Antigen detection ELISAs: pretreatment of serum to reduce interference by specific host antibodies

The pretreatment of serum to reduce interference by specific host antibodies was investigated as a means of improving the sensitivity of antigen detection ELISAs whilst screening serum samples. Four antigen detection assays based on monoclonal antibodies directed against antigens of the bovine filariid Onchocerca gibsoni were used in this study and of these, three assays suffered a dramatic drop in sensitivity when detecting male O. gibsoni antigen in the presence of bovine serum as compared with antigen in buffer. A number of methods for pretreating serum to eliminate the problem of antibody interference with antigen detection were attempted, including heat and alkali treatments, detergent treatment of heat treated samples and the use of a reducing agent. The pretreatment of serum by boiling for 5 minutes in the presence of an equal volume of 0.1 M Na2EDTA pH 4.0 and recovery of the supernatant fluid following centrifugation at 16000 g was the most effective method of restoring the sensitivity of each of these three assays whilst screening bovine serum. Pretreatment of serum using this method produced up to a 512-fold increase in sensitivity compared with results obtained in assays with non-treated serum.Edna McConnell Clark Foundatio

The use of monoclonal antibody-based ELISAs to monitor the efficacy of drugs against male Onchocerca gibsoni in vitro

Four monoclonal antibodies directed against antigens of Onchocerca gibsoni were used in antigen detection ELISAs to monitor the efficacy of CGP 20309, CGP 20376, CGP 21833, CGP 24589 and CGP 26702 at 5 μg/ml against male O. gibsoni in vitro. No significant differences (P < 0.05) in antigen output between treated and control groups of parasites were recorded. However, consistently higher levels of antigen from treated (CGP 21833) as compared to control parasites were measured with all four assays, with differences being higher in the first 2 to 3 days post treatment than subsequently. The sensitivity of comparisons between groups was reduced by the high variability in output of antigen both between worms and also from the same worm, in part as a result of mechanical damage to worms sustained during collection or manipulation in vitro. This problem was reduced by zero handling once worms were established in vitro and it is recommended that future work should include a 24 to 48 hour period before treatment commences to detect raised antigen levels associated with physically damaged parasites so they can be excluded. It was concluded that this type of assay has no intrinsic technical or logistical advantage over other published methods of assessing drug-related damage in in vitro filarial screens. Nevertheless, further work using antigen detection ELISAs in this context is justified since these assays, unlike all other methods of assessing drug-induced damage in vitro, have direct application for use in identifying chemotherapeutic effects against similar parasites in vivo.Edna McConnell Clark Foundatio

A highly specific and sensitive monoclonal antibody-based ELISA for the detection of circulating antigen in bancroftian filariasis

A monoclonal antibody, Og4C3, directed against antigens of Onchocerca gibsoni (but not phosphorylcholine) has been used in a sandwich ELISA to detect a circulating antigen of Wuchereria bancrofti in human serum. The interfering effect of host antibody was reduced by first boiling one part of serum for 5 min in the presence of three parts of 0.1 M Na2EDTA, pH 4.0. A total of 119 sera from individuals and 8 pooled sera from clinical and/or parasitologically defined cases of filariasis, plus 8 individual and 1 pooled endemic control sera, all from the filariasis serum bank of the World Health Organisation, as well as 20 non-endemic control sera, were screened with the assay. Circulating antigen was detected in serum from people infected with W. bancrofti but not Brugia malayi, B. timori, O. volvulus or Loa loa, and not in endemic or non-endemic controls. Of the 68 sera from W. bancrofti-infected subjects, 55/55 parasitologically confirmed and 12/13 clinically confirmed but amicrofilaraemic cases reacted in the assay. A weak but significant correlation (r2 = 0.4016) was found between numbers of microfilariae in blood and detectable levels of circulating antigen from patients with bancroftian filariasis.Edna McConnell Clark Foundatio

The use of monoclonal antibody-based ELISAs to monitor chemotherapeutic effects in the bovine-Onchocerca gibsoni drug screen

Three monoclonal antibodies directed towards antigens of Onchocerca gibsoni were used in antigen detection ELISAs to detect parasite antigens in sera from 100 cattle infected with O. gibsoni, in trials with the filaricidal compounds CGP 6140, CGP 20309, CGP 20376, CGP 21833, CGP 24589 and CGP 26702. Measurable levels of parasite antigens were highly variable, both within and between treatment and control groups of animals, with no consistent trends which related to time after treatment, micro or macrofilaricidal effects against O. gibsoni, or dose rate for any of the compounds used. It was concluded that these assays were unsuitable as a method of identifying drug-induced damage to O. gibsoni following the administration of these compounds. A detailed protocol for selecting suitable assays is discussed.Edna McConnell Clark Foundatio

Cord blood banking – bio-objects on the borderlands between community and immunity

Umbilical cord blood (UCB) has become the focus of intense efforts to collect, screen and bank haematopoietic stem cells (HSCs) in hundreds of repositories around the world. UCB banking has developed through a broad spectrum of overlapping banking practices, sectors and institutional forms. Superficially at least, these sectors have been widely distinguished in bioethical and policy literature between notions of the ‘public’ and the ‘private’, the commons and the market respectively. Our purpose in this paper is to reflect more critically on these distinctions and to articulate the complex practical and hybrid nature of cord blood as a ‘bio-object’ that straddles binary conceptions of the blood economies. The paper draws upon Roberto Esposito’s reflections on biopolitics and his attempt to transcend the dualistic polarisations of immunity and community, or the private and the public. We suggest that his thoughts on immunitary hospitality resonate with many of the actual features and realpolitik of a necessarily internationalised and globally distributed UCB ‘immunitary regime’

CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

The loss of AP-2alpha and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2alpha during melanoma progression remains unknown.Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2alpha protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2alpha. Loss of AP-2alpha expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2alpha promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2alpha expression following CREB silencing increases endogenous p21(Waf1) and decreases MCAM/MUC18, both known to be downstream target genes of AP-2alpha involved in melanoma progression.Since AP-2alpha regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21(Waf1), our data identified CREB as a major regulator of the malignant melanoma phenotype

Vitamin C Enhances Vitamin E Status and Reduces Oxidative Stress Indicators in Sea Bass Larvae Fed High DHA Microdiets

Docosahexaenoic acid (DHA) is an essential fatty acid necessary for many biochemical, cellular and physiological functions in fish. However, high dietary levels of DHA increase free radical injury in sea bass (Dicentrarchus labrax) larvae muscle, even when vitamin E (&alpha;-tocopherol, &alpha;-TOH) is increased. Therefore, the inclusion of other nutrients with complementary antioxidant functions, such as vitamin C (ascorbic acid, vitC), could further contribute to prevent these lesions. The objective of the present study was to determine the effect of vitC inclusion (3,600 mg/kg) in high DHA (5 % DW) and &alpha;-TOH (3,000 mg/kg) microdiets (diets 5/3,000 and 5/3,000 + vitC) in comparison to a control diet (1 % DHA DW and 1,500 mg/kg of &alpha;-TOH; diet 1/1,500) on sea bass larvae growth, survival, whole body biochemical composition and thiobarbituric acid reactive substances (TBARS) content, muscle morphology, skeletal deformities and antioxidant enzymes, insulin-like growth factors (IGFs) and myosin expression (MyHC). Larvae fed diet 1/1,500 showed the best performance in terms of total length, incidence of muscular lesions and ossification degree. IGFs gene expression was elevated in 5/3,000 diet larvae, suggesting an increased muscle mitogenesis that was confirmed by the increase in the mRNA copies of MyHC. vitC effectively controlled oxidative damages in muscle, increased &alpha;-TOH larval contents and reduced TBARS content and the occurrence of skull deformities. The results of the present study showed the antioxidant synergism between vitamins E and C when high contents of DHA are included in sea bass larvae diets