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Y Frobert

Publications and source records attributed to Y Frobert.

At least 37 records · Page 2Linked to original sources
PubMed1999

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.

Amino Acid Sequence↗
PubMed1998

Two-site enzyme immunometric assays for determination of native and denatured beta-lactoglobulin.

Two enzyme immunometric assays suitable for measuring native and denatured beta-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cow's milk and cow's milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cow's milk in goat's milk.

Animals↗
PubMed1998

Substance P receptor immunodetection in the spinal cord: comparative use of direct anti-receptor antibody and anti-complementary peptide antibody.

The immunolocalization of substance P (SP) receptors was compared in the rat spinal cord using either a direct anti-substance P NK1-receptor antibody (anti-SPR) or an anti-complementary peptide antibody (anti-CP). The first antibody recognizes an intracellular epitope, the C-terminal tail of the NK1-receptor. The second antibody recognizes an extracellular epitope located at or near the ligand-binding domain because anti-CP antibody and SP were previously shown to compete for binding to the receptor. At the light microscope level, it was observed that anti-CP antibody labels both laminae I and II of the dorsal horn, while anti-SPR antibody labels exclusively lamina I, except at the lumbar level. This could suggest that spinal NK1 receptors are heterogeneous. Anti-SPR antibodies may recognize an NK1 receptor subclass confined to lamina I. Conversely, anti-CP antibody may recognize either another receptor subclass or two different subclasses present in laminae I and II. At the electron microscope level, labeling was localized either on the intracellular or the extracellular face of the plasma membrane depending on the location of the epitope recognized by both antibodies on the transmembrane receptor. However, using either antibody, the ultrastructural labeling was found at non-junctional sites, suggesting that SP may act in a non-synaptic manner on all putative receptor subclasses.

Animals↗
PubMed1997

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Acetylcholinesterase↗
PubMed1997

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P.

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclonal antibodies, we selected the mAb12 antibody which specifically crossreacted, through its paratope, with a binding site present on membranes from rat parotid gland cells, with an affinity close to 2 x 10(-7) M and with membranes from CHO cells expressing human brain NK1 receptors. Immunocytochemical investigations using mAb12 revealed immunostaining whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and immunocytochemical experiments, the competition occurring between the antibody and substance P, or a substance P-protein conjugate, indicates that mAb12 recognizes a membrane epitope located at or near the substance P binding domain on the NK1 receptor. Immunization of mice with mAb12 led to the production of specific anti-substance P antibodies, again suggesting that mAb12 shares common structural features with the neuropeptide. This monoclonal antibody can now be used in further biochemical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for designing peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulation, inflammation or chronic pain.

Animals↗
PubMed1997

Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples.

A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 micromol/L, a CV <10% in the 1.25-10 micromol/L range, and intra- and interassay CVs of <10%. Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for other structurally related catecholamine metabolites. Parallelism of the EIA was shown in dilution studies and the correlation with routine HPLC assay in 62 normal and pathologic samples was EIA = 1.492 (HPLC) - 3.46, S(y/x), = 47.52, range = 4-1800 micromol/L, r2 = 0.977. Additional data concerning the validity of this assay were provided by HPLC analysis of urinary immunoreactive material.

Acetylcholinesterase↗
PubMed1996

Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.

Several proinflammatory cytokines can increase prostaglandin E2 (PGE2) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE2 synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE2 and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.

Blotting, Northern↗
PubMed1996

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Allergens↗
PubMed1996

Thymosin beta4, inhibitor for normal hematopoietic progenitor cells.

Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.

Amino Acid Sequence↗
PubMed1996

Two different approaches for developing immunometric assays of haptens.

To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

Amino Acid Sequence↗
PubMed1995

Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

Acetylcholinesterase↗
PubMed1995

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay.

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

Amino Acid Sequence↗
PubMed1995

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.

Amino Acid Sequence↗
PubMed1995

Humoral and cellular immune responses to synthetic peptides from the Plasmodium falciparum blood-stage antigen, Pf155/RESA, in Cameroonian women.

T- and B-cell responses to the Plasmodium falciparum blood-stage antigen Pf155/RESA were investigated in 104 Cameroonian women, half of whom were pregnant. We used purified protein and six synthetic peptides representing T- and B-cell epitopes. In vitro T-cell responses were measured by proliferation and IL2, IFN-gamma, and IL4 release. B-cell responses were assessed by plasma antibodies. All peptides induced a cellular response in some individuals. A proliferative response was induced in 25% of the donors by Pf155/RESA, and in 7 to 11% by any peptide. Cytokine release occurred in 23 to 30% of the Pf155/RESA-stimulated cultures, and in 8 to 25% of the peptide-stimulated cultures. Overall, each peptide induced a cellular response (proliferation and/or cytokine release) in 44% of the donors. T-cells from 23% of the donors failed to respond to any peptide. Responding cells did not usually respond in all readouts, and proliferation and release of any of the three cytokines were not correlated. Similarly, antibody and T-cell responses were not related. Selected epitopes of Pf155/RESA, an important vaccine candidate, are well recognized in naturally exposed individuals and are able to activate T-cells to proliferate and to produce various lymphokines in numerous individuals from a malaria endemic area.

Adult↗
PubMed1995

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody.

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10(-6)M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non-junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.

Amino Acid Sequence↗
PubMed1995

Malaria and pregnancy in Cameroonian primigravidae: humoral and cellular immune responses to Plasmodium falciparum blood-stage antigens.

To investigate the mechanisms underlying the increased susceptibility to malaria in pregnant women, we determined the level of malaria-specific immunity in primigravidae. Humoral and cellular in vitro responses to unpurified (a crude schizont extract and a gametocyte preparation) and purified (affinity-purified Pf155/ring-infected erythrocyte surface antigen [RESA]) Plasmodium falciparum proteins, an immunodominant 45/47-kilodalton antigen from Mycobacterium bovis, and leucoagglutinin were compared between 52 primigravidae and 52 nonpregnant women from a semirural area of Cameroon. In vitro cellular responses were investigated in terms of lymphocyte proliferation, as well as production of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and IL-4. Cells from primigravidae exhibited a reduced proliferative response to schizont and gametocyte antigens, as well as to the M. bovis antigen. Conversely, the IL-2 response to Pf155/RESA was reduced. Interleukin-4 and IFN-gamma production did not appear to be affected in primigravidae. Antibody levels were also similar between pregnant and nonpregnant women. Our results underline the importance of examining several parameters of T cell activation with different types of antigens for a correct evaluation of the ability of lymphocytes to respond to malaria.

Adolescent↗