Dilute Buffer Research Articles

A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

With alkaline phosphatase (ALP)–adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR–McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350–1000pg/mL and the detection limit of 0.24ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%–96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

Identification of HnRNP-A2/B1 as a Target Antigen of Anti-Endothelial Cell IgA Antibody in Behçet's Disease

Behçet's disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease. We detected the target protein using western blotting and immunoprecipitation and determined the amino-acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/TOF). Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by ELISA. The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino-acid sequences of heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP-A2/B1). Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), 4 of 30 systemic lupus erythematosus patients (13.3%), 8 of 30 rheumatoid arthritis patients (26.7%), 9 of 30 Takayasu's arteritis patients (30%), 6 of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 were correlated with those against the recombinant streptococcal hsp-65.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 μg L(-1)) and an IC(50) of 3.2 nM (0.64 μg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

Effects of Macromolecular Crowding on Protein Biophysics

Proteins fold and function inside cells which are environments very different from that of a dilute buffer solution most often used in in vitro experiments. The cell compartments are full of other proteins, membranes and DNA; it is estimated that up to 40 % of the available volume in a cell can be occupied by other biomolecules. The crowded environment results in increased viscosity, excluded-volume effects and amplified opportunity for specific and non-specific inter-molecular interactions. These environmental factors are not accounted for in the mechanistic studies of protein folding and function that have been executed during the last decades. The question thus arises as for how these effects - present when polypeptides normally fold in vivo - modulate protein biophysics? To take a step closer to understanding the in vivo scenario, we here assess how crowded environments affect protein biophysical properties. For this we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with the target proteins, in combination with strategically selected proteins and a range of biophysical/spectroscopic methods. We have found that in the presence of macromolecular crowding in vitro, proteins become more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, protein folded states may change both secondary structure content and overall shape. For a protein with a complex folding mechanism involving an off-path intermediate, excluded volume effects make the folding energy landscape smoother (i.e., less misfolding) than in buffer. Our findings demonstrate that excluded volume effects tune protein biophysical parameters: this is of mechanistic relevance since proteins have evolved to fold and function in crowded environments.

Effects of Pretreating Serum Samples on the Performance of a Latex Agglutination Test for Serodiagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a fungal disease caused by Paracoccidioides brasiliensis, and Brazil is one of the principal countries where it is endemic. Diagnosis is based on the observation of budding P. brasiliensis yeast in clinical specimens from patients; however, the sensitivity of the visualization of fungi is low, indicating that serological tests are used for early diagnosis. The double-immunodiffusion test (ID) is the "gold standard" test for serology in PCM, although the execution of this test requires the availability of laboratorial infrastructure. We report the improved performance of a latex agglutination test (LAT) by pretreating 30 serum samples from PCM patients and 71 controls (histoplasmosis and aspergillosis patients, patients with bacterial infections, and normal human sera) with a dilution buffer incubated at 37°C for 30 min. The sensitivity and specificity of the LAT test in the nonpretreated samples were 73% and 79%, respectively. However, when samples were pretreated, the sensitivity and specificity of the test increased to 90%. In this study, we did not observe cross-reactivity with histoplasmosis patient sera, but some reactions to sera from patients with aspergillosis and bacterial infections were noted. Normal human sera were not reactive in our tests. These results indicate the need for the elimination of heterologous reactions so that we can adequately use this method for screening cases of PCM.

Calcitonin measurement on fine needle washouts: Preanalytical Issues and Normal Reference Values

Calcitonin (CT) measurement on fine-needle aspiration cytology (FNAC) washouts (FNAC-CT) is a promising tool in the diagnosis of medullary thyroid carcinoma (MTC). Guidelines for the method with codified cut-off are needed to use this technique in clinical routine. This study was undertaken to assess the best pre-analytical procedure and to define a reliable reference value for FNAC-CT. Fifty-four patients underwent thyroid surgery, so MTC was excluded by surgical pathology examination and CT immunostains. Before surgery, FNAC-CT was measured on 78 thyroid nodules from such 54 patients. Needles were rinsed by normal saline and specific CT-free dilution buffer, and CT was measured by a fully automated immunochemiluminometric assay. FNAC-CT levels were not significantly different in normal saline or specific buffer. The calculated 97.5th upper FNAC-CT value was 8.5 pg/mL (saline) and 7.43 pg/mL (buffer), respectively. Seeing its relatively large sample size, rigorous selection criteria and inclusion of CT immunostaining of thyroid nodules, the present study provides a reliable guideline and a clinically relevant reference value for FNAC-CT measurement in thyroid nodules.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

The Ever Folding Protein Landscape

The Ever Folding Protein Landscape

Conjunctival provocation with airborne allergen in patients with atopic keratoconjunctivitis

Atopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exacerbations. Many patients experience no obvious seasonal variation, although a majority of patients are allergic to common airborne allergens. To investigate the allergic reaction, to conjunctival provocation with airborne allergens, in patients with AKC. Eleven patients with AKC and birch and/or grass pollen allergy participated in the study, which was performed outside the pollen season. Five patients with seasonal allergic conjunctivitis (SAC) and five healthy subjects were included for validation purposes. The challenge was performed in one eye with the allergen, to which the patient was reactive, and with dilution buffer in the other eye. Signs and symptoms from both eyes were graded at baseline and at 10 min, 8 and 48 h after provocation. Tear fluid was collected from both eyes for cytokine analyses at baseline and at 8 and 48 h. A significant change in clinical symptoms and signs, (redness and chemosis) was evident 10 min after provocation compared with baseline (P = 0.005) and compared with the unprovoked eye (P = 0.005) in AKC subjects. These parameters were normalized after 8 and 48 h. A significant increase for IFN-γ (P = 0.021) and IL-6 (P = 0.015), and a near significant increase for IL-10 (P = 0.066) were seen in the tear fluid of the challenged eye at 48 h after provocation vs. baseline and vs. the control eye for IFN-γ (P = 0.005), IL-6 (P = 0.028) and IL-10 (P = 0.008) in AKC subjects. In this single dose allergen provocation study, AKC patients responded with a typical IgE-mediated allergic reaction. An increase in cytokines at 48 h after the challenge was demonstrated and might, with further studies, give us a better understanding of the nature of inflammation in AKC.

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.

Development of a specific ELISA to measure BACE1 levels in human tissues

The aspartyl protease BACE1 is the rate limiting enzyme in the synthesis of amyloid beta, which accumulation in the human brain is a hallmark of Alzheimer's disease (AD). BACE1 has been proposed as a surrogate marker of AD; however, very few BACE1 immunoassays have been reported. In the present study we have screened ten BACE1 antibodies by Western blot and several antibody pairs to develop a new BACE1 sandwich ELISA procedure. We identified one pair that showed little background and good reproducibility. Several dilution buffers and sample denaturation methods were tried to partially unfold BACE1 before capture. We found that dilution in PBS followed by 10 min incubation at 50 °C critically improves the performance of the assay. Finally, we successfully measured BACE1 levels in a few human brain and platelet lysates as well as in plasma and AD CSF. We anticipate that this assay will lay the ground to accurately measure BACE1 levels in human tissues, which could facilitate the molecular diagnosis of AD in the near future.

Optimisation of in ovo electroporation of the chick neural tube

Chick in ovo neural tube electroporation has become a widely used method for assaying gene function during embryonic development. Since its first description, many variants of this technique have been described, with varying values for specific parameters such as electrode type and spacing, voltage, pulse duration and plasmid DNA concentration. Here we examine the influence of some of these variables and derive a detailed and optimal protocol for electroporating the caudal neural tube during the third day of embryonic development. Our findings highlight the importance of electrode placement and DNA dilution buffer for optimal expression and absence of electroporation artifacts.

P.1.c.005 Protective effect of Platycodon grandiflorum in a middle cerebral artery occlusion and reperfusion model of cerebral ischemia in rats

The effect of malonate on the dissolution rate of the reservoir minerals: albite, quartz, and microcline has been examined. The effect of pH on rate has been decoupled from the effect of the ligand by controlling pH using dilute buffer solutions at values of pH 4 through pH 10 at 70°C. Any effect of the buffer ligands (acetate and borate) or alkali cation (Na) on rate was accounted for by comparing the measured dissolution rates in buffer solutions with and without malonate. Flowing reactors were used and malonate concentration was varied from 1 to 100 mmolal.As malonate anion concentration is increased, the dissolution rate of these minerals also increases. At moderately acidic to near neutral pH in the solutions containing the highest malonate concentration, malonate most significantly increases the dissolution rate of the silicates over that expected due to simple hydrolysis. At alkaline pH the effect of malonate on the dissolution rate of these silicates is not significant, compared to the dissolution rate due to simple hydrolysis alone.

Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis

BackgroundImmunoprecipitation and subsequent 2D-PAGE/mass spectrometry are powerful tools to study post-translational protein modifications. Often disregarded in this workflow is the impact of the chemical cross-linker upon antibody affinity, as well as incomplete elution of primary target protein in buffers commonly used in 2D-PAGE. This may impede detection of non-abundant protein isoforms.ResultsHere we have compared cross-linking of antibodies to Dynabeads® Protein A by using DMP or BS3, as well as the efficiency of various target elution buffers prior to 2D-PAGE separation. BS3 cross-linking generally resulted in less non-specific binding than DMP, whereas DMP cross-linking gave overall higher yield of target protein. Regardless of the cross-linker used, incomplete elution of target protein was observed with conventional glycine- or urea-based buffers. Conversely, complete elution was obtained with 2% hot SDS and subsequent dilution in urea buffer containing 4% CHAPS, to 0.2% final SDS yielded perfectly focused gels suitable for mass spectrometry analysis.ConclusionCareful choice of Ig cross-linker as well as efficient elution of target protein in SDS prior to downstream 2D-PAGE may be key factors to analyze low-abundance proteins enriched by magnetic bead immunoprecipitation.

A Survey of Antiprion Compounds Reveals the Prevalence of Non-PrP Molecular Targets

Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593⁵·⁵⁰) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

Simultaneous Determination of Bromhexine and Amoxicillin in Pharmaceutical Formulations by Capillary Electrophoresis

A simple and time-efficient method, based on capillary zone electrophoresis, was developed for the simultaneous determina- tion of bromhexine (BMX) and amoxicillin (AMX) in pharmaceuti- cal formulations. The optimized electrophoretic conditions comprise 50 mM sodium phosphate plus 50 mM citric acid as running buffer (pH 3.0), cartridge temperature 25°C, hydrodynamic injection (5 s. at 0.5 psi), 30 kV separation voltage, and UV detection at 214 nm. The analytes were separated in less than 5 min. Formulation samples were processed by successive treatment with methanol, hydrochloric acid, filtering and dilution in the running buffer. Loperamide was used as internal standard for quantitation. A linear detection range of 10-85 μg/mL for BMX (R 2 = 0.997) and 250-2010 μg/mL for AMX (R 2 = 0.995) was observed, with LOD for BMX as low as 2 μg/mL. The developed method enabled high analyte recoveries (99-104%) and excellent run-to-run reproducibility. Moreover, it was successfully employed for the determination of BMX and AMX in several phar- maceutical formulations, demonstrating its applicability to the routine quality control.

Involvement of Jun Dimerization Protein 2 (JDP2) in the Maintenance of Epstein-Barr Virus Latency

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the BZLF1 viral immediate-early protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found that Jun dimerization protein 2 (JDP2) plays a significant role in suppressing Zp activity. Reporter, EMSA, and ChIP assays of a Zp mutant virus revealed JDP2 association with Zp at the ZII cis-element, a binding site for CREB/ATF/AP-1. Suppression of Zp activity by JDP2 correlated with HDAC3 association and reduced levels of histone acetylation. Although introduction of point mutations into the ZII element of the viral genome did not increase the level of BZLF1 production, silencing of endogenous JDP2 gene expression by RNA interference increased the levels of viral early gene products and viral DNA replication. These results indicate that JDP2 plays a role as a repressor of Zp and that its replacement by CREB/ATF/AP-1 at ZII is crucial to triggering reactivation from latency to lytic replication.

Macromolecular Crowding Fails To Fold a Globular Protein in Cells

Proteins perform their functions in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules stabilizes globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells using a variant of protein L, a 7 kDa globular protein with seven lysine residues replaced by glutamic acids; 84% of the variant molecules populate the denatured state in dilute buffer at room temperature, compared with 0.1% for the wild-type protein. We then used in-cell NMR spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (∼1 kcal/mol) free-energy deficit. The data are consistent with the idea that nonspecific interactions between cytoplasmic components can overcome the excluded-volume effect. Evidence for these interactions is provided by the observations that adding simple salts folds the variant in dilute solution but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with the results of other studies of protein stability in cells and suggest that stabilizing excluded-volume effects, which must be present under crowded conditions, can be ameliorated by nonspecific interactions between cytoplasmic components.

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s −1, respectively. The k cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s −1, respectively, at 1.0 M. On the other hand, the k cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100–600 s −1. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.