Allergy Therapeutics

Allergy Therapeutics click here market aluminium-free SCIT products. “
“Conventional aluminium-containing adjuvants have been used in vaccine formulations for decades but promote poor induction of Th1 or cell-mediated immunity [1] and [2]

and require refrigeration during transportation and storage. Approximately 50% of vaccines are discarded globally, largely due to cold chain disruption [3] and [4]. Therefore, a major objective of vaccine formulation t is to develop a safe, immunogenic composition which addresses the issues of immune bias and stability. Protein-coated microcrystals (PCMCs) are a recent advance in vaccine formulation [5] and have the potential to by-pass the cold chain. Originally developed to stabilise enzymes for

industrial applications [5], [6], [7], [8] and [9], PCMCs are formed by rapid co-precipitation of protein(s) with an amino acid or sugar, producing particles with an inert core microcrystal coated with protein(s) [6], [8] and [9]. Vaccine antigens, loaded onto PCMCs, exhibited much higher resistance to heat stress compared to native antigens [5] and [7]. These reports used PCMC formulations which were instantly soluble in aqueous buffer [5], [6], [7], [8] and [9]. In this study, novel sustained-release PCMCs have been used which are poorly soluble due to modification of their outer surface with sparingly soluble CaP. CaP served as an adjuvant in some early acellular vaccines [10] and [11], and is well-tolerated in man [11], [12], [13], [14], [15] and [16]. CaP also XL184 enhances Th1-biased immunity although this may be antigen-dependent [11], [17] and [18]. Here, the immunogenicity of CaP-modified PCMCs loaded with different model antigens was investigated. DT, a formaldehyde-toxoided antigen [19], [20] and [21], and BSA have been used extensively as model antigens when validating new vaccine formulations [22], [23], of [24] and [25]. The DT preparation was the 2nd international standard

for use in flocculation tests (02/176, NIBSC, UK). CyaA* was purified and characterised as described previously [26], [27] and [28]. BSA was from Sigma and BSA-FITC was from Life Technologies, UK. All reagents were of the highest grade available and were used at rt. The aqueous solution was prepared in endotoxin-free, sterile water (Sigma) and contained 30 mg/ml l-glutamine as the core component of the PCMCs, trehalose and the test antigens, sufficient to give final loadings of 10% and 0.2–0.4%, respectively, in the PCMC preparation. To precipitate PCMCs, 3 ml of the aqueous solution was added drop-wise to 60 ml of rapidly stirred isopropanol and stirring continued for 1 min at 1500 rpm. For CaP-modified PCMCs, the required concentration of NaH2PO4 was included in the aqueous solution and CaCl2 was included in the isopropanol at a 2-fold molar excess compared to NaH2PO4. PCMCs were collected by vacuum filtration onto PVDF hydrophilic 0.

Dans certains cas exceptionnels, il correspond à un syndrome para

Dans certains cas exceptionnels, il correspond à un syndrome paranéoplasique (thymome, cancer pulmonaire) associé à des AC dirigés contre le canal potassique voltage-dépendant.

La myasthénie est systématiquement évoquée devant une forme bulbaire. Le diagnostic repose sur RAD001 le test aux anticholinestérasiques, l’ENMG, le dosage des AC antirécepteurs à l’acétylcholine. Plus de 50 cas de lymphomes associés à un tableau de type SLA sont rapportés. Il peut s’agir d’une atteinte isolée du système nerveux périphérique, mais dans la majorité des cas il existe une atteinte conjointe du NMP et du NMC. Ils justifient la réalisation systématique d’une électrophorèse des protéines, d’une CRP et d’un hémogramme. Une biopsie ostéo-médullaire, un scanner thoraco-abdominal complètent ces premiers examens si besoin. Syndromes paranéoplasiques et cancers associés : l’association d’une SLA et d’un cancer est le plus souvent fortuite. Il peut être justifié de réaliser un dosage d’AC antineuronaux (AC anti-HU), un scanner thoracique, voire thoraco-abdominal, une échographie prostatique ou encore une mammographie devant certaines présentations cliniques (altération

marquée de l’état général, atteinte diffuse du système nerveux, atteinte prédominante du NMC). L’association rare de cas d’atteinte du neurone moteur et de syndrome de Gougerot-Sjögren primitif peut justifier la recherche d’un syndrome sec et le dosage des AC anti-ENA. Certaines maladies infectieuses sont concernées : • infection par le VIH : un selleck inhibitor tableau prenant le masque d’une SLA a été décrit justifiant la réalisation d’une sérologie VIH ; La recherche d’une hyperparathyroïdie est habituelle en raison de rares cas d’amélioration des symptômes de la maladie après normalisation du bilan hormonal. Il ne semble pas exister d’association avec une hyperthyroïdie.

Font aussi partie des diagnostics différentiels, certaines maladies : • métaboliques : gangliosidoses GM2 (dosage de l’hexoaminidase A), adrénoleucodystrophie (dosage des acides gras à très longue chaîne), sclérose combinée de la moelle (vitamine B12 et folates) ; Le diagnostic de myosites à inclusions pourra être évoqué devant un tableau atypique où le déficit moteur prédomine sur les muscles fléchisseurs des PAK6 doigts et les quadriceps. Le diagnostic de certitude est alors apporté par la biopsie musculaire. Le bilan paraclinique fait appel à l’ENMG, l’imagerie et les tests biologiques complémentaires [64]. L’objectif de ces examens est, en complément de l’examen neurologique qui formule les hypothèses, de permettre un diagnostic positif rapide et d’éliminer d’autres affections proches. Il n’existe pas, à ce jour, de guide pratique validé. Il apparaît donc difficile d’imposer ou non la réalisation systématique de certains examens. Le choix des explorations revient au neurologue qui adapte le bilan en fonction du contexte clinique et de son expérience.

g , EC50, ED50, LD50, IC50), and d is the slope at the steepest p

g., EC50, ED50, LD50, IC50), and d is the slope at the steepest part of the curve, also known as the Hill slope. The model HIF inhibitor may be written to represent an ascending sigmoid curve of the type in Fig. 1 or a descending curve, depending on the sign of d. Specifically, positive d values yield ascending curves while negative values yield descending curves. Eq. (1) represents one of a family of Hill equations that have been used to describe specific non-linear relationships under diverse scenarios, including, but not limited to, quantitative pharmacology (Gesztelyi et

al., 2012), ligand binding (Poitevin and Edelstein, 2013 and Siman et al., 2012), plant growth modeling (Zub, Rambaud, Bethencourt, & Brancourt-Hulmel, 2012), and modeling patterns of urban electricity usage (To, Lai, Lo, Lam, & Chung, 2012). Computer programs have been available since the early 1970s to estimate the parameters of different versions of the Hill equation, most of which are specific to fitting kinetic data (Atkins, 1973, Knack and Rohm, 1977, Leone et al., 2005 and Wieker et

al., 1970). None of these uses Eq. (1) specifically, although commercial software exists that can be made to fit the four-parameter logistic curve in Eq. (1) (e.g., GraphPad Prism,; The MiraiBio Group of Depsipeptide price Hitachi Solutions at Eq. (1) can also be fit to data using a computer program written using the open-access language, R, or the Solver Add-in next in Microsoft Excel. In addition, some of these also permit the computation of confidence and prediction bands around the curve. However, the existing tools either require an investment in commercial software, which are also typically opaque to the user

as to the code and algorithms used to generate the results, or require the ability of the user to write computer code in order to accomplish these tasks. A long-term goal of the Call laboratory is to determine the mechanism of action of inhaled anesthetics (IAs), for which Drosophila melanogaster is used as the model system for providing in vivo responses to IAs in the presence of various genetic manipulations. Drosophila represents a good model for working with anesthetics as fruit flies follow the Meyer–Overton rule of anesthetics and display physiological responses to IAs similar to those in humans ( Allada and Nash, 1993 and Tinklenberg et al., 1991). Additionally, flies provide an inexpensive, yet robust model with access to a variety of genetic tools available to answer many scientific questions in vivo. The Call laboratory has recently adapted an apparatus for the quantification of the Drosophila response to IAs ( Dawson, Heidari, Gadagkar, Murray, & Call, 2013). Known as the inebriometer, it was originally designed to quantitatively measure the flies’ response to ethanol vapors ( Weber, 1988).

More screening criteria were listed in Supplementary Fig 1 At t

More screening criteria were listed in Supplementary Fig. 1. At the end of this process 26 individuals from the cohort recruited were defined as authentic non-responders based on producing

MDV3100 anti-HBs levels of less than 10 mIU/ml after having received a total of six doses of vaccine administered over two consecutive rounds of vaccination schedule. DNA samples from 20 of these non-responders were available for use in this study. For comparative purpose, after considering almost the same criteria for screening non-responders, a group of vaccine responders were identified on the basis of having produced anti-HBs levels equal to or more than 100 mIU/ml after having received the standard 3 doses of vaccine. Finally 45 responders were randomly selected and there are no significant differences between the responders and non-responders in age (age range 25–60 for responders vs. age range 30–59 for non-responders, P = 0.0512) and gender (23F/22M for responders vs. 7F/13M for non-responders, P = 0.2291). The detailed demographic data of the Selleckchem Epacadostat 20 non-responders and 45 responders is shown in Supplementary Table 1. Since no peripheral blood mononuclear cells (PBMC) were available from the non-responders and responders, 29 healthy adults who had physical examination in Peking University Third Hospital without evidence of prior HBV

infection were also enrolled for further experiments. This study was approved by the Ethics Committee of the Peking University Health Science Center and all subjects provided signed informed consent. Six TfH associated molecules CXCR5,

ICOS, CXCL13, IL-21, BCL6 and CD40L were selected for SNP analysis. Altogether 24 SNPs within these genes were chosen for the analysis (Supplementary Table 2), according to the following 2 criteria: first, the minor allele frequency (MAF) obtained from NCBI SNP database ( or the SNP browser software 4.0 (Applied Biosystems) should be higher than 10% in the ethnic Han Chinese population. Second, there should be published evidence showing that the enough SNP is associated with some disease. Genomic DNA extracted as previously described was dissolved in sterile double distilled water and stored at −20 °C [4]. SNP genotyping was undertaken by Bioyong Technology using Sequenom MassARRAY technology (Bioyong Technology Co., Beijing, China). Peripheral Blood Mononuclear Cells were isolated using Histopaque-1077 (Sigma, 10771) according to the manufacturer’s instructions and stored at −80 °C. For flow cytometry assays, recovered cells were incubated for 30 min with a cocktail of antibodies that included eFluor450 conjugated anti-CD3 mAb (eBioscience, 48-0038), PE-Cy7 conjugated anti-CD4 mAb (BD, 557852), APC conjugated anti-CD19 mAb (BD, 555415) and PE conjugated anti-CXCR5 mAb (eBioscience, 12-9185). Following incubation the cells were washed with PBS and fixed with 2% paraformaldehyde.

In case of detection of amylase, the starch agar medium plate was

In case of detection of amylase, the starch agar medium plate was flooded with 1% iodine solution, to observe the zone of hydrolysis. The bacterium, 2b, found to produce maximum zone of hydrolysis around the colony on the casein agar medium and on starch agar medium was selected for further study. The isolate was maintained Obeticholic Acid concentration on Horikoshi medium slants (pH 10.0) and stored at 4 °C. The morphological characteristics of the selected isolate 2b obtained

on Horikoshi’s –I (pH 10.0) agar plates were studied. The shape, size and arrangement of the cells were studied in Gram-stained preparations. Endospore staining was carried out according to the method of Schaeffer and Fulton.8 Motility of 12 and 24 h old cells was observed by phase contrast microscopy of hanging-drop preparations. Growth experiments at pH 7–11 were performed on Horikoshi I broth adjusted to various pH Selleckchem Anticancer Compound Library values: pH 7–9 (adjusted by adding NaHCO3) and pH 10–11 (adjusted by adding). Growth at various NaCl concentrations (2–10%) and at various temperatures (4–55 °C) was investigated in Horikoshi I broth (pH 10.0). Acid production from carbohydrates was determined by the method of using thymol blue instead of bromothymol blue at pH 10.0 9 and 10. Physiological and biochemical tests such as indole production from tryptophan, methyl-red and Voges–Proskauer

tests, Simmons’ citrate utilization test, catalase and oxidase activity, urea hydrolysis, production of H2S from cysteine, nitrate reduction to nitrite, hydrolysis of casein, gelatin and starch were examined

according to Smibert and Krieg.11 The taxonomic status of the selected bacterium 2b was identified following the criteria laid down by Bergey’s Manual of Systematic Bacteriology.12 The identification was further confirmed by Microbial Type Culture Collection Center and Gene Bank (MTCC), Institute of Microbial Technology, (IMTECH), Chandigarh, India. The 16S RNA gene sequencing of the isolate was performed by National Center for Cell Sciences (NCCS), Pune, India. The purified PCR product of 16sr RNA was sequenced using ABI Prism. The sequence obtained was BLAST searched Histone demethylase and compared with sequences of other closely related members of genus Bacillus retrieved from GenBank database. Phylogenetic tree was constructed from 16S rRNA gene sequences of members of genus Bacillus using neighbour-joining method. 13 The analysis involved 39 nucleotide sequences of genus Bacillus. The sequence so obtained was taken up for running NCBI BLAST against nonredundant nucleotide database using megablast algorithm for getting homologous sequences14 and 15. Sequences showing a relevant degree of similarity were imported into the CLUSTAL W program16 and multiple sequence alignment was performed. Phylogenetic trees were constructed by different treeing algorithms: neighbour-joining,13 maximum parsimony tree17 and maximum-likelihood18 and UPGMA method19 using MEGA5.

These women were older, and were

These women were older, and were high throughput screening assay not all in school and inequalities in coverage have been observed and reported [21]. Bowyer et al. quantitatively assessed the knowledge and awareness of HPV and the vaccine, amongst schoolgirls who had already been offered the HPV vaccine in the targeted UK vaccination programme [23]. In this cohort, knowledge about HPV infection was relatively

low, and only 53.1% participants were aware that HPV could cause cervical cancer. Approximately half of the participants were aware that cervical screening was still required after HPV vaccination. In our data analyses, although the women studied were from the catch-up arm of the programme, we observed approximately half of the vaccinated cohort attending cervical screening (55.2%). Analysis of factors potentially affecting uptake

of health services available for primary cervical cancer prevention VE 822 in the UK, highlighted that women who originate from more socially deprived areas are less likely to engage with the services available. Moreover, 9758/30,882 (31.6%) had neither attended for screening nor received the HPV vaccine. However, although social deprivation affected the initial engagement, once women engaged, at least in this age group, there was no significant difference in clinical outcome. Cervical cancer rates are higher in women from more socially deprived backgrounds [24]. However, data from

our study suggests that this is a consequence of women from more socially deprived areas not Dipeptidyl peptidase engaging with the current primary cervical cancer prevention strategies in the UK. In women offered HPV vaccination through the catch-up arm of the programme, this study shows a protective effect with a reduction in cytological abnormalities from 16.7% in unvaccinated women to 13.9% in vaccinated women. However, the level of abnormalities detected in the vaccinated women is still relatively high, potentially reflecting acquisition of the virus prior to vaccination. This data suggests that the catch-up arm of the vaccination programme has not had a substantial protective effect and a higher impact on cytological abnormalities is anticipated in the target group, who may not have been exposed to the virus prior to vaccination. Women who have chosen to receive the HPV vaccination and attend for cervical screening may be more health conscious, and this may be reflected in their sexual behaviours. It is therefore possible that they may be less likely to become infected with HPV, accounting for the reduction seen in the proportion of cytological abnormalities.

During a 1-h scan, we observed that GF primarily affected the pha

During a 1-h scan, we observed that GF primarily affected the phase between the initial rapid washout of the peptide after renal uptake and the final retention of peptide. This process was presented as slow decline in renal radioactivity (an indication of strong tubular reabsorption) in the absence of GF, which was replaced by relatively faster decline of the

radioactivity in the presence of GF, suggesting impairment of tubular reabsorption. Dynamic PET images clearly showed that radioactivity was predominantly found in the cortex of Imatinib in vivo the kidneys in control mice as early as 20–25 min p.i. and was retained for long periods thereafter. In addition to reduced radioactivity in the Gefitinib renal cortex, radioactivity in mice co-injected with GF could be clearly visualized in the renal pelvic area even up to 35–40 min p.i., which is indicative of the active transit of the radioactivity into the urinary bladder. Co-injection of GF resulted in increase in urinary bladder radioactivity, which corresponded to a decrease in total renal radioactivity, indicating that

decreased renal uptake was due to the blockade of renal reabsorption of 64Cu-cyclam-RAFT-c(-RGDfK-)4, the predominant radioactive component detected in the urine samples of mice with or without co-injection of GF ± Lys at 1 h p.i. In addition, neither PET nor biodistribution studies showed the effect of GF on the blood clearance of 64Cu-cyclam-RAFT-c(-RGDfK-)4, science and in vivo metabolite analysis did not reveal the effect of GF on the metabolism of 64Cu-cyclam-RAFT-c(-RGDfK-)4. Taken together, these data strongly suggest that co-injection with GF can result in reduced renal accumulation of 64Cu-cyclam-RAFT-c(-RGDfK-)4, which is possibly achieved through suppression of tubular reabsorption. Megalin, a multiligand receptor expressed exclusively on the apical membrane of proximal tubular cells, can bind to a variety of structurally distinct proteins, peptides, drugs, and other molecules [24], [25], [26] and [27]. Megalin-mediated endocytosis has been reported to play a significant

role in the renal reabsorption of several radiolabeled peptides irrespective of their molecular targets, molecular weights, numbers of amino acid residues (AARs), or numbers of charged AARs (CAARs) [24] and [26]. Based on these studies, we consider that megalin may also be involved in the renal reabsorption of 64Cu-cyclam-RAFT-c(-RGDfK-)4. The number of CAARs in a radiolabeled peptide has been shown to be related to its renal uptake levels [26] and [28]. Gotthardt et al. reported a positive relationship between the renal uptake levels of radiolabeled peptides and the numbers of CAARs (Glu, Lys, Asp, or Arg) contained in the peptides in the following order: exendin (10 CAARs) > minigastrin (7 CAARs) > octreotide (1 CAARs) > bombesin (0 CAARs) [28].

This article reviews the role of coronary computed tomography (CT

This article reviews the role of coronary computed tomography (CT) angiography in the assessment of coronary risk, and its usefulness in the emergency department in facilitating appropriate disposition decisions. Also discussed is coronary artery calcification incidentally found on CT scans when done for indications such as evaluation of pulmonary embolism or lung cancer. The evidence base and clinical applications for both techniques are described, together with cost-effectiveness and radiation exposure considerations. Ozlem Soran Medically refractory angina pectoris (RAP) is defined by presence of severe angina with objective evidence of ischemia and failure to relieve

symptoms with coronary revascularization. Medication and invasive revascularization are the most common approaches for selleck chemical treating coronary artery disease (CAD). Although symptoms are eliminated or alleviated by these invasive approaches, the disease and its causes are present after treatment. New treatment approaches are needed to prevent the disease from progressing and symptoms from recurring. External enhanced counterpulsation therapy provides a treatment modality in the management of CAD and can complement invasive revascularization procedures. Data support that it should be considered as a first-line treatment of RAP. Doron Aronson and Elazer R. Edelman Diabetes mellitus (DM) is a major GSK1120212 purchase risk factor for cardiovascular

disease. Near-normal glycemic control does not reduce cardiovascular events. For many patients with 1- or 2-vessel coronary artery disease, there is little benefit from any revascularization procedure over optimal medical therapy. For multivessel coronary disease, randomized trials demonstrated the superiority of coronary artery bypass grafting over multivessel percutaneous coronary intervention in patients with treated DM. However, selection of the optimal myocardial revascularization strategy requires a multidisciplinary team approach (‘heart team’). This review summarizes the current evidence regarding the effectiveness of various medical

therapies and revascularization strategies in patients with DM. A. Pieter Kappetein, Nicolas M. van Mieghem, and Stuart J. Head Coronary artery bypass grafting (CAGB) is superior to percutaneous coronary intervention (PCI) in reducing mortality in certain patients unless and improving the composite end points of angina, recurrent myocardial infarction, and repeat revascularization procedures. However, CABG is associated with a higher perioperative stroke risk. For patients with less complex disease or left main coronary disease, PCI is an acceptable alternative to CABG. Lesion complexity is an essential consideration for stenting, whereas patient comorbidity is an essential consideration for CABG. All patients with complex multivessel coronary artery disease should be reviewed by a heart team including a cardiac surgeon and interventional cardiologist. Shilpa Agrawal, Puja K.

Copper proteins have diverse roles in biological electron transpo

Copper proteins have diverse roles in biological electron transport

and oxygen transportation, processes that exploit the easy interconversion of Cu(I) and Cu(II).1 In the field of bioinorganic chemistry, the development of reagents RG7204 in vitro that can specifically recognize and cleave DNA under physiological conditions via oxidative and hydrolytic mechanisms has been attracting a great interest.2, 3 and 4 In DNA strand scission chemistry, the intermediates responsible for DNA cleavage, active oxygen species, particularly the hydroxyl radical and perhaps their adducts with metal complexes, have been directly or indirectly demonstrated.5, 6, 7, 8, 9 and 10 A number of metal complexes have been reported as anticancer agents in the literature; however, copper, iron, cobalt and nickel complexes are also regarded as promising alternatives to platinum complexes, particularly biocompatible copper(II) complexes

that bind MLN2238 cell line and cleave both DNA and protein under physiological conditions and on the use of these synthetic nucleases and proteases for potential anticancer drug development. Copper complexes, which possess biologically accessible redox potentials and demonstrate high nucleobase affinity, are potential reagents for cleavage of biomolecules. Sadler and co-workers have reported mixed ligand bis(salicylato)copper(II) complexes with diimines as co-ligands exhibit cytotoxic and antiviral activities.11 Very recently, Reedijk and co-workers

have reported [CuII(pyrimol)Cl] complex, which shows efficient self-activated DNA cleavage and cytotoxic effects on 11210 murine leukaemia and A2780 human ovarian carcinoma cell lines. The biological studies of metal complexes highlighted the potential of antioxidant activity of copper(II) complex with bioactive ligand.12 and 13 In the present work, we synthesized and characterized a copper(II) complex of the ligand 1-(1H-benzimidazol-2-yl)-N-(tetrahydrofuran-2-ylmethyl)methanamine and also the DNA cleavage and in vitro-antioxidant activities were explored. The synthetic route for the present complex is shown in Scheme 1. 1-(tetrahydrofuran-2-yl)methanamine, copper(II) chloride, agarose (molecular biology grade) and ethidium bromide were procured from Sigma Aldrich, USA and used as received. Other materials like sodium borohydride and solvents like methanol, Calpain acetonitrile and dichloromethane were of reagent grade. Benzimidazole carbaldehyde was prepared using published procedure.14 Buffers were prepared using deionised and sonicated triple distilled water. Tris (hydroxymethyl) aminomethane–HCl (Tris–HCl) buffer (pH, 7.2) was used for DNA cleavage studies. UV–visible spectrum of the complex was recorded on a Perkin–Elmer Lambda 35 double beam spectrophotometer at 25 °C. Electron paramagnetic resonance spectrum of the copper(II) complex was obtained on a Varian E 112 EPR spectrometer.

To differentiate monocytes into immature DCs 250 U/ml granulocyte

To differentiate monocytes into immature DCs 250 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF) and 100 U/ml IL-4 (Invitrogen) was selleck products added. Medium was refreshed after 3 days. DC were incubated for 48 h at 37 °C in RPMI 1640 containing 500 U/ml GM-CSF with OVA (highest

concentration 5 μg/ml), either free or encapsulated into liposomes with and without PAM or CpG (highest concentration 10 μg/ml), keeping the lipid:OVA:TLR ligand ratio 50:2:1 (w/w). OVA, OVA liposomes and mixtures of OVA with PAM or OVA with CpG were used as controls and LPS (100 ng/ml, Invivogen) was added as a positive control. Cells were washed 3 times with PBS containing 1% (w/v) bovine serum albumin and 2% (v/v) FCS and incubated for 30 min with a mixture of 20× diluted anti-HLADR-FITC, anti-CD83-PE and anti-CD86-APC (Becton Dickinson) in the dark at 4 °C. Cells were washed and the expression of MHCII, CD83 and CD86 was quantified using flow cytometry (FACSCanto II, Becton Dickinson) relative to LPS, assuming 100% maturation for LPS-treated DC. Live cells were gated based on forward and side scatter. Groups of 8 mice were immunised with the OVA-loaded liposomes with and without PAM or CpG by ID injection into the abdominal skin as described

previously [30]. Besides the liposomes, solutions of OVA or OVA with PAM or CpG in PBS were injected and subcutaneous (SC) injection of OVA served as a control. The mice were vaccinated twice with three weeks intervals

with a dose of 5 μg selleck screening library OVA and 10 μg PAM or CpG in a total volume of 30 μl. To maintain this Terminal deoxynucleotidyl transferase ratio between antigen and immune potentiator, liposomes used for the immunisation study were not filtered to remove free antigen and TLR ligand. Blood samples were collected from the tail vein 1 day before each immunisation. Three weeks after the last vaccination the mice were sacrificed. Just before euthanasia total blood was collected from the femoral artery. Afterwards the spleens were removed. Blood samples were collected in MiniCollect® tubes (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) till clot formation and centrifuged 10 min at 10,000 × g to obtain cell-free sera. The sera were stored at −80 °C until further use. OVA specific antibodies (IgG, IgG1 and IgG2a) in the sera were determined by sandwich ELISA as described previously [30]. Briefly, plates were coated overnight with 100 ng OVA/well. After blocking, two-fold serial dilutions of sera from individual mice were applied to the plates. HRP-conjugated antibodies against IgG, IgG1 or IgG2a were added and detected by TMB. Antibody titres were expressed as the reciprocal of the sample dilution that corresponds to half of the maximum absorbance at 450 nm of a complete s-shaped absorbance-log dilution curve.