Specific Rabbit Polyclonal Antibodies Research Articles

ОПТИМИЗАЦИЯ ПРОТОКОЛА ИММУНОФЕРМЕНТНОГО АНАЛИЗА ДЛЯ ИНДИКАЦИИ Т-2 ТОКСИНА

T-2 toxin is one of the most common and toxic trichothecene mycotoxins – secondary metabolites of molds that develop on cereals and some other crops. This article discusses the development stage of a test system for the determination of T-2 toxin based on enzyme-linked immunosorbent assay (ELISA), which uses an enzyme label – a conjugate of anti-rabbit goat antibodies with peroxidase. An important step in the creation of any ELISA-based test system is the preliminary titration of reaction components. The aim of the work was to determine the optimal concentration of the conjugate of antispecies antibodies in an indirectly competitive enzyme-linked immunosorbent enzyme immunoassay with the indication of T-2 toxin. A number of dilutions of the antivirus conjugate were examined: 1 : 1000; 1 : 2000; 1 : 3000; 1 : 4000; 1 : 5000; 1 : 7500; 1 : 10000; 1 : 12 500; 1 : 15,000; 1 : 17,500; 1 : 20,000; 1 : 30 000. In the ELISA staging protocol, calibration solutions of T-2 toxin at concentrations of 0.0 were used; 2.5; 5, 10, 20, 40, 80, 160 ng/ml and specific polyclonal rabbit antibodies to the T-2-BSA conjugate. Based on the average optical density values, calibration plots were constructed using the percentage of signal absorption from the zero standard. When evaluating the results of the study, the criterion for choosing the dilution of the conjugate of anti-species antibodies was considered the greatest, at which the best linearity of the grading plot is achieved and the level of its non-specific reaction with the zero standard would be the lowest. It was established that the optimal variants of dilutions of the conjugate of anti-species antibodies under the same experimental conditions tested were 1: 4000, 1 : 5000 and 1 : 12 500. Dose-dependent signal absorption was observed in all concentrations of anti-species antibodies. Dilutions of the conjugate of anti-species antibodies 1 : 1000–1 : 3000 and 1 : 17 500–1 : 30 000 were not taken into account, since the optical density of most wells was higher than the optimal boundaries in the first case (> 3.9) and lower in the second (< 0.4). Based on the foregoing, optimal dilution of the conjugate of anti-species antibodies was selected 1 : 12 500.

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis invitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

In VitroAnalyses Reveal the Effect of Synthetic Cytokinin Forchlorfenuron (FCF) on a Septin-Like Protein of Taeniid Cysticerci

Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro, FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.

Immunoblotting Quantification Approach for Identifying Potential Hypoallergenic Citrus Cultivars

The inherent allergens of citrus fruits, such as Cit s 1, Cit s 2, Cit s 3 can cause allergic reactions. A better understanding of the genetic factors (cultivar to cultivar) affecting the allergenic potential of citrus fruits would be beneficial for further identification of hypoallergenic genotypes. In the present study, an immunoblotting quantification approach was adopted to assess the potential allergenicity of 21 citrus cultivars, including nine subgroups (tangerine, satsuma, orange, pummelo, grapefruit, lemon, kumquat, tangor, and tangelo). To prepare highly sensitive and specific rabbit polyclonal antibodies, antigenicity of purified rCit s 1.01, rCit s 2.01, and rCit s 3.01 peptides were enhanced with high epitope density in a single protein molecule. The data integration of three citrus allergen quantifications demonstrated that the four pummelo cultivars (Kao Phuang Pummelo, Wanbai Pummelo, Shatian Pummelo, and Guanxi Pummelo) were potential hypoallergenic, compared with other 8 subgroups. Moreover, the immunological analyses with sera of allergic subjects revealed that Shatian Pummelo and Guanxi Pummelo showed the lowest immunoreactivity in 8 representative citrus cultivars. These potential hypoallergenic genotypes are of great significance to not only allergic consumers but also citrus breeders in the genetic improvement of hypoallergenic citrus as breeding resources.

A rational approach to obtaining high-specific polyclonal antibodies against recombinant alpha-synuclein

The approach for the quick and efficient production ofpolyclonal antibodies tothe target antigen alpha-synuclein has been proposed. Two methods have been employed to purify specific rabbit polyclonal antibodies against recombinant human alpha-synuclein, produced by subcutaneous immunization with complete Freund's adjuvant. It was shown that purification on CNBr-activated Sepharose with immobilized alpha-synuclein resulted in antibody preparation with rabbit serum histidine-rich glycoprotein as a contaminant. Two-stage antibody purification procedure first on Sepharose with immobilized protein G, and then on alpha-synuclein immobilized column helps to avoid contamination and to obtain homogenous antibody preparation. Antibodies recognize different conformations of alpha-synuclein and can be used in a variety of immunochemical approaches, including immunocytochemistry.

Identification and Characterization of Novel Rare Nucleophosmin (NPM1) Gene Mutations in Acute Myeloid Leukemia (AML) By a Combinatorial Approach of Immunohistochemistry and Molecular Analyses

Abstract Introduction. Nucleophosmin (NPM1) gene mutations occur in 50-60% of adult AML with normal karyotype (Falini et al, NEJM 2005). About 50 NPM1 mutations have been so far identified, all clustering in exon-12 (Falini et al, Blood 2007) but few sporadic cases involving either exon-9 (one) (Mariano et al, Oncogene 2006) or exon-11 (two) (Albiero et al, Leukemia 2007). In spite of molecular heterogeneity, all mutations cause common changes at the C-terminus of NPM1 mutants, i.e. loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES) motif (Falini et al, Blood 2006a).As a consequence, all NPM1 mutants aberrantly accumulates in the cytoplasm of leukemic cells and can be detected by immunohistochemistry, which is fully predictive of NPM1 gene mutations (Falini et al, Blood 2006b). Methods. From 2005 to 2015, 702 AML patients samples were analyzed at diagnosis by both immunohistochemistry (IHC) for NPM1 subcellular localization and western blot (WB) with anti-NPM1 mutant specific rabbit polyclonal antibodies antibodies, produced in our laboratory (Martelli et al, Leukemia 2008). Discordant cases were further analyzed by NPM1 gene Sanger sequencing. Newly discovered NPM1 mutated genes were subcloned in pEGFP-C1 vector and transiently expressed in NIH-3T3 adherent cells to study the NPM1 mutant subcellular localization by immunofluorescence microscopy. The NESbase version 1.0 program was used to identify putative NES within the new protein sequence, and their efficiency was evaluated by the pREV1.4-based NES efficiency assay, as previously described (Bolli et al, Cancer Res 2007). Results. At IHC and WB analyses, concordance in diagnosis was obtained in 695/702 samples (291 NPM1-mutated and 404 NPM1-unmutated AML). In 7/702 (1%), IHC detected cytoplasmic NPM1 whilst WB with anti-NPM1 mutant antibodies was negative. Unfortunately, in 3 out of these cases, the original patient sample was not available for further analyses. In the other 4, exon-12 NPM1 gene sequence was wild-type (WT), in keeping with the negative WB results. One of these cases harbored the previously described exon-11 NPM1 mutation, in 1 case no mutation was detected (further studies are ongoing), and in 2 cases new mutations involving exon-6 were discovered. Strikingly, in the latter cases, WB analysis with different anti-NPM1 antibodies revealed a new band at different molecular weight (MW) than NPM1-WT. Indeed, in 1 case an in frame 21 nucleotides insertion at exon-6 lead to a 7 aa longer than WT protein, whilst in the other a 19 nucleotides insertion created a new stop codon leading to a truncated protein. In both cases, a new NES motif was created. Importantly, cell transfection experiments confirmed that the new NPM1 mutants localized at least partly in the cytoplasm, and the pREV1.4-based NES efficiency assay showed the new NES were active. Conclusions. Here, we report on the identification and functional characterization of two novel NPM1 mutations in AML. Our observations further support the view that cytoplasmic NPM1 dislocation is a critical step in leukemogenesis, and that immunohistochemistry, that detects, through cytoplasmic dislocation on NPM, 'all types' of NPM1 mutations, might be used as first step for directing further molecular studies. Disclosures Di Raimondo: Janssen-Cilag: Honoraria.

Abstract 1370: A new RAS G12D specific rabbit polyclonal antibody for immunohistochemical FFPE application

Abstract Background: Determining RAS mutation status is considered as prognostic factor for colorectal cancer patients in anti-epidermal growth factor receptor (EGFR) antibody therapy. Because genetic testing remains costly and time-consuming, it is necessary to develop mutation specific antibody for immunological analysis. However RAS G12D mutation antibody for immunohistochemical (IHC) staining is not available. Results: Here we report a novel RAS G12D rabbit polyclonal antibody (GTX132407) for immunochemistry (IHC) testing, which was developed by cooperating with GeneTex In. Based on analyzing 15 clinical colorectal cancer specimens, the result of IHC staining using RAS G12D mutation specific antibody (GTX132407) was correlated with genetic testing of RAS G12D mutation using ultra-sensitive total RAS mutation screen kit [Hong Jing Biotechnology Inc. (FemtoPath)]. Conclusion: In summary, the result showed that IHC testing with RAS G12D specific mutation antibody could be a reliable tool for predicting RAS genetic mutation status. Citation Format: Chi-Kuan Chen. A new RAS G12D specific rabbit polyclonal antibody for immunohistochemical FFPE application. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1370.

An ELISA for quantifying quail IgY and characterizing maternal IgY transfer to egg yolk in several quail strains

In avian species, maternal blood immunoglobulin Y (IgY) is transferred to the egg yolks of maturing oocytes, but the mechanism underlying this transfer is unknown. To gain insight into the mechanism of maternal IgY transfer in quail, we established an enzyme-linked immunosorbent assay (ELISA) for the quantitation of quail IgY. We characterized strain differences in blood and egg yolk IgY concentrations and exogenously injected IgY-Fc uptakes into egg yolks. A specific rabbit polyclonal antibody to quail IgY was raised for the ELISA. Blood and egg yolk IgY concentrations were determined in six quail strains (one inbred strain, L; four closed population strains, AWE, DB, PS, WE; one commercial strain, Commercial). The birds were also injected with digoxigenin-labeled quail IgY-Fc, and its uptakes into laid eggs were compared. The strain difference in blood and egg yolk IgY concentrations was at most 2.5-fold, between PS and AWE. The rank order of IgY concentrations was AWE, Commercial, DB, L≥WE≥PS. A significant positive correlation (|R|=0.786) between individual blood IgY and egg yolk IgY and the concentrated egg yolk IgY (1.5–2-fold) against blood IgY was observed. Interestingly, there was a significant inverse correlation (|R|=0.452) between injected IgY-Fc uptakes and the blood IgY concentration, implying competition of the injected IgY-Fc and blood IgY in the process of IgY uptake into egg yolks. In conclusion, we successfully determined blood and egg yolk IgY concentrations in various quail strains by a quail IgY-specific ELISA. The concentrated egg yolk IgY against the blood IgY and the inverse relationship of exogenous IgY-Fc uptake against the blood IgY supports the existence of a selective IgY transport mechanism in avian maturing oocytes.

Regulator of Calcineurin 1 in Periodontal Disease.

Nuclear factor of activated T-cells (NFAT) and NF-kB pathway associated processes are involved in the pathogenesis of various inflammatory disorders, for example, periodontal disease. The activation of these pathways is controlled by the regulator of calcineurin 1 (RCAN1). The aim of this study was to elucidate the role of RCAN1 in periodontal disease. Healthy and inflamed periodontal tissues were analyzed by immunohistochemistry and immunofluorescence using specific rabbit polyclonal anti-RCAN1 antibodies. For expression analysis human umbilical vein endothelial cells (HUVEC) were used. HUVEC were incubated for 2 h with Vascular Endothelial Growth Factor (VEGF) or with wild type and laboratory strains of Porphyromonas gingivalis (P. gingivalis). Expression analysis of rcan1 and cox2 was done by real time PCR using specific primers for rcan1.4 and cox2. The expression of rcan1 was found to be significantly suppressed in endothelial cells of chronically inflamed periodontal tissues compared to healthy controls. Rcan1 and cox2 were significantly induced by VEGF and wild type and laboratory P. gingivalis strains. Interestingly, the magnitude of the rcan1 and cox2 induction was strain dependent. The results of this study indicate that RCAN1 is suppressed in endothelial cells of chronically inflamed periodontal tissues. During an acute infection, however, rcan1 seems to be upregulated in endothelial cells, indicating a modulating role in immune homeostasis of periodontal tissues.

Immunocapture of circulating Schistosoma mansoni cathepsin B antigen (Sm31) by anti-Sm31 polyclonal antibodies

Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms. Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients. Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA). To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography. Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr.~31kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis. CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections.

Nicotinamidephosphoribosyltransferase (NAMPT) Induces Ubiquitination of LEF-1 Transcription Factor By Deacetylation

We and others demonstrated, that transcription factor lymphoid enhancer-binding factor 1 (LEF-1) is crucial for neutrophil granulocytopoiesis and lymphopoiesis. LEF-1 expression is severely diminished in myeloid progenitors of severe congenital neutropenia (CN) patients, leading to "maturation arrest" of granulopoiesis.In the present study we evaluated the mechanisms of LEF-1 regulation. Previously we identified elevated levels of the enzyme Nicotinamide phosphoribosyltransferase (NAMPT) and its target Sirtuin 1 (SIRT1) in myeloid cells of CN patients. SIRT1 is NAD+-dependent protein deacetylase and in this study we analyzed whether LEF-1 could be regulated by NAMPT/SIRT1-triggered deacetylation. We identified lysine residue in LEF-1 protein, which could be de-/acetylated, generated specific rabbit polyclonal antibody recognizing this acetyl-Lys and confirmed NAMPT/SIRT1-triggered deacetylation of LEF-1 protein on this lysine in T-cell leukemia cell line Jurkat and in AML cell line LW/SO.We first studied the effects of LEF-1 deacetylation by NAMPT/SIRT1 on its expression and functions. We found markedly diminished levels of total LEF-1 protein as well as its nuclear translocation in Jurkat and LW/SO cells treated with NAMPT. At the same time, inhibition of NAMPT using specific NAMPT inhibitor FK866 resulted in elevated mRNA and protein levels of LEF-1 and its target genes survivin, c-myc and cyclin D1.We also performed reporter gene assay using WT or acetyl-Lys LEF-1 mutant and TOP promoter construct, containing six LEF-1/TCFs binding sites. Acetyl-Lys LEF-1 mutant was not able to activate TOP promoter, as compared to its strong activation by WT LEF-1. Moreover, co-transfection with NAMPT- or SIRT1-expression constructs also showed inhibitory effects on LEF-1-triggered activation of TOP promoter. These experiments clearly demonstrated that NAMPT/SIRT1-triggered deacetylation of the LEF-1 protein inhibited its expression and functions.We further evaluated the mechanism of NAMPT-triggered reduction of LEF-1 expression. In silico analysis of LEF-1 protein using CPLM software revealed that lysine residue in LEF-1 protein, which is de-/acetylated, could be also ubiquitinated. We therefore studied the effects of deacetylation on the ubiquitination of LEF-1 protein in Jurkat cells. We evaluated the effects of 26S proteasome inhibitor, bortezomib, on LEF-1 protein levels after treatment with NAMPT. Indeed, upon treatment with bortezomib the level of LEF-1 protein was restored. This confirmed, that deacetylation of LEF-1 by NAMPT led to degradation of LEF-1 by ubiquitination. We assumed, that in CN patients, hyperactivation of NAMPT/SIRT1 pathway might lead to downregulation of LEF-1 by deacetylation and ubiquitination. Indeed, we observed decreased G-CSF-triggered granulocytic differentiation and proliferation of CD34+ cells from healthy individuals transduced with LEF-1 acetyl-lysine mutant, as compared to cells transduced with WT LEF-1. Moreover, treatment of CD34+ cells of CN patients with bortezomib resulted in restoration of defective LEF-1 expression ultimately leading to normalization of granulocytic differentiation.In conclusion, NAMPT/SIRT1-triggered deacetylation of LEF-1 resulted in its degradation by ubiquitination. DisclosuresNo relevant conflicts of interest to declare.

Expression of ENaC, SGK1 and Nedd4 isoforms in the cochlea of guinea pig.

It has been demonstrated that the epithelial sodium channel (ENaC) may play critical roles in (re)absorbing Na+ from apical plasma membrane in various tissues and cells. Moreover, the serum glucocorticoid-inducible kinase 1 (SGK1) and the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated isoforms Nedd4 are involved in ENaC regulation in response to hormones such as aldosterone, vasopressin and insulin. The aim of the study was to investigate the cellular localizations of ENaC subunits, SGK1, and Nedd4 isoforms in the cochlea of guinea pig. Ten adult guinea pigs were sacrificed and their cochleas were collected. The expression patterns of ENaC subunits, SGK1 and Nedd4-2 isoforms in the guinea pig cochlea were studied by immunohistochemistry with the specific polyclonal rabbit antibodies against rat α-, β- and γ-ENaC, SGK1, Nedd4-1/2 and Nedd4-2. The results showed that all these proteins were extensively expressed in various regions of the cochlea. They were found in the spiral ligament, organ of Corti, spiral limbus, spiral ganglion and Reissner's membrane with different staining patterns. The results indicated that a Na+ transport system may exist in the cochlea of guinea pig consisting of ENaC, SGK1 and Nedd4, which may work in concert to transport Na+ and to maintain homeostasis in inner ear as it does in other epithelia.

Expression of TMEM166 Protein in Human Normal and Tumor Tissues

Transmembrane protein 166 (TMEM166) is a novel human regulator involved in both autophagy and apoptosis. In this study, we generated a specific rabbit polyclonal antibody against human TMEM166 and assessed the expression of this protein in various human normal and tumor tissue samples by tissue microarray-based immunohistochemical analysis. Varying TMEM166 protein levels were expressed in a cell-type and tissue-type-specific manner in detected tissues or organs. Strong TMEM166 expression was shown in the glomerular zona of the adrenal cortex, chromophil cells of the pituitary gland, islet cells, squamous epithelium of the esophagus mucosa, the fundic gland, and hepatocytes. Moderate or weak TMEM166 staining was identified in the parathyroid gland, the testis, vaginal stratified squamous cells, lung macrophages, hematopoietic cells, renal tubular epithelial cells, macrophages in the spleen red pulp, and neuronal cells in the cerebral cortex. Some tissues failed to stain for TMEM166, such as adipose tissue, colon, cerebellum, lymph node, mammary gland, ovary, prostate, rectum, skin, small intestine, thyroid gland, tonsil, and thymus. In comparing human normal and tumor tissues, TMEM166 expression was widely downregulated in the cancer tissues. Our studies provide the basis for future investigations into cell-type-specific functions of this protein in human normal and tumor tissues.

Determination of allergenic load and pollen count ofCupressus arizonicapollen by flow cytometry usingCup a1polyclonal antibody

There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement.Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured.We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003.Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load.

Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing -lactamase derived from Haemophilus influenzae

Group A streptococci (GAS) cause, among other infections, pharyngotonsillitis in children. The species is frequently localized with the Gram-negative respiratory pathogens non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, which both produce outer membrane vesicles (OMVs). The aim of this study was to investigate whether OMVs isolated from NTHi contain functional β-lactamase and whether the OMVs hydrolyse amoxicillin and thus protect GAS from killing by the antibiotic. The antibiotic susceptibility of isolates was determined using the Etest. The resistance genes blaTEM-1 (encoding NTHi β-lactamase), bro-1 (encoding M. catarrhalis β-lactamase) and ftsI (encoding NTHi penicillin-binding protein 3) were searched for by PCR, followed by sequencing. OMVs were isolated by ultracentrifugation and the presence of β-lactamase was detected by western blots including specific rabbit polyclonal antibodies. The chromogenic substrate nitrocefin was used to quantify and compare the β-lactamase enzyme activity in the OMVs. The hydrolysis of amoxicillin by β-lactamase was estimated by an agar diffusion method. We showed that OMVs released from β-lactam-resistant M. catarrhalis and NTHi contain functional β-lactamase that hydrolyses amoxicillin and protects GAS from killing by amoxicillin. This is the first report of the presence of β-lactamase in NTHi OMVs. We suggest that OMV-derived β-lactamase from coinfecting pathogens such as NTHi and M. catarrhalis may contribute to the occasional treatment failures seen in GAS tonsillitis.

Endogenous morphine‐6‐glucuronide (M6G) is present in the plasma of patients: Validation of a specific anti‐M6G antibody for clinical and basic research

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers. While endogenous morphine levels are detectable using enzyme-linked immunosorbant assay (ELISA), mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. This study describes the preparation of a specific anti-M6G rabbit polyclonal antibody and its validation. The specificity of this antibody was assessed against 30 morphine-related compounds. Then, a M6G-specific ELISA-assay was tested to quantify M6G in the plasma of healthy donors, morphine-treated, and critically ill patients. The antibody raised against M6G displays a strong affinity for M6G, codeine-6-glucuronide, and morphine-3-6-glucuronide, whereas only weak cross-reactivities were observed for the other compounds. Both M6G-ELISA and LC-MS/MS approaches revealed the absence of M6G in the plasma of healthy donors (controls, n = 8). In all positive donors treated with morphine-patch (n = 5), M6G was detected using both M6G-ELISA and LC-MS/MS analysis. Finally, in a study on critically ill patients with circulating endogenous morphine (n = 26), LC-MS/MS analysis revealed that 73% of the positive-patients (19 of 26), corresponding to high M6G-levels in M6G-ELISA, contained M6G. In conclusion, we show that endogenous M6G can be found at higher levels than morphine in the blood of morphine-naive patients. With respect to the interest of measuring endogenous M6G in pathologies, we provide evidences that our ELISA procedure represents a powerful tool as it can easily and specifically detect endogenous M6G levels.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

Background: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled with AlexaFluor® 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter® ). Optical microscopy study was realized with a Leica optical microscope. Bland & Altman was used to determine agreement between both techniques measured. Results: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a p-value: 0.0008E-2 and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. © 2013 Clinical Cytometry Society.

Investigation of specific interactions between microbial cells and polyclonal antibodies using a resonator with lateral electric field

The interaction between polyclonal antibodies and Azospirillum brasilense Sp7 cells was studied using a resonator with lateral electric field. To this end, specific polyclonal rabbit antibodies against the O-antigen epitopes of the strain A. brasilense Sp7 were obtained and the possibility of their application for detection of microbial cells using a piezoelectric resonator with lateral electric field was shown. It was established that frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with the suspension of A. brasilense Sp7 cells and antibodies substantially differed from those of the resonator with the control suspension of cells without antibodies. It was shown that the obtained antibodies interacted with azospirilla cells, and the marker was accumulated all over the cell surface. The limit of possible detection of microbial cells during their interaction with antibodies was found to be 104 cells/mL. Detection of A. brasilense Sp7 cells using antibodies proved to be possible in the presence of foreign bacteria. The presented results demonstrate the possibility of recording the interaction between microbial cells and antibodies and developing a biosensor for quantitative detection of microbial cells.

Molecular Markers of Early-Stage Mycosis Fungoides

The lack of a specific marker differentiating early mycosis fungoides (eMF) from benign inflammatory dermatitis presents significant difficulties in the assessment and management of suspected MF patients, which often leads to delayed diagnosis and improper medical approaches. To address this, an investigation was carried out to characterize positive identification markers for eMF by comparing eMF lesions with healthy skin and benign inflammatory dermatitis, using high-throughput genomic transcription profiling. A total of 349 genes were differentially expressed in eMF lesions compared with normal skin. These genes belong to pathways associated with inflammation, immune activation, and apoptosis regulation. Most of them (N=330) also demonstrated significant upregulation in chronic dermatitis, making them nonideal markers for eMF. Among them, 19 genes with specific enrichment in eMF lesions were identified that showed no significant upregulation in chronic dermatitis. Two of them, TOX and PDCD1, showed high discrimination power between eMF lesions and biopsies from benign dermatitis by RNA expression. Furthermore, TOX demonstrated highly specific staining of MF cells in eMF skin biopsies in immunohistochemistry and immunofluorescence, including the early epidermotropic cells in Pautrier's microabscesses. This study demonstrates the potential of eMF-enriched genes, especially TOX, as molecular markers for histological diagnosis of eMF, which currently is a major diagnostic challenge.

The expression and localization of a novel protein phosphatase inhibitor 2810408A11Rik in mouse testis and sperm

This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in spermatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysis and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408A11Rik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.