Bmax Values Research Articles

Surface plasmon resonance immunosensor for the detection of Salmonella typhi antibodies in buffer and patient serum

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip was developed first time for the detection of flagellin specific antibodies of Salmonella typhi (S. typhi). Flagellin protein of S. typhi was prepared by recombinant DNA technology. The modification of gold chip with 4-MBA was in-situ characterized by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, KD and Bmax values were calculated and found to be 26.3fM and 62.04m°, respectively, for the immobilized monoclonal antibody (Moab) of recombinant flagellin (r-fla) protein of S. typhi (r-fla S. typhi). In addition, thermodynamic parameters such as ΔG, ΔH and ΔS were determined first time for r-fla S. typhi and Moab of r-fla S. typhi interactions and the values revealed the interaction between r-fla S. typhi and Moab of r-fla S. typhi as spontaneous, endothermic and entropy driven one. Moreover, healthy human serum samples and patient sera (Widal positive and Widal negative) were subjected to SPR analysis. The present SPR based approach provides an alternative way for S. typhi detection in less than 10min.

Measurement of 5-HT1A Receptor Density and in-vivo Binding Parameters of [18F]mefway in the Nonhuman Primate

The goal of this work was to characterize the in-vivo behavior of [(18)F]mefway as a suitable positron emission tomography (PET) radiotracer for the assay of 5-hydroxytryptamine(1A) (5-HT(1A)) receptor density (B(max)). Six rhesus monkeys were studied using a multiple-injection (M-I) protocol consisting of three sequential bolus injections of [(18)F]mefway. Injection times and amounts of unlabeled mefway were optimized for the precise measurement of B(max) and specific binding parameters k(off) and k(on) for estimation of apparent K(D). The PET time series were acquired for 180 minutes with arterial sampling performed throughout. Compartmental analysis using the arterial input function was performed to obtain estimates for K(1), k(2), k(off), B(max), and K(Dapp) in the cerebral cortex and raphe nuclei (RN) using a model that accounted for nontracer doses of mefway. Averaged over subjects, highest binding was seen in the mesial temporal and dorsal anterior cingulate cortices with B(max) values of 42±8 and 36±8 pmol/mL, respectively, and lower values in the superior temporal cortex, RN, and parietal cortex of 24±4, 19±4, and 13±2 pmol/mL, respectively. The K(Dapp) of mefway for the 5-HT(1A) receptor sites was 4.3±1.3 nmol/L. In conclusion, these results show that M-I [(18)F]mefway PET experiments can be used for the in-vivo measurement of 5-HT(1A) receptor density.

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na+, K+-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na+, K+-ATPase, peptide effect on high affinity [3H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1×10−4M concentration. On the other hand, the single addition of 1×10−6M, 1×10−5M and 1×10−4M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [3H]-ouabain binding (in %) to 87±16; 74±16 and 34±17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [3H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased Kd value whereas failed to modify Bmax value, indicating a competitive type interaction of the peptide at Na+, K+-ATPase ouabain site. At variance, SR 48692 decreased Bmax value whereas it did not modify Kd value. [3H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30min earlier with 100μg and 250μg/kg SR 48692. It was observed that the 250μg/kg SR 48692 dose led to 19% decrease in basal [3H]-ouabain binding. After SR 48692 treatments, addition of 1×10−6M led to additive or synergic effect. Results suggested that [3H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

Structure of the community of planktic algae of the white sea in relation to availability of oxidized or reduced forms of nitrogen and level of illumination

Phytoplankton sampled in Kandalaksha Bay of the White Sea in late June 2006 was exposed in situ for 14 days under two levels of illumination (E1 > E2) with the addition of 180 μM of nitrogen as nitrate, urea, ammonium, and glycine. Every 3 days, species composition, abundance, and biomass of algae were estimated. The maximum biomass (Bmax) of phytoplankton depended on nitrogen substrates and level of illumination. The differences in Bmax between phytoplankton assimilating different nitrogen substrates became more apparent under E2. Phytoplankton assimilating nitrates, urea, and ammonium had higher Bmax under E1, but algae grown with glycine had higher Bmax under E2. Although the values of Bmax differed, the structures of all assemblages were similar except for that grown on ammonium under E1. Competitive parameters of algae populations depended on the form of nitrogen and level of illumination in a species-specific manner. Comparison of the present data with results of similar experiments in 2005 revealed that structure of assemblages formed after nitrogen pulse additions depended not only on nitrogen forms and illumination level but also on initial (before pulse) structure of community and availability of nitrogen for algae.

Synthesis of homoveratric acid-imprinted polymers and their evaluation as selective separation materials.

A bulk polymerization method was used to easily and efficiently prepare homo-veratric acid (3,4-dimethoxyphenylacetic acid)-imprinted polymers from eight basic monomers: 2-vinylpyridine, 4-vinylpyridine, 1-vinylimidazole, N-allylaniline, N-allyl-piperazine, allylurea, allylthiourea, and allylamine, in the presence of homoveratric acid as a template in N,N-dimethylformamide as a porogen. The imprinted polymer prepared from allylamine had the highest affinity to the template, showing an imprinting factor of 3.43, and allylamine polymers MIP8/NIP8 were selected for further studies. Their binding properties were analyzed using the Scatchard method. The results showed that the imprinted polymers have two classes of heterogeneous binding sites characterized by two pairs of Kd, Bmax values: Kd(1) = 0.060 μmol/mL, Bmax(1) = 0.093 μmol/mg for the higher affinity binding sites, and Kd(2) = 0.455 μmol/mL, Bmax(2) = 0.248 μmol/mg for the lower affinity binding sites. Non-imprinted polymer has only one class of binding site, with Kd = 0.417 μmol/mL and Bmax = 0.184 μmol/mg. A computational analysis of the energies of the prepolymerization complexes was in agreement with the experimental results. It showed that the selective binding interactions arose from cooperative three point interactions between the carboxylic acid and the two methoxy groups in the template and amino groups in the polymer cavities. Those results were confirmed by the recognition studies performed with the set of structurally related compounds. Allylamine polymer MIP8 had no affinity towards biogenic amines. The obtained imprinted polymer could be used for selective separation of homoveratric acid.

Muscarinic Receptor Binding of Imidafenacin in the Human Bladder Mucosa and Detrusor and Parotid Gland.

The current study aimed to characterize comparatively the binding of imidafenacin to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland. The muscarinic receptor in homogenates of human tissues (bladder mucosa and detrusor muscle and parotid gland) was measured using a radioligand binding assay with [N-methyl-(3) H]scopolamine methyl chloride ([(3) H]NMS). Imidafenacin competed with [(3) H]NMS for binding sites in the bladder mucosa and detrusor muscle and parotid gland, and its affinity was significantly (2.6-8.7 times) higher than that of oxybutynin. Also, the affinity of imidafenacin for muscarinic receptors was approximately two-fold higher in the parotid gland than bladder tissue. The affinity of imidafenacin in the mucosa was similar to that in the detrusor muscle, suggesting that this agent exhibits therapeutic effects by blocking muscarinic receptors in the mucosa as well as detrusor muscle. Scatchard analysis revealed that imidafenacin increased significantly (approximately four-fold) Kd values for [(3) H]NMS binding in the human detrusor muscle and parotid gland, with little effect on Bmax values. This observation indicates that imidafenacin binds to the muscarinic receptors in human tissues in a competitive and reversible manner. Imidafenacin binds to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland with high affinity. This agent was considered to exhibit therapeutic effects on the lower urinary tract symptoms due to an overactive bladder by blocking muscarinic receptors in the urothelium as well as detrusor muscle.

Distribution of Protein Kinase C in the Hippocampus of the Gerbil and Rat: Autoradiographic Analysis by [3H]Phorbol 12,13-Dibutyrate

In-vitro quantitative receptor autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu) was used to determine the affinity constant (Kd) and the maximum number of receptor sites (Bmax) for protein kinase C (PKC) in subregions of the gerbil hippocampus, and to compare the distribution of [3H]PDBu binding sites in the gerbil hippocampus with that in the rat hippocampus. The Kd and Bmax values in the subregions of the gerbil hippocampus were estimated at 2.6-3.8 nM and 2.38-2.54 pmol (mg tissue)-1, respectively. The distribution of hippocampus [3H]PDBu binding sites was uniform in the gerbil but not in the rat. The [3H]PDBu binding activities in the strata oriens of the CA1 and CA3 subfields and the molecular layer of the dentate gyrus in the rat hippocampus were significantly higher than in the gerbil hippocampus. However, binding activity in the stratum lacunosum-moleculare of the rat CA1 subfield was statistically lower. These data demonstrate a difference in the distribution of [3H]PDBu binding activity in the hippocampus between the gerbil and rat.

Muscarinic receptor density is reduced in diabetic rat atria, an effect prevented by the aldose reductase inhibitor, Statil.

Binding assays using [3H]quinuclidinyl benzilate (QNB) as a muscarinic receptor ligand were carried out on atrial tissue from 6 control and 11 six-week streptozocin-diabetic rats. Five of the latter had received the aldose reductase inhibitor, Statil (25 mg kg-1 day-1 orally). Dissociation constants for specific (atropine displaceable QNB) binding were not changed either by diabetes or Statil treatment. Muscarinic receptor numbers, as estimated by Bmax values, were, however, significantly depressed in tissues from the untreated diabetic group, compared with tissues from either the controls or the Statil-treated diabetic group. These results may explain the reduced sensitivity to muscarinic agonists previously observed in atria from similarly diabetic rats and indicate the involvement of the sorbitol pathway in the change. Possible mechanisms are discussed.

Binding of [3H]haloperidol to dopamine D2 receptors in the rat striatum.

The present study was designed to examine the properties of [3H]haloperidol binding to dopamine D2-receptors in rat striatum membranes, displacement potencies of various chemicals and differences between the affinities of various chemicals and two new 5-hydroxytryptamine (5-HT2) receptor antagonists, MCI-9042, (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenetyl)phenoxy]methyl]et hyl hydrogen succinate hydrochloride and one of its metabolites. The plots of specific binding for the striatum membranes obtained from the Scatchard analysis using [3H]haloperidol were monophasic when non-specific binding was determined with 10 microM chlorpromazine, and the Kd and Bmax values were 7.42 +/- 1.03 nM and 1.58 +/- 0.20 pmol (mg protein)-1 (n = 10), respectively. The displacement potencies of D2 receptor, 5-HT2 receptor, histamine H1-receptor, and adrenoceptor antagonists were characterized by [3H]haloperidol binding to D2 receptors. The pKi values of a new antiplatelet agent, MCI-9042, and its metabolite were 5.02 and 5.53, respectively, and these values were lower than those of the D2-receptor antagonists, fluphenazine, spiperone, haloperidol, prochlorperazine, chlorpromazine, thioridazine, and sulpiride. They were also lower than the pKi values of the 5-HT2-receptor antagonists, pirenperone, ketanserin, methysergide, and mianserin. We conclude that the binding site of [3H]haloperidol in the rat striatum is the D2 receptor, that MCI-9042 and its metabolite have lower affinities for D2 receptors than for 5-HT2 receptors, and that this radioreceptor assay is useful for assessing the affinities of various agents.

The Effect of Acute Dyflos (DFP) Treatment on [3H]Nicotine Binding to Mouse Brain Homogenate

One possible adaptive mechanism that might arise due to inhibition of cholinesterase in the brain is a down regulation of central cholinergic receptors. Therefore studies were performed to determine the effect of acute dyflos exposure on [3H]nicotine binding. Specific [3H] nicotine binding was demonstrated to be saturable, reversible, stereospecific, and inhibited by a number of nicotinic compounds. Scatchard analysis of specific [3H] nicotine binding produced a curvilinear plot that was resolved into high- and low-affinity sites with Kd values of 6.1 +/- 2.5 and 114 +/- 13 nM, and Bmax values of 11.8 +/- 3.5 and 182 +/- 24 fmol (mg protein)-1, respectively. The nicotinic binding sites in brain homogenate from dyflos-treated mice that were killed 20 min or 10 h after exposure did not exhibit significant alterations in binding parameters from control mouse brain homogenate. However, brain homogenate from treated mice that were killed 24 h after exposure resulted in statistically significant differences in the low-affinity K(D) and Bmax values from controls. Since no alterations were found in the high-affinity binding parameters and dyflos had only a minimal effect on the low-affinity site at 24 h, it was concluded that nicotinic receptor down regulation does not appear to be the mechanism through which the mouse functionally adapts to cholinesterase inhibition caused by acute dyflos treatment.

Alpha 2-adrenoceptor changes during cerebral ageing. The effect of Ginkgo biloba extract.

[3H]Rauwolscine binding to alpha 2-adrenoceptors in cerebral cortex and hippocampus membranes of young (4 months) and aged (24 months) Wistar rats has been investigated. In aged rats, Bmax values of [3H]rauwolscine binding were significantly reduced (25-32%) in the cerebral cortex and hippocampus, as compared with the number of alpha 2-adrenoceptors found in young rats. Chronic treatment with Ginkgo biloba extract did not alter [3H]rauwolscine binding in the hippocampus of young rats, but significantly increased (28%) the [3H]rauwolscine binding density in aged rats. These data confirm the previously described age-related noradrenergic alteration and suggest that noradrenergic activity in aged rats is more susceptible to Ginkgo biloba extract treatment.

Apparent down-regulation of rat brain mu- and kappa-opioid binding sites labelled with [3H]cycloFOXY following chronic administration of the potent 5-hydroxytryptamine reuptake blocker, clomipramine.

This study examined the effect of chronic clomipramine administration on opioid mu- and kappa-binding sites. Clomipramine (5 mg kg-1 day-1) or saline was administered to rats via osmotic minipumps for 3 days or 28 days. Lysed-P2 brain membranes were prepared and preincubated for 60 min without (control membranes) or with 1 microM of the mu-selective acylating agent, 2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid azole-HC1 (BIT), to deplete membranes of mu-binding sites. [3H]6-Desoxy-6 beta-fluoronaltrexone ( [3H]cyclo FOXY) was used to label mu and kappa-binding sites. Weighted nonlinear least squares analysis of cycloFOXY binding surfaces permitted determination of the Kd and Bmax values of mu- and kappa-binding sites in control and treated rats. Subacute (3 days) administration of rats with clomipramine had no significant effect on [3H]cycloFOXY binding. Chronic (28 days) administration of clomipramine produced a small (approximately 10%) but statistically significant decrease in the Bmax. These findings are discussed in reference to other studies that have examined the effect of chronic antidepressant administration on opioid receptors, and speculate that the endogenous opioid systems may play a role in obsessive-compulsive disorder.

Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule β-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing β-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized β-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

Abstracts

Background: Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. Methodology/Principal Findings: Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). Conclusions: In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism. Citation: Livolsi A, Niederhoffer N, Dali-Youcef N, Rambaud C, Olexa C, et al. (2010) Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome. PLoS ONE 5(3): e9464. doi:10.1371/journal.pone.0009464 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received October 7, 2009; Accepted February 8, 2010; Published March 1, 2010 Copyright: 2010 Livolsi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the French Ministery of Research. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Pascal.Bousquet@medecine.u-strasbg.fr

Deterioration in brain and heart functions following a single sub-lethal (0.8 LCt 50) inhalation exposure of rats to sarin vapor: : A putative mechanism of the long term toxicity

The main injuries among victims of the terrorist act in the Tokyo subway resulted from sub-lethal inhalation and whole body exposure to sarin vapor. In order to study the long term effects of such exposure and to simulate these conditions, freely moving rats were exposed to sarin vapor (27.2 ± 1.7 μg/l) for 10 min. About 50% of the rats showed no overt symptoms and the rest had mild to moderate clinical symptoms that subsided within 4 h following exposure. A reduction of weight was noted during the first 3 days with full recovery on the 4th day. Rat's heart was challenged with epinephrine 1 and 6 months post exposure. A significant reduction in the threshold for epinephrine-induced arrhythmia (EPIA) was noted in rats exposed to sarin. A time dependent increase in the kD and Bmax values of muscarinic auto receptors (M2) was recorded in the rat's cortex and striatum. No changes were recorded in the rats' brain trans locator protein (TSPO) levels, concomitant with no observed changes in the animals' performance in A Morris water maze test. A significant increase in open field activity was noted 6 months following exposure to sarin vapor as well as a significant decrease in prostaglandin E 2 (PGE 2) production in the brain. It is speculated that down regulation of the M2 auto receptor function, caused hyper reactivity of the cholinergic system which leads to the changes described above. The continuous reduction in M2 auto-receptor system through an unknown mechanism may be the cause for long lasting decline in sarin-exposed casualties' health.

Modulation of benzodiazepine receptor, adrenoceptor and muscarinic receptor by diazepam in rat parotid gland

This study investigated the influence of diazepam on the binding characteristics of adrenoceptor, muscarinic and benzodiazepine receptors in rat parotid gland membrane using a radioligand binding assay. At a concentration of > 10 − 6 M, diazepam competed with [ 3H]dihydroalprenolol for β-adrenoceptor, but not [ 3H]prazosin for α-adrenoceptor or [ 3H]quinuclidinyl benzilate for muscarinic receptor. Continuous administration of diazepam at doses of 0.4 mg/kg/day, i.p. for 7 days in rat significantly decreased pilocarpine (4.0 mg/kg, i.p.)-induced parotid salivary flow. Diazepam also produced a significant increase in the dissociation constant (Kd) value for [ 3H]dihydroalprenolol binding, but no change in the maximal binding capacity (Bmax) value, and a decrease in the Kd value for [ 3H]diazepam binding to benzodiazepine receptors, but no change in the Kd or Bmax values for [ 3H]prazosin or [ 3H]quinuclidinyl benzilate binding. These results suggest that continuous administration of diazepam modifies affinity for β-adrenoceptor and benzodiazepine receptor binding sites in parotid gland membrane and that changes in these binding sites may be closely related to diazepam-induced suppression of salivary secretion.

In vitro assessment of the effectiveness of non-nutritive sorbent materials as binding agents for boar taint compounds

Boar taint, an off-odor and an off-flavor in the meat from some uncastrated male pigs, is due to high levels of the testicular steroid hormone, androstenone, and the indole, skatole. Thus far, there are no known methods for controlling both androstenone and skatole through dietary means. We tested the adsorbent agents, cholestyramine (CH), activated carbon (AC), tween-60 (Tween), bentonite (BNT) and polyvinylpolypyrrolidone (PVPP) for binding androstenone, estrone (E1), estrone sulfate (E1S) and skatole from buffer solutions in an in vitro system. The goal was to determine the potential utility of these binding agents as feed additives to control boar taint. Michaelis–Menten analysis was utilized to determine the effectiveness of the adsorbents. At pH 7.4, E1S was bound to AC and CH with the highest Bmax (maximum binding), whereas Tween and AC had the greatest Bmax for E1. The Bmax for skatole at pH 7.4 was highest for AC, CH and PVPP. AC had a higher Bmax for androstenone than CH and Tween. The Bmax values at pH 3.0 with E1S for AC and CH were essentially 100%, whereas the binding of Tween to E1S at pH 3.0 decreased by 49.5% from binding at pH 7.4 (P < 0.05). The Adint values, which represent efficiency of binding, illustrated that AC bound E1, androstenone and skatole with greater efficiency than the other binding agents at pH 7.4, whereas AC bound E1S as efficiently as CH. We conclude that AC was the most effective adsorbent agent for binding E1, E1S, androstenone and skatole in vitro, followed by CH, Tween, PVPP and lastly BNT. These adsorbent agents may be useful for binding boar taint compounds in in vivo studies to decrease the risk of boar taint.

Increased binding of cortical and hippocampal group II metabotropic glutamate receptors in isolation-reared mice

Post-weaning social isolation in rodents induces behavioral alterations, including hyperlocomotion, depression- and anxiety-like behaviors, aggression, and learning and memory deficits. These behavioral abnormalities may be related to the core symptoms in patients with neuropsychiatric disorders, such as schizophrenia and depression. In view of the recent studies that the group II metabotropic glutamate receptor (mGluR2/3) is involved in neuropsychiatric disorders, the present study examined the effect of isolation rearing on the binding of the mGluR2/3 antagonist [3H]LY341495 to mGluR2/3 in the mouse brain by in vitro autoradiography. The [3H]LY341495 binding in the prefrontal cortex, cerebral cortical layers I–III and hippocampus was significantly increased by rearing in social isolation while the binding in other brain regions was not altered. A saturation binding study of hippocampal membranes from isolation-reared mice revealed that the Bmax value increased significantly without any changes in the Kd value. Moreover, the mGluR2/3 antagonist MGS0039 (1.0mg/kg, intraperitoneally) decreased the immobility time of isolation-reared mice in the forced swim test. These results suggest that isolation rearing causes an increase in mGluR2/3 densities in the prefrontal cortex and hippocampus and that the increased receptor function may contribute to pathogenic mechanisms for depression-like behavior of the isolation-reared mice.

Surface plasmon resonance detection of biological warfare agent Staphylococcal enterotoxin B using high affinity monoclonal antibody

A novel sensitive method was developed for the detection as well as quantification of Staphylococcal enterotoxin B (SEB) using surface plasmon resonance (SPR). It is well known that the amount of SEB needed to cause the intoxication to human beings is very less and this concentration (0.02 μg/kg) is highly dangerous, hence, it is used as biological warfare agent. Thus, the need to develop a reliable and potential detection system against SEB is warranted. In the present work, SEB antibody was immobilized on carboxymethyldextran modified gold chip. The immobilization of SEB antibody and interaction of antigen with immobilized antibody were in-situ characterized by SPR and electrochemical impedance spectroscopy. A sample solution containing SEB antigen was injected in a working channel and the results revealed linearity in the concentration from 2.0 to 32.0 pM with a detection limit of 1.0 pM. By using kinetic evaluation software, K D (equilibrium constant) and Bmax (maximum binding capacity of analyte) values were calculated and found to be 13 pM and 424.23, respectively. Moreover, the thermodynamic parameter, change in Gibb's free energy was deduced and found to be −62.08 kJ/mol and this value shows the spontaneous interaction between SEB antigen and SEB antibody. In order to optimize the detection method, temperature and pH variation studies were also performed. Interference study was conducted to know the selectivity for the antigen–antibody interaction of SEB. The selectivity efficiency of SEB, SEC, SEA and SED were 100, 27.15, 20.01 and 12.05%, respectively towards SEB antibody.

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT3A receptors but leads to differential increases of 5-HT-induced maximum response (Emax) on cells expressing different subunits. Increases of Emax were due to analogously enhanced Bmax values for binding of the 5-HT3 receptor antagonist [3H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT3A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.