Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

Polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of paclobutrazol in fruits.

Paclobutrazol (PBZ) is a plant growth regulator (PGR) widely used in fruit and vegetable cultivation. However, due to the severe toxicity of PBZ, a sub-ppm level maximum residue limit (MRL) was established worldwide. Therefore, it is significant to propose a rapid, sensitive and high throughput screening method for monitoring the PBZ residues in foods. In this study, a simple and sensitive indirect competitive Enzyme-linked immunosorbent assay (icELISA) was established for PBZ detection in fruits basing polyclonal antibody. For both economy and pollution prevention, a microwave-solvent-free method was used to synthesize the PBZ hapten with high efficiency. The detection conditions, such as coating antigen concentration, antibody concentration, organic reagent concentration, ionic strength and pH, were optimized. Under the optimized conditions, this method showed high sensitivity and specificity. The detection range is 1.27-138.23 ng/mL, half-maximum inhibition concentration (IC50) is 13.26 ng/mL, and the IC20 was lower than the reported ELISAs for PBZ. Additionally, this method had high accuracy and precision. The recoveries were ranged from 88.78% to 96.80% in PBZ spiked apple samples with RSD below 4%. All the results showed that the polyclonal antibody based icELISA could be useful for PBZ screening in fruit samples.

Boosting Adaptive Immunity: A New Role for PAFR Antagonists.

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.

The efficacy and safety of maraviroc addition to a stable antiretroviral regimen in subjects with suppressed plasma HIV-RNA is not influenced by age.

There are few data about the immunovirological efficacy, safety/tolerability, and durability of maraviroc (MVC) addition to aging patients on suppressive antiretroviral therapy (cART) and undetectable viral load (<50 copies/ml). The aging population is underrepresented in most HIV clinical trials. This study included 80 patients aged ≥50 years and 161 aged <50 years and showed that after 48 weeks of treatment, there was no between-group differences in the median increase of CD4(+) T cells or the virological suppression rate. Safety and tolerability were also comparable. In multivariable analysis, the effect of age was not modified and was independent of the response to MVC. An immunological recovery of ≥100 CD4(+) T cells was significantly less common in those with a longer HIV history (≥15 years) (OR 0.43; p=0.016) or having <200/mm(3) CD4(+) T cells at MVC initiation (OR 0.27; p=0.004). Meanwhile, achieving a CD4/CD8 ratio ≥0.5 at week 48 was less likely in those with CD4(+) T cell counts <200 at MVC initiation (OR 0.09; p<0.0001) or with a previous AIDS event (OR 0.43; p=0.028). In summary, the immunovirological efficacy, safety/tolerability, and durability of MVC addition in patients virologically suppressed were independent of the patient's age at treatment onset.

Immunological evaluation of ß-thalassemia major patients receiving oral iron chelator deferasirox.

OBJECTIVE: To determine the immune abnormalities and occurrence of infections in transfusion-dependent ß-thalassemia major patients receiving oral iron chelator deferasirox (DFX). STUDY DESIGN: An observational study. PLACE AND DURATION OF STUDY: Hematology Clinics, King Khalid University Hospital, Riyadh, Saudi Arabia, from July to December 2010. METHODOLOGY: Seventeen patients with ß-thalassemia major (12 females, median age 26 years) receiving deferasirox (DFX) for a median duration of 27 months were observed for any infections and had their immune status determined. Immune parameters studied included serum immunoglobulins and IgG subclasses, serum complement (C3 and C4) and anti-nuclear antibody (ANA) level, total B and T-lymphocytes, CD4+ and CD8+ counts, CD4+/CD8+ ratio, and natural killer (NK) cells. Immunological parameters of the patients were compared with age, gender, serum ferritin level and splenectomy status. Lymphocyte subsets were also compared with age and gender matched normal controls. RESULTS: A considerable reduction in serum ferritin was achieved by DFX from a median level of 2528 to 1875 µmol/l. Serum IgG levels were increased in 7 patients. Low C4 levels were found in 9 patients. Total B and T-lymphocytes were increased in 14 patients each, while CD4+, CD8+ and NK cells were increased in 13, 12 and 11 patients respectively. Absolute counts for all lymphocyte subsets were significantly higher compared to the normal controls (p ² 0.05 for all parameters). Raised levels of IgG were associated with older age, female gender, splenectomized status and higher serum ferritin levels but this did not reach statistical significance except for the higher ferritin levels (p=0.044). Increased tendency to infections was not observed. CONCLUSION: Patients with ß-thalassemia major receiving DFX exhibited significant immune abnormalities. Changes observed have been described previously, but could be related to DFX. The immune abnormalities were not associated with increased tendency to infections.

Is there a role for the incretin system in blood pressure regulation?

Incretin-based therapies are now well established for diabetes management and are among the frontline agents for control of hyperglycemia. In addition to their antihyperglycemic effects, evidence is emerging on the role of these agents on blood pressure regulation, cardioprotective and renoprotective properties. Because of the pleiotropic nature of these affects, these agents could offer significant benefits with regards to the cardiorenal metabolic complications that are part of the diabetes and obesity epidemic in the United States and worldwide. We review the various known mechanisms or pathways by which incretin based therapy exerts its regulation of blood pressure with emphasis on novel mechanisms such as inflammation/immunomodulation and oxidative stress.

Extracellular adenosine controls NKT-cell-dependent hepatitis induction.

Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.

Determination of diniconazole in agricultural samples by sol-gel immunoaffinity extraction procedure coupled with HPLC and ELISA.

BACKGROUND: In the European Union (EU), the use of diniconazole-M is no longer authorized. However, residues of diniconazole-M occur in various plant commodities. METHODOLOGY/PRINCIPAL FINDINGS: A selective and simple analytical method for the trace level determination of diniconazole in soil, fruit, vegetables and water samples was developed based on immunoaffinity extraction followed by Enzyme-linked immunosorbent assay (ELISA) and the high-performance liquid chromatography (HPLC) analysis. The ELISA was based on monoclonal antibodies highly specific to diniconazole and was a fast, cost-effective, and selective screening method for the detection of diniconazole. The results of the ELISA correlated well with gas chromatography (GC) results, with the correlation coefficient of 0.9879 (n = 19). A simple gel permeation chromato- graphy clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The immunoaffinity column (IAC) capacity was 0.180 mg g(-1). The columns could be re-used approximately 20 times with no significant alteration in capacity. The recoveries from complex samples were in the range of 89.2% to 96.1% with a relative standard deviation (RSD) of 0.770%-6.11% by ELISA. The results were in good agreement with those obtained by HPLC method. CONCLUSION/SIGNIFICANCE: The IAC extraction procedure coupled with HPLC and ELISA analysis could be also used as alternative effective analytical methods for the determination of diniconazole concentrations in complex samples.

[Preparation of immunoaffinity chromatographic column specific to diniconazole and its applications to the pretreatment of samples].

The purified anti-diniconazole antibody was polymerised to hydrolytic tetramethoxysilane (TMOS) to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to diniconazole. The optimized conditions of the IAC were as follows: water as equilibrium and adsorbent medium, 30% and 50% (v/v) methanol aqueous solutions as eluents. The results showed that the dynamic column capacity was up to 125.4 microg/g of bed volume. The river water and fruit samples spiked with diniconazole were cleaned up and enriched by the IAC, and the diniconazole in eluant was determined by high performance liquid chromatography. The average recoveries (n = 5) of diniconazole in river water sample were 90.36%-100.14% with relative standard deviations (RSDs) of 2.03%-6.08%, and the average recoveries in fruit samples were 85.55%-94.02% with RSDs of 3.38%-6.78%. The IAC cleanup procedure provided an effective pretreatment method for the determination of diniconazole in sample media such as river water and fruits.

Immunologic effectiveness of maraviroc- and raltegravir-containing regimens (R+M+) versus raltegravir-based regimens that do not include maraviroc (R+M-).

OBJECTIVE: To compare the immunologic effectiveness of raltegravir-maraviroc (R+M+)-based regimens with raltegravir-based regimens that do not include maraviroc (R+M-) in treatment-experienced patients in clinical practice. METHODS: We conducted a retrospective study of treatment-experienced HIV-infected adults receiving either R+M+- or R+M--based therapy. Longitudinal CD4 counts were analyzed using a linear mixed model. RESULTS: One hundred and fifty-six patients were included in the analysis, of whom 32 were receiving R+M+ and 124 R+M-. Mean baseline CD4 counts in patients on R+M+ and R+M- were 463.8 and 442.3 cells/mm(3), respectively (P = .67). In multivariable mixed models, a baseline viral load ≥50 copies/mL was significantly associated with CD4 change during follow-up (P < .0001). No difference between R+M+ and R+M- was observed during follow-up (P = .81). CONCLUSION: CD4 cell recovery was similar among patients receiving either R+M+- or R+M--based therapy during a 24-month period of follow-up.

Synergistic inhibition of R5 HIV-1 by maraviroc and CCR5 antibody HGS004 in primary cells: implications for treatment and prevention.

CCR5 blockers inhibit CCR5-tropic (R5) HIV-1, including strains resistant to other antiretrovirals. We demonstrate that the CCR5 antibody HGS004 and the CCR5 antagonist maraviroc have potent antiviral synergy against R5 HIV-1, translating into dose reductions of more than 10-fold for maraviroc and more than 150-fold for HGS004. These data, together with the high barrier of resistance to HGS004, suggest that combinations of maraviroc and HGS004 could provide effective preventive and therapeutic strategies against R5 HIV-1.

Pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal effect.

Isavuconazole is a triazole with broad-spectrum activity against medically important fungal pathogens. We investigated the pharmacokinetics and pharmacodynamics of isavuconazole in a murine model of disseminated candidiasis. We determined the pharmacokinetics in both plasma and kidney. The relationship between tissue concentrations and the resultant antifungal effect was described using a mathematical model. The pharmacodynamic parameter that optimally links drug exposure with the antifungal effect was determined using dose fractionation studies. The impact of the immune status of mice receiving isavuconazole was determined in persistently and temporarily neutropenic animals. The pharmacokinetics of 1.6 to 28 mg isavuconazole/kg of body weight were linear. Exposure-response relationships demonstrated near-maximal effect following the administration of >15 mg/kg. The mathematical model showed that exposures in the kidney were 5.77 times higher than those in plasma, and there was persistence of the drug at this site despite concentrations in plasma falling to undetectable levels. The in vitro and in vivo postantifungal effects were 2 to 5 and 8.41 h, respectively. The area under the concentration-time curve (AUC)/MIC ratio was the parameter that optimally linked drug exposure with the observed antifungal effect. The total drug AUC/MIC ratios associated with a 90% probability of survival in temporarily and persistently neutropenic mice were 270 and 670, respectively. Once corrected for protein binding, these values are similar to the magnitude of drug exposure associated with a high probability of a successful therapeutic outcome for other triazoles. This study provides the experimental foundation for the use of isavuconazole in patients with disseminated candidiasis.

Gold immunochromatographic assay for simultaneous detection of carbofuran and triazophos in water samples.

Using a simple test for rapid identification and quantification of pesticide multiresidues in food and environmental samples is a long-cherished approach for practical monitoring purposes. Here two gold-based lateral-flow strips (strip A and strip B) were investigated for simultaneous detection of carbofuran and triazophos. For the strip A format, a bispecific monoclonal antibody (BsMcAb) against both carbofuran and triazophos was employed to prepare the immunogold probe. For the strip B format, anti-carbofuran monoclonal antibody (McAb) and anti-triazophos McAb separately labeled with colloidal gold were combined as detector reagents. By comparison of visual results from pesticide standard tests between the two formats, the strip B assay manifested higher sensitivities for both pesticides. Analysis of spiked water samples by the preferable strip indicated that the detection limits for carbofuran and triazophos were 32 and 4 microg/L, respectively. The strength of the portable one-step strip assay was in the simultaneous screening for two pesticides within a short time (8-10 min) without any equipment.

Development of a bispecific monoclonal antibody to pesticide carbofuran and triazophos using hybrid hybridomas.

A mouse hybrid hybridoma (tetradoma) was derived from fusing hybridomas producing monoclonal antibody to N-methylcarbamate pesticide carbofuran with hybridomas producing monoclonal antibody to organophosphorus pesticide Triazophos. The prepared tetradoma line (12C1 to 2H12) secreted hybrid immunoglobulin exhibiting parental and bispecific binding characteristics. The effect of relevant physicochemical factors on the immunoassay based on the 12C1 to 2H12 bispecific monoclonal antibody had been studied to optimize the ELISA performance. The developed immunoassay showed that the detection limit (I(20)) were 0.36 and 1.89 ng/mL for triazophos and carbofuran, respectively, without obvious cross-reactivity to other related compounds. Water samples spiked with triazophos at 0.5, 1, and 5 ng/mL or carbofuran at 5, 10, and 20 ng/mL were directly analyzed by the developed ELISA format. The mean recovery of triazophos and carbofuran were 108.1% and 107.5%, with variation coefficient of 15.9% and 17.7%, respectively.