the MAbs could specifically bind to DRV infected BHK-21 cells. The MAbs were delineated as three groups by a competitive binding assay. By using 12-mer peptide phage display and mutagenesis, MAb 4E2 was identified to recognize minimal epitope 56EAPYPG61 and MAb 1A7 recognize 341WVV/MAGLI/V347, residues 341V/M and 347I/V are replaceable. Dot blotting and sequence analysis confirmed that EAPYPG and WVV/MAGLI/V are cross-reactive epitopes in both DRV and avian reovirus (ARV). An enzyme-linked immunosorbent assay (ELISA) based on two expressed EAPYPG and WVVAGLI as antigen demonstrated its diagnostic potential by specific reacting with serum samples from DRV- or ARV-infected birds. Based on these observations, an epitope-based ELISA could be potentially used for DRV or ARV surveillance. These findings provide insights into the organization of epitopes on σA protein that might be valuable for the development of epitope-based serological diagnostic tests for DRV and ARV infection.

scholarly journals Characterization of RNA damage under oxidative stress in Escherichia coli

2012 ◽  
Vol 393 (3) ◽  
pp. 123-132 ◽  
Author(s):  
Min Liu ◽  
Xin Gong ◽  
Ravi Kumar Alluri ◽  
Jinhua Wu ◽  
Tene’ Sablo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Short Exposure ◽  
Dependent Manner ◽  
Ribosomal Rnas ◽  
Rna Oxidation ◽  
Folded Rna ◽  
Dose Dependent

Abstract We have examined the level of 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine, in RNA in Escherichia coli under normal and oxidative stress conditions. The level of 8-oxo-G in RNA rises rapidly and remains high for hours in response to hydrogen peroxide (H2O2) challenge in a dose-dependent manner. H2O2 induced elevation of 8-oxo-G content is much higher in RNA than that of 8-hydroxydeoxyguanosine (8-oxo-dG) in DNA. Under normal conditions, the 8-oxo-G level is low in RNA isolated from the ribosome and it is nearly three times higher in non-ribosomal RNAs. In contrast, 8-oxo-G generated by a short exposure to H2O2 is almost equally distributed in various RNA species, suggesting that although ribosomal RNAs are normally less oxidized, they are not protected against exogenous H2O2. Interestingly, highly folded RNA is not protected from oxidation because 8-oxo-G generated by H2O2 treatment in vitro increases to approximately the same levels in tRNA and rRNA in both native and denatured forms. Lastly, increased RNA oxidation is closely associated with cell death by oxidative stress. Our data suggests that RNA is a primary target for reactive oxygen species and RNA oxidation is part of the paradox that cells have to deal with under oxidative stress.

scholarly journals Inhibitory action of polyamines on protein kinase C association to membranes

1987 ◽  
Vol 247 (1) ◽  
pp. 175-180 ◽  
Author(s):  
M Moruzzi ◽  
B Barbiroli ◽  
M G Monti ◽  
B Tadolini ◽  
G Hakim ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Process ◽  
Dependent Manner ◽  
Kinase C ◽  
Association Reaction ◽  
Dose Dependent ◽  
Inside Out

Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

scholarly journals Production and characterization of a monoclonal antibody to biotin

1986 ◽  
Vol 237 (2) ◽  
pp. 477-482 ◽  
Author(s):  
K Dakshinamurti ◽  
R P Bhullar ◽  
A Scoot ◽  
E S Rector ◽  
G Delespesse ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Biotin Deficiency ◽  
Keyhole Limpet Haemocyanin ◽  
Limiting Dilution

Monoclonal antibodies to biotin have been prepared by using biotin linked to keyhole limpet haemocyanin (KLH) as the antigen. Spleen cells obtained from mice immunized with biotin-KLH were fused with the myeloma cell line NS-1. The resulting hybridomas were screened for the production of antibodies to biotin using an enzyme-linked immunosorbent assay. Clones producing antibodies to biotin were isolated by limiting dilution methods. Four cell lines, each derived originally from a different fusion, were chosen for the production of monoclonal antibodies. The monoclonal antibodies obtained have been characterized with respect to their ability to interact with biotin, biotin-bovine serum albumin, biotin-KLH and biocytin as well as to inhibit biotin-dependent enzymes. They have been used to produce cellular biotin deficiency in vitro for studies of biotin function.

Fibronectin production by chicken granulosa cells in vitro: effect of follicular development

1992 ◽  
Vol 127 (5) ◽  
pp. 466-470 ◽  
Author(s):  
Elikplimi K Asem ◽  
Jacqueline A Carnegie ◽  
Benjamin K Tsang
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Ovarian Granulosa Cells ◽  
Preovulatory Follicles ◽  
Vitro Effect ◽  
Dose Dependent

In vitro studies were conducted to investigate the role of chicken ovarian granulosa cells in the production of fibronectin, a component of the basal lamina of ovarian follicles. Collagenase dispersed granulosa cells obtained from the first (F1; about 35 mm in diameter) and third (F3; 15–20 mm in diameter) largest preovulatory follicles, as well as from a pool of small yellow follicles (SF; 6–10 mm in diameter), were incubated in serum-free medium-199 for 24 to 96 h in the absence and presence of luteinizing hormone (LH) or forskolin. Fibronectin secreted in the medium was quantitated by enzyme linked immunosorbent assay. Basal fibronectin production (which increased with the duration of incubation) was significantly greater (p<0.001) in granulosa cells derived from mature follicle (F1) than in F3 or SF cells. Both LH and forskolin stimulated fibronectin production in SF and F3 cells in a dose-dependent manner; however, they were without effect in F1 cells. The magnitude of increase in fibronectin production elicited by LH or forskolin was greater in SF cells than in F3 cells. The cytoplasm of cultured granulosa cells taken at all stages of follicular development stained positively for fibronectin. These findings indicate that chicken granulosa cells produce fibronectin. This ability is acquired early in follicular development and the stimulatory effect of the gonadotropin (LH) diminished as the follicle approached ovulation.

scholarly journals Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung

1994 ◽  
Vol 304 (1) ◽  
pp. 131-137 ◽  
Author(s):  
R Wang ◽  
C R Dodia ◽  
M K Jain ◽  
A B Fisher
Keyword(s):  
Phospholipase A2 ◽  
Dependent Manner ◽  
Rat Lung ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
New Class ◽  
Terminal Amino ◽  
Dose Dependent

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.

Somatostatin blocks the potentiation of TRH-induced TSH secretion from perifused pituitary fragments and the change in intracellular calcium concentrations from dispersed pituitary cells elicited by prepro-TRH (PS4) or by tri-iodothyronine

1997 ◽  
Vol 19 (1) ◽  
pp. 87-97 ◽  
Author(s):  
J-P Roussel ◽  
E Grazzini ◽  
H Astier
Keyword(s):  
Primary Cultures ◽  
Inhibitory Effect ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Physiological Concentration ◽  
Rat Pituitary ◽  
Tsh Secretion ◽  
Intracellular Ca ◽  
Dose Dependent

ABSTRACT TRH and somatostatin (SRIH) are well known to stimulate and to inhibit TSH secretion respectively. However, the mechanisms underlying the effect of SRIH on thyrotrophs are still not understood. We have previously shown in vitro that the TSH response to TRH is potentiated in a Ca2+-dependent fashion through the activation of dihydropyridine (DHP)-sensitive Ca2+ channels by the prepro-TRH (160-169) cryptic peptide (PS4) and tri-iodo-l-thyronine (T3), when the hormone was added shortly before a TRH pulse in order to avoid its genomic effect. Using perifused rat pituitary fragments, the present study has shown that SRIH inhibits, in a dose-dependent manner, the TSH response to physiological concentration of TRH (10 nm) and reverses the Ca2+-dependent potentiation of that response induced either by PS4 or by T3. We have also demonstrated that the inhibition by SRIH of the T3 potentiation of TRH-induced TSH secretion is pertussis toxinsensitive. Our data suggest that SRIH inhibits the PS4 and T3 potentiation of TRH-induced TSH secretion through the inactivation of DHP-sensitive Ca2+ channels. Using primary cultures of rat anterior pituitary cells and videomicroscopy, we have already demonstrated that TRH, as well as PS4 and T3, are able to increase intracellular Ca2+ concentration ([Ca2+]i) rapidly, in 15 s. Our study has shown that SRIH is able to abolish the acute rise in [Ca2+]i induced either by PS4 or by T3. Since [Ca2+]i responses to PS4 and T3 are also abolished by the DHP nifedipine, our results suggest that [Ca2+]i changes in PS4- or T3-sensitive pituitary cells depend directly or indirectly on the activation of DHP-sensitive Ca2+ channels and that the inhibitory effect of SRIH may be mediated by inactivation of this type of channel.

Corticotrophin-Releasing Factor Augments the IH in Rat Hypothalamic Paraventricular Nucleus Parvocellular Neurons In Vitro

2005 ◽  
Vol 94 (1) ◽  
pp. 226-234 ◽  
Author(s):  
De-Lai Qiu ◽  
Chun-Ping Chu ◽  
Tetsuro Shirasaka ◽  
Hiromasa Tsukino ◽  
Hiroyuki Nakao ◽  
...  
Keyword(s):  
Single Cell ◽  
Paraventricular Nucleus ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Cation Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Parvocellular Neurons ◽  
Dose Dependent

The goal of this study was to characterize the effects of corticotrophin-releasing factor (CRF) on rat paraventricular nucleus (PVN) putative parvocellular neurons using whole cell patch-clamp recordings and single-cell reverse transcription-multiplex polymerase chain reaction (single-cell RT-mPCR) techniques. Under current clamp, CRF (10–600 nM) increased the neuronal basal firing rate and depolarized neurons in a dose-dependent manner. CRF-induced depolarization was unaffected by co-perfusion with TTX, 6-cyano-7-nitroquinoxaline-2 3-dione (CNQX), and bicuculline but was completely inhibited by ZD7288. Under voltage clamp, 300 nM CRF significantly increased the hyperpolarization-activated cation current ( IH) in a voltage-dependent manner, shifted the IH conductance-voltage relationship ( V1/2) toward depolarization by ∼7.8 mV, and enhanced the IH kinetics without changing the slope constant (k). Extracellular application of ZD7288 completely blocked IH and the CRF-induced increase in IH. Furthermore, CRF-induced effects were completely blocked by extracellular application of 1 μM α-helical CRF-(9–14) (α-hCRF), a nonselective CRF receptor antagonist, but were not affected by extracellular application of antisauvagine-30, a selective CRF-receptor 2 antagonist. Single-cell RT-mPCR analysis showed that these neurons co-expressed CRF receptor 1 mRNA and CRF receptor 2 mRNA. Furthermore, CRF-sensitive neurons co-expressed HCN1 channel mRNA, HCN2 channel mRNA, and HCN3 channel mRNA, but not HCN4 channel mRNA. These results suggest that CRF modulates the subpopulation of PVN parvocellular neuronal function by CRF-receptor 1–mediated potentiation of HCN ion channel activity.

Characterization of anti-Citrobacter 036 specific polysaccharide monoclonal antibodies

1984 ◽  
Vol 30 (1) ◽  
pp. 91-97 ◽  
Author(s):  
P. J. Shearman ◽  
D. R. Bundle ◽  
E. Romanowska ◽  
C. Lugowski ◽  
M. Bogulska
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Passive Hemagglutination ◽  
Specific Polysaccharide ◽  
Whole Cell Vaccine ◽  
Antibody Class ◽  
Culture Supernatants ◽  
Dose Dependent

Monoclonal mouse antibodies specific for the 0 antigen of Citrobacter 036, a homopolymer of β(1 → 2)-linked 4-deoxy-D-arabinohexose, were generated by the hybridoma technique. Balb/c mice were immunized with killed whole-cell vaccine and initial selection of active clones was based on enzyme-linked immunosorbent assay (ELISA) employing purified lipopolysaccharide (LPS). Concentrated culture supernatants from selected hybrid cultures were used to identify 10 0-antigen specific monoclonal antibodies using the multiple criteria of immunoprecipitation of 0 chains and LPS, inhibition by acid hydrolyzed 0 chains in the screening ELISA, and antibody class analysis. Four monoclonal antibodies were chosen for further study using dose-dependent 0-chain inhibition of ELISA and passive hemagglutination, passive hemolysis, and bacterial agglutination titres. When screened with Citrobacter serotypes known to contain the sugar 4-deoxy-D-arabinose, passive hemagglutination tests showed that the two monoclonal antibodies examined possessed titres which could be correlated with the reported 4-deoxy-D-arabinohexose content of the respective LPS's. This sugar is an antigenically important unit of several Citrobacter serotypes as defined by these well-characterized monoclonal antibodies.

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