HIV Med 2005; 6(Suppl 2): 96–106 5  Brook G, Main J, Nelson M et

HIV Med 2005; 6(Suppl 2): 96–106. 5  Brook G, Main J, Nelson M et al. British HIV Association guidelines for the management of coinfection with HIV-1 and selleck chemicals llc hepatitis B or C virus 2010. HIV Med 2010; 11: 1–30. 6  Tedder RS, Rodger AJ, Fries L et al. The diversity and management of chronic hepatitis B virus infections in the United Kingdom: a wake-up call. Clin Infect Dis 2013; 56: 951–960. 7  Kim BK, Revill PA, Ahn SH. HBV genotypes: relevance to natural history, pathogenesis and treatment of chronic hepatitis B. Antivir Ther 2011; 16: 1169–1186. 8  Tanwar S, Dusheiko

G. Is there any value to hepatitis B virus genotype analysis? Curr Gastroenterol Rep 2012; 14: 37–46. 9  Flink HJ, van Zonneveld M, Hansen BE GSK1120212 purchase et al. Treatment with peg-interferon alpha-2b for HBeAg- positive chronic hepatitis B: HBsAg loss is associated with HBV genotype. Am J Gastroenterol 2006; 101: 297–303. 10  Soriano V, Mocroft A, Peters L et al. for EuroSIDA. Predictors of hepatitis B virus genotype and viraemia in HIV-infected patients with chronic hepatitis B in Europe. J Antimicrob Chemother 2010; 65: 548–555. 11  Hepatitis B (chronic): Diagnosis and management

of chronic hepatitis B in children, young people and adults. National Clinical Guideline Centre, 2013. Final draft for consultation. Available at (accessed May 2013). 12  Price H, Bansi CYTH4 L, Sabin CA et al. for the UK Collaborative HIV Cohort Hepatitis Group, Steering Committee. Hepatitis B virus infection in HIV-positive individuals in the UK collaborative HIV cohort (UK CHIC) study. PLoS One 2012; 7: e49314. 13  French AL, Lin MY, Evans CT et al. Long-term serologic follow-up of isolated hepatitis B core antibody in HIV-infected and HIV-uninfected women. Clin Infect Dis 2009; 49: 148–154. 14  Sheng WH, Kao JH, Chen PJ et al. Evolution of hepatitis B serologic markers in HIV-infected patients receiving highly

active antiretroviral therapy. Clin Infect Dis 2007; 45: 1221–1229. 15  Gandhi RT, Wurcel A, Lee H et al. Response to hepatitis B vaccine in HIV-1 positive subjects who test positive for isolated antibody to hepatitis B core antigen: implications for hepatitis B vaccine strategies. J Infect Dis 2005; 191: 1435–1441. 16  Wiedman M, Liebert UG, Oeson U et al. Decreased immunogenicity of recombinant hepatitis B vaccine in chronic hepatitis C. Hepatology 2000; 31: 230–234. 17  Leroy V, Bourliere M, Durand M et al. The antibody response to hepatitis B virus vaccination is negatively influenced by the HCV viral load in patients with chronic hepatitis C: a case-control study. Eur J Gastroenterol Hepatol 2002; 14: 485–489. 18  Navarro JF, Teruel JL, Mateos ML, Marcen R, Ortuno J. Antibody level after hepatitis B vaccination in hemodialysis patients: influence of hepatitis C infection. Am J Nephrol 1996; 16: 95–97.

FMD changes rapidly in response to beneficial or noxious stimuli,

FMD changes rapidly in response to beneficial or noxious stimuli, making it a useful tool for

assessing the immediate impact of interventions on the vasculature. Studies in healthy volunteers have used changes in FMD as an end-point for vaccine assessment. We have previously shown that vaccination adversely affects FMD, and this effect is mitigated by pretreatment with statins [14]. Consistent with and extending selleck compound previously published data, the novel influenza A/H1N1 vaccine significantly impaired FMD in HIV-infected patients, and this effect lasted for at least 48 h. The clinical implications of our study pertain to cardiovascular risk in HIV-infected patients. Viraemia represents a low-grade stimulus; vaccination selleck inhibitor may be superimposed as an acute insult, thus creating a highly pro-inflammatory milieu accompanied by worsening endothelial function. In the presence of an already dysfunctional endothelium, as is the case for HIV infection [17], the combination could result in untoward events. However, no short-term adverse cardiovascular events

have been reported following vaccinations in the setting of HIV infection [22]. In the general population, conflicting data exist regarding the potential of seasonal vaccination to defer acute myocardial infarction [23,24]. A number of studies have linked influenza infection with elevated cardiovascular risk [25]. Such a link has been found for the seasonal influenza strains

in the general population; indeed, the prevalence of acute myocardial infarction rises following an influenza infection. In the light of such reports, seasonal influenza infection has been acknowledged as a novel risk factor for cardiovascular events [26]. However, no such data exist on the pandemic H1N1 influenza strain, or for HIV-infected patients [27]. Regarding vaccination against the seasonal strains of influenza, it has been reported Palbociclib purchase that vaccination reduces the risk of myocardial infarction in the general population [28], as well as in patients with coronary heart disease [29]. To date, however, there is a paucity of studies regarding vaccination in HIV-infected patients [30]. Apart from providing clinical insights into the effects of vaccination in a high-risk group, the combination of HIV infection and the novel influenza A/H1N1 vaccine in our study has utility as a new model for studying endothelial responses to vaccination. Vaccines may not be equal with respect to their inflammatory and endothelial effects. The inclusion of different antigens (bacterial or viral) and the use of booster substances may result in different degrees of vascular reactivity. However, it should be noted that people with different immunological backgrounds may respond in different ways to vaccination, and our results cannot be directly extrapolated to the general population.

The TDF is a useful tool for grouping adherence barriers; patient

The TDF is a useful tool for grouping adherence barriers; patients have endorsed the relevance of literature-identified adherence barriers and provided useful anecdotes to further inform practice. Non-adherence to prescribed Small molecule library mouse medicines is of notable concern and a priority for pharmacy practice research. Whilst there is ample literature to report barriers to medication adherence in chronic conditions, a recent synthesis

of this literature is lacking, as is a cohesive, theory-based strategy for grouping medication adherence barriers. The Theoretical Domains Framework (TDF) is a composite of health psychology theory, designed to offer a structured approach for exploring the determinants of behaviour. Within this framework, key determinants of behaviour are grouped into behavioural domains such as skills, beliefs about capabilities and emotions.1 Medication ICG-001 order adherence barriers were identified through a literature review and mapped to the behavioural domains of the TDF. University ethical approval was granted for the consultation exercises. Members of the public taking medicines for the prevention of cardiovascular disease (CVD) were purposively sampled from volunteers responding to a university-based recruitment strategy using

internal communication systems and social media that extended to the local community. The participants discussed the relevance of the literature-identified adherence barriers and the mapping of these to the TDF in two consultation exercises. These consultations Methocarbamol were audio-recorded, transcribed, and themed according to the behavioural domains of the TDF. Sixty medication adherence barriers were discussed across the following TDF behavioural domains; knowledge, skills, memory attention

and decision making processes, social influences, environmental constraints, emotions, motivation and goals, beliefs about consequences, beliefs about capabilities and the newly created goal conflicts. Two consultation exercises were undertaken, with five participants in the first and nine in the second. All participants were prescribed at least one medication for the prevention of CVD; the median number of medicines prescribed was 2 (IQR = 2–5). Eight (57%) participants were male and the median age was 66 (52 to 74) years. Participants understood the concept of grouping the adherence barriers according to the theoretical domains of the TDF and could relate the majority of the literature-identified barriers to their own experiences. The exceptions to this were barriers relating to fear of discrimination (emotions behavioural domain) and feeling embarrassed by taking medicines (social norms behavioural domain. Patient narratives provided an enhanced understanding about the ways in which medication adherence barriers can manifest.

Patients with autoimmune, vascular, biliary or tumoral liver dise

Patients with autoimmune, vascular, biliary or tumoral liver disease were excluded from the study. Liver fibrosis was staged according to the Scheuer system as follows: No or mild fibrosis (no fibrosis or portal fibrosis without septa; F0 and F1), moderate fibrosis (portal fibrosis and few septa; F2), severe fibrosis (numerous septa with architectural

distortion, without cirrhosis; F3), and cirrhosis (F4) [20]. Liver biopsies that were AZD3965 supplier <15 mm long (except in the case of cirrhosis) were excluded from the histological analysis. The length of each liver biopsy specimen was established. A single experienced pathologist staged the liver biopsies. The serum levels of TIMP-1 and MMP-2 were determined in frozen sera stored at −80 °C, which had not previously been thawed. Commercial assays based on the quantitative sandwich enzyme immunoassay technique were used to measure serum

click here levels of TIMP-1 (Quantikine®; Human TIMP-1 Immunoassay; R&D Systems, Minneapolis, MN, USA) and MMP-2 (Quantikine®; Human/Mouse/Rat MMP-2 (total) Immunoassay; R&D Systems). These assays were carried out following the manufacturer’s instructions. Briefly, a monoclonal antibody specific for TIMP-1 or MMP-2 was pre-coated onto a microplate. Standards and samples were pipetted into the wells and any TIMP-1 or MMP-2 present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TIMP-1 or MMP-2 was added to the the wells. Following a

wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and colour developed in proportion to the amount of TIMP-1 or MMP-2 bound in the initial step. The colour development was stopped and the intensity of the colour was measured. The AST to platelet ratio index (APRI) was calculated by dividing the AST concentration (IU/L), expressed as the number of times above the upper limit of normal (ULN), by the platelet count (109 cells/L): AST (/ULN) × 100/platelet count. This index has been validated in HIV/HCV-coinfected patients [3,4]. If APRI is ≥1.5, patients can be classified as having significant fibrosis [fibrosis stage (F) ≥2], with a positive predictive value (PPV) of 87% [3]. The low cut-off of APRI <0.5 was inaccurate to exclude F≥2 [3]. The main outcome variables were the detection of F≥2 and of F4 with a combination of variables at the date of liver biopsy.

These features make NDH-2 a promising target for the development

These features make NDH-2 a promising target for the development of new drug candidates. High-resolution structural data and deeper understanding of phenothiazine action may facilitate structure-based design of small-molecule NDH-2 inhibitors with improved efficacy and selectivity. Diarylquinolines represent a novel class of antimycobacterial drugs with strong in vitro and in vivo activity against different mycobacterial species (Andries et al., 2005; Ji et al., 2006). Diarylquinolines block ATP synthesis and cause a

decrease of cellular ATP levels (Koul et al., 2007). As the bacterial ATP stores are depleted over a period of time, subsequently pronounced bacterial killing is observed (Koul et al., 2008). Diarylquinolines specifically interact with the oligomeric transmembrane subunit c of mycobacterial ATP synthase (Koul et al., 2007, see also Fig. 2). During enzymatic catalysis, this oligomeric subunit, together with subunits ɛ SAR245409 cost and γ, rotates relative to subunits α3β3δab and in this way couples proton flow to the synthesis of

ATP (Boyer, 1993; Junge et al., 1997). Protons enter from the periplasmic space via an entry channel in subunit a and are then transferred to an essential acidic residue in the membrane-spanning part of subunit c (Fig. 2). After a close to 360° rotation of the cylindrical subunit c oligomer relative to subunit a, the protons are released on the cytosolic side of the membrane via an exit channel in subunit a (Vik & Antonio, 1994; Diez Dabrafenib mw et al., 2004). Mutagenesis studies indicate that diarylquinoline lead compound TMC207 binds to the central region of subunit c, close to the essential acidic residue (Koul

et al., 2007). TMC207 may compete with protons for binding to subunit c or may alternatively interfere with the extensive conformational changes of this subunit during catalysis. Whereas typical inhibitors SSR128129E of ATP synthase subunit c, such as dicyclohexyl-carbodiimide and oligomycin, are not selective and highly toxic (Matsuno-Yagi & Hatefi, 1993; Wallace & Starkov, 2000; Amacher, 2005), TMC207 displays a surprising selectivity, with only an extremely low effect on human ATP synthesis (Haagsma et al., 2009). Although several residues of subunit c are reported to modulate diarylquinoline sensitivity (Koul et al., 2007), the molecular basis for the observed selectivity needs to be further investigated. No high-resolution structure is available for mycobacterial ATP synthase or its subunits, and structural models for mycobacterial subunit c have only been built based on the known structure of the homologous subunit from E. coli, Ilyobacter tartaricus or Bacillus PS3 (de Jonge et al., 2007; Koul et al., 2007). High-resolution structural data for mycobacterial subunit c and biochemical investigations on drug/target interaction would help to explain drug selectivity and would provide input for docking studies to design new compound derivates.

Mutations in 23S rRNA gene at positions 2058 and 2059 are most co

Mutations in 23S rRNA gene at positions 2058 and 2059 are most commonly associated with macrolide resistance Palbociclib cell line in many species including M. gallisepticum (Vester & Douthwaite, 2001; Wu et al., 2005). Not unexpectedly, M. gallisepticum mutants with the A2058G or the A2059G mutation exhibited cross-resistance to

erythromycin, tilmicosin and tylosin in this study. This finding is important as macrolides are also common drugs for the treatment of M. gallisepticum infection. In conclusion, our study showed that mutations in 23S rRNA gene were responsible for resistance to pleuromutilin antibiotics in M. gallisepticum. Some of these mutations led to cross-resistance to other classes of antibiotics including macrolides, lincosamides and phenicols. Notably, no mutations in ribosomal protein L3 were found in this study. This is the first report of pleuromutilin resistance mechanisms in Mycoplasma spp. Further study should be carried out to investigate whether clinical isolates of M. gallisepticum NVP-AUY922 order also contain these mutations in 23S rRNA gene. If so, the cross-resistance mediated by 23S rRNA gene mutations should be considered in the treatment of M. gallisepticum

infection. We wish to thank Dr Jin Zhu (University of Canberra, Australia) for suggested revisions of the manuscript. This study was supported by a grant from the National Natural Science Foundation of China (No. 30571401) and the Program for Chang Jiang Scholars and the Innovative Research Team at the University of China (No. IRT0866). “
“Autophagy is a degradation system in which cellular Montelukast Sodium components are digested via vacuoles/lysosomes, and involved in differentiation in addition to helping cells to survive starvation. The autophagic process is composed of several steps: induction of autophagy, formation of autophagosomes, transportation to vacuoles, and

degradation of autophagic bodies. To further understand autophagy in the filamentous fungus Aspergillus oryzae, we first constructed A. oryzae mutants defective for the Aoatg13, Aoatg4, and Aoatg15 genes and examined the resulting phenotypes. The ΔAoatg13 mutant developed conidiophores and conidia, although the number of conidia was decreased compared with the wild-type strain, while conidiation in the ΔAoatg4 and ΔAoatg15 mutants was not detected. The ΔAoatg15 mutants displayed a marked reduction of development of aerial hyphae. Moreover, autophagy in these mutants was examined by observation of the behavior of enhanced green fluorescent protein (EGFP)–AoAtg8.

All had fasting low-density lipoprotein (LDL) cholesterol ≤ 130mg

All had fasting low-density lipoprotein (LDL) cholesterol ≤ 130mg/dL. Seventy-eight per cent of patients were men and 65% were African-American. Median (interquartile range) age and CD4 count were 47 (43, 52) years and 648 (511, 857) cells/μL, respectively. All had HIV-1 RNA < 400 HIV-1 RNA copies/mL. Mean CCA-IMT BIBW2992 manufacturer was correlated with log-transformed CD8+CD38+HLA-DR+ percentage (r = 0.326; P = 0.043), and concentrations of interleukin-6 (r = 0.283; P = 0.028), soluble vascular cell

adhesion molecule (sVCAM; r = 0.434; P = 0.004), tumour necrosis factor-α receptor-I (TNFR-I; r = 0.591; P < 0.0001) and fibrinogen (r = 0.257; P = 0.047). After adjustment for traditional cardiovascular disease (CVD) risk factors, the association with TNFR-I (P = 0.007) and fibrinogen (P = 0.033) remained significant. Subjects with plaque (n = 22; 37%) were older [mean (standard deviation) 51 (7.7) vs. 43 (9.4) years, respectively; P = 0.002], and had

a higher CD8+CD38+HLA-DR+ percentage [median (interquartile range) 31% (24, 41%) vs. 23% (20, 29%), respectively; P = 0.046] and a higher sVCAM concentration [mean (standard deviation) 737 (159) vs. 592 (160) ng/mL, respectively; P = 0.008] compared with those without plaque. Pro-inflammatory monocyte subsets and serum markers of monocyte activation (soluble CD163 and soluble CD14) were not associated with CCA-IMT or plaque. Participants in SATURN-HIV have a high level of inflammation and immune activation that is associated with subclinical vascular disease despite low serum LDL cholesterol. “
“The incidence of sexually transmitted hepatitis C virus (HCV) reinfection is on the rise in HIV-infected men who have sex with men (MSM). Data on natural history of acute

hepatitis C and possible factors associated with spontaneous clearance are limited. The Casein kinase 1 aim of this study was to analyse the outcome of HCV reinfections in HIV-positive MSM. A retrospective analysis was carried out on patients with more than one sexually acquired HCV infection who were diagnosed at four major German HIV and hepatitis care centres. Reinfection was defined by genotype or phylogenetic clade switch, detectable HCV RNA after a sustained virological response (SVR) or after spontaneous clearance (SC). In total, 48 HIV-positive MSM were identified with HCV reinfection, among them 11 with a third episode and one patient with four episodes. At the first episode, 43 and five patients had an SVR and SC, respectively. The second episode was accompanied by a genotype switch in 29 patients (60%). Whereas 30 and nine patients showed an SVR and SC, respectively, eight patients developed chronic hepatitis. Neither HCV genotype switch nor interleukin-28B genotype was associated with SC. However, SC rates at the second episode were higher for patients with SC at the first episode compared with patients without SC (60 vs. 14%, respectively; P = 0.03).

, 1963) It consists of two domains; a hydroxylase N-terminal dom

, 1963). It consists of two domains; a hydroxylase N-terminal domain with one molecule of noncovalently bound PQQ and Ca2+ at its active site as cofactors and a cytochrome c C-terminal binding domain with one covalently bound molecule of c-type haem which acts as an electron acceptor following lupanine dehydration (Hopper et al., 2002). Periplasmic targeting of the recombinant LH enzyme in Escherichia coli requires the co-expression of cytochrome c maturation factors and complex post-translational modifications that include signal peptide processing, covalent haem attachment to the C-terminal cytochrome c domain and putative disulphide bond formation

PD0325901 nmr (Stampolidis et al., 2009). Sequence analysis

with Clustal W (Larkin et al., 2007) reveals many common features of LH to members of the quinohaemoprotein family such as methanol dehydrogenase from Methylobacterium extorquens and particularly, ethanol dehydrogenase (EDH) from Comamonas testosteroni (Fig. 1). Some of the highly conserved residues among quinohaemoproteins involved in PQQ binding and at the active site of the enzyme are present Selleckchem Ku 0059436 in LH as is the invariant amino acid, Trp, which forms the floor of the active site cavity (Anthony, 1996; Hopper & Kaderbhai, 2003). In quinohaemoproteins, PQQ is usually sandwiched between a disulphide bond formed by two neighbouring Cys (Chen et al., 2002), for example, in methanol dehydrogenase 103,104Cys (Afolabi et al., 2001) and ethanol dehydrogenase 116,117Cys (Mennenga et al., 2009). The role of this bond is still a mystery. One hypothesis is that the disulphide bridge could potentially serve as an intraprotein redox centre, acting as a functional switch by relaying electrons from PQQ to the terminal acceptor in a similar manner to ferredoxin:thioredoxin reductase (Dai et al., 2000), glutathione reductase and lipoamide

dehydrogenase (White et al., 1993). A second theory claims that the bond could have a structural role for proper positioning of PQQ within the active site of the enzyme (Oubrie et al., 2002). However, LH possesses, in total, four Cys residues, two are part of the cytochrome c motif (586Cys and 589Cys), and the remaining two are separated by Methamphetamine 18 amino acids (124Cys and 143Cys). In this study, we attempted to establish the presence of the disulphide bond using chemical means and role in recombinant LH using site-directed mutagenesis with 143CysSer and 124,143CysSer mutations in E. coli. All chemicals were purchased from Sigma, Qiagen Ni-NTA agarose from Qiagen, and electrophoresis reagents were obtained from Bio-Rad and BDH (UK). Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs and Promega (UK). Escherichia coli TB1 and pINK-LH-His4 construct were from Dr M. A. Kaderbhai Laboratory.

2c) No cleavage was observed when the XerSY314F mutant was used

2c). No cleavage was observed when the XerSY314F mutant was used instead of the wild-type protein (data not shown). The vector pBEA756 possesses both gram-positive thermosensitive (Ts) and ColE1 replication origins. An internal fragment of the S. suis xerS gene was generated by PCR and cloned into this vector, generating the plasmid pBEA756XerCint. This plasmid was then successfully introduced into S. suis by electroporation

as described in section ‘Growth conditions and DNA manipulations’. At the restrictive temperature (37 °C), homologous recombination events were selected for by maintaining growth in the presence of kanamycin. Torin 1 molecular weight A single crossover event between the cloned xerS gene on the plasmid and the chromosomal copy of xerS resulted in the inactivation of the xerS gene, which was confirmed by PCR and by Southern blot (data not shown). Microscopic analysis of xerS mutant cells showed a significant increase in average chain length, with most of wild-type cells being 5–10 cells long, while mutants were more

than 10 cells long; in addition, extremely long chains, containing more than 30 cells, were also observed (Fig. 3). The re-introduction of a functional xerS with pGXerCF (pGhost9) restored the wild-type phenotype (data not shown). In this report, we described the purification and inactivation of the S. suis xerS gene and its MBP-fused product. The S. suis XerS recombinase was overexpressed and purified as a maltose-binding of protein fusion, as previous work with XerCD recombinases has indicated that the N-terminal MBP moiety has no significant effect on Xer binding, cleavage BI 6727 solubility dmso or strand transfer activity (Blakely et al., 1997, 2000; Neilson et al., 1999; Villion & Szatmari,

2003). The difSL site was located about 50 bp before the start of the xerS coding region, as was found for most of the lactococci and streptococci (Le Bourgeois et al., 2007). In addition, XerS of S. suis displays 70% identity and 82% similarity to XerS of Lactococcus lactis (Le Bourgeois et al., 2007). Specific binding of difSL was detected at MBP-XerS concentrations of 3.43 nM and above, in the presence of a 1000-fold molar excess of poly dIdC competitor (Fig. 1a). The observation of more than one complex suggests that MBP-XerS is binding to both half-sites of difSL, which is consistent with other systems using one recombinase like Flp and Cre. Binding to the left half-site was detected, while virtually no retarded bands were visible in reactions on the right half-site (Fig. 1b,c), in agreement with results found by Nolivos et al. (2010) on the lactococcal difSL site. The faster migrating bands correspond to the binding of a single XerS monomer on the DNA, while the slower migrating forms correspond to the binding of two or more XerS protomers on the DNA. The additional retarded complexes seen with the difSL left half-site are most likely additional monomers binding to the complex via protein–protein interactions.

The calibrated

The calibrated this website standard serum 89-SF was absorbed with 10 μg/mL CWPS. At least two internal controls were added to each plate. For serotypes 14 and 19F, a high-titre and a low-titre serum sample from

pneumococcal vaccine responders were used as internal controls, respectively. For serotype 23F, two high-titre and two low-titre serum samples from pneumococcal vaccine responders were used as internal controls. The coefficients of variation (CVs) for the high-titre controls were 19.53 and 16.55% for serotypes 14 and 19F and the CVs were 18.34 and 17.91% for serotype 23F. The CVs for the low-titre controls were 17.12 and 14.91% for serotypes 14 and 19F and the CVs were 15.39 and 14.23% for serotype 23F. Sera from persons with AIDS who had no antibodies to S. pneumoniae serotype 14, 19F, 23F or 6B capsules and <1 μg of antibody to CWPS per mL were used as negative controls. Capsular polysaccharides from S. pneumoniae serotypes 14 (catalogue number 197-X; lot #2038310), 19F (catalogue number 205-X; lot #2033178), 23F (catalogue number 217-X; lot #2048448) and 6B (catalogue number 225-X; lot #2045655) were obtained from the American

Type Culture Collection (ATCC; Manassas, VA, USA). These capsular polysaccharides were suspended in phosphate-buffered saline (PBS; pH 7.4) containing 0.02% NaN3 at a concentration of 10 μg/mL and used directly to coat wells

GSI-IX solubility dmso by incubation at 4 °C overnight. After washing of the antigen-coated plates, the pre-absorbed patient sera or controls were added to plates and incubated at room temperature for 2 h. After thorough washing to remove antibodies not bound to the wells, horseradish peroxidase (HRP)-conjugated goat antibody to human IgG (Zymed Laboratories Inc., San Francisco, CA, USA) at a dilution of 1:2000 was used to detect IgG, and the reaction was developed for 10 min in the dark by the addition of K-blue substrate (Neogen Demeclocycline Corporation, Lexington, KY, USA), followed by the addition of 1N sulphuric acid to stop the reaction. Between incubations, all washings were performed with Tris buffered saline (TBS) buffer containing 0.1% Brij solution. Optical density was read in an ELISA reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm, with subtraction of the optical density of the appropriate blank. The antibody concentrations were calculated relative to the 89-SF control serum, using the US Centers for Disease Control and Prevention (CDC) program elisa for Windows (version 2.15) [28]. The detection limits for our assay for 14, 19F, 23F and 6B, determined using the control serum 89-SF, were 0.02, 0.015, 0.02 and 0.50 μg/mL, respectively.