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Recombinant Antigens (recombinant + antigen)
Selected AbstractsRecombinant proteins in the diagnosis of toxoplasmosisAPMIS, Issue 8 2010DUPADAHALLI KOTRESHA Kotresha D, Rahmah N. Recombinant proteins in the diagnosis of toxoplasmosis. APMIS 2010; 118: 529,42. Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient's serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis. [source] Antigen co-encapsulated with adjuvants efficiently drive protective T cell immunityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2007Antje Heit Abstract Compared to "live" vaccines, the immunogenicity of "subunit" vaccines based on recombinant antigen (Ag) is poor, presumably because exogenous Ag fails to effectively access the endosomal Ag-processing pathways of Ag-presenting cells (APC). To overcome this limitation, we exploited biodegradable poly(lactic-co-glycolic) microspheres (MP) co-entrapping Ag and Toll-like receptor (TLR) 9 or 7 ligands as an endosomal delivery device. In vitro, microspheres were rapidly phagocytosed by APC and translocated into phago-endosomal compartments, followed by degradation of the Ag and concurrent activation of endosomal TLR. As a consequence, full maturation of and cytokine secretion by APC as well as Ag-cross-presentation ensued. In vivo, "loaded" microspheres triggered clonal expansion of primary and secondary Ag-specific CD4 and CD8 T cells. The efficacy of CD8 T cell cross-priming was comparable to that of live vectors. The potency of T cell vaccination was demonstrated by protective and therapeutic interventions using infection- and tumor-model systems. These preclinical "subunit" vaccination data thus recommend MP as a generally applicable and powerful endosomal delivery device of exogenous Ag plus TLR-based adjuvants to vaccinate for protective and therapeutic CD4 and CD8 T cell immunity. [source] Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coliLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Norsyahida Abstract Aim:, To investigate the effects of feeding and induction strategies on the production of BmR1 recombinant antigen. Methods and Results:, Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of BmR1 recombinant antigen. Cells were grown at a controlled specific growth rate (,set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l,1) was obtained during fed-batch process operated at ,set of 0·15 h,1, whereby lower ,set (0·075 h,1) gave the highest protein production (9·82 mg l,1). The yield of BmR1 was increased by 1·2-fold upon induction with 1 mmol l,1 IPTG (isopropyl-,- d -thiogalactoside) compared to using 5 mmol l,1 and showed a further 3·5-fold increase when the culture was induced twice at the late log phase. Conclusions:, Combination of feeding at a lower ,set and twice induction with 1 mmol l,1 IPTG yielded the best result of all variables tested, promising an improved method for BmR1 production. Significance and Impact of the Study:, This method can be used to increase the production scale of the BmR1 recombinant antigen to meet the increasing demand for Brugia RapidÔ, a commercial diagnostic test for detection of brugian filariasis. [source] Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2008Alessandra Barbante Summary The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions. [source] Immunoprophylactic evaluation of a 37-kDa Brugia malayi recombinant antigen in lymphatic filariasisCLINICAL MICROBIOLOGY AND INFECTION, Issue 4 2006P. Dabir Abstract The Brugia malayi filarial antigens recognised preferentially by sera from an endemic normal population are considered to be potential vaccine candidates. By immunoscreening the cDNA library of the infective L3 stage of B. malayi with pooled endemic normal sera, a cDNA clone Bm-SL3 was identified. Analysis of sera from different patient groups with the rBm-SL3 protein showed it to be highly reactive with endemic normal sera compared to its reactivity with microfilaraemic and non-endemic normal sera. The immunoprotective efficacy of the rBm-SL3 antigen against B. malayi filarial infection was evaluated in susceptible host jirds (gerbils) (Meriones unguiculatus). Jirds immunised with the rBm-SL3 antigen showed 68% cytotoxicity against microfilariae and 67,69% cytotoxicity against infective larvae in in-vitro antibody-dependent cellular cytotoxicity assays and in-situ micropore chamber methods. Analysis of IgG subclasses against Bm-SL3 revealed a significant increase in IgG1 and IgG2 antibodies in endemic normal sera compared with other groups. Lymphocyte proliferation to Bm-SL3 was significantly higher in the endemic normal group compared with that in clinical and microfilarial carriers (p < 0.001). Significantly enhanced levels of IFN-, in the culture supernatant of peripheral blood mononuclear cells of endemic normal sera after stimulation with Bm-SL3 suggest that the cellular response in this group may have a Th1 bias. [source] Antigen-specific oligoclonal bands in cerebrospinal fluid and serum from patients with anti-amphiphysin- and anti-CV2/CRMP5 associated paraneoplastic neurological syndromesEUROPEAN JOURNAL OF NEUROLOGY, Issue 6 2007O. Stich Using isoelectric focusing and affinity blotting employing paraneoplastic recombinant antigens, we investigated cerebrospinal fluid (CSF) and sera from three patients with positive anti-CV2/CRMP5- and one patient with positive anti-amphiphysin serology. CSF and sera were previously adjusted to total IgG concentrations of 20 mg/l. All patients suffered from paraneoplastic neurological syndromes (PNS) with predominant involvement of the central nervous system (CNS). Using affinity blot preloaded with paraneoplastic antigen, we detected in three of four patients more or stronger specific oligoclonal bands (OCB) in the CSF than in the corresponding serum, providing qualitative evidence of antigen specific intrathecal antibody synthesis. These results are in line with previous studies demonstrating specific OCB predominantly in CSF from patients with anti-Hu-, anti-Yo- and anti-Ri-associated PNS, supporting the hypothesis of autoimmunity in the pathogenesis of PNS. One patient harboured extensive anti-amphiphysin specific OCB, although OCB of total IgG could not be detected, indicating a higher sensitivity for detection of intrathecal antibody synthesis of the affinity blot preloaded with the paraneoplastic antigen, compared with investigation of total IgG OCB. These results could have implications concerning pathophysiological autoimmune aspects in other inflammatory diseases of CNS associated with total IgG OCB, provided that the target antigen is known. [source] Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUSINTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Klaus-Dieter Preuss Abstract Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 × 108 paraprotein,antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely. © 2007 Wiley-Liss, Inc. [source] Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial diseaseMICROBIOLOGY AND IMMUNOLOGY, Issue 7 2009Eun-Ju Do ABSTRACT In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA1350-1784, OmpB801-1269, and OmpB1227-1634 regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii. For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA1350-1784 (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB801-1269 and OmpB1227-1634 were 90% and 95%, respectively. The specificities of the OmpB801-1269 and the OmpB1227-1634 were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii, and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease. [source] Antigenic cross-reactivity between different alleles of the Plasmodium falciparum merozoite surface protein 2PARASITE IMMUNOLOGY, Issue 11-12 2003Ingrid Felger SUMMARY The polymorphic domain of the gene encoding Plasmodium falciparum merozoite surface protein 2 (MSP2) was PCR amplified from blood of malaria patients, genotyped, and 19 distinct fragments were cloned and expressed in E. coli. The reactivity of naturally occurring antibodies against this panel of recombinant MSP2 antigens was tested using 67 homologous or heterologous sera from a serum bank of travel clinic patients. Sera from semi-immune individuals strongly recognized almost all recombinant antigens. Sera from primary infected patients either did not react at all (9 sera), or reacted weakly against varying numbers of antigens (39 sera). The antigens that showed reactions were mostly of the allelic family corresponding to the infecting clone, but in very few cases also of the alternative allelic family. Single clone infections and repeated samples from the same individual were analysed in greater detail. Thus, we were able to quantify cross-reactivity induced by a single P. falciparum infection. Within the two allelic families of MSP2, cross-reactivity was observed between some but not all alleles of the same family, whereas antibodies cross-reactive between variants belonging to different allelic families were detected in only a few cases. [source] Humoral immune response against 38- and 16-kDa mycobacterial antigens in childhood tuberculosis,PEDIATRIC PULMONOLOGY, Issue 9 2009Gunes Senol MD Abstract Several enzyme-linked immunosorbent assays (ELISAs) based on mycobacterial antigens have been tried for the rapid diagnosis of tuberculosis (TB). In this study, the value of the 16 and 38-kDa mycobacterial antigens in the diagnosis of TB was investigated in pediatric patients in Izmir, Turkey in whom they were found using clinical and/or bacteriological methods. A commercial ELISA kit was used for measuring IgG against 38 and 16-kDa recombinant antigens. The humoral immune response was analyzed in a group of 32 TB patients (24 pulmonary, 3 lymphadenitis and 2 pleuritis, 2 meningitis and a disseminated TB) and in control groups consisting of 20 healthy children and 20 pulmonary diseases other than TB cases. The sensitivity, specificity, positive predictive value, and the negative predictive value of the test were found to be 25%, 90%, 66.7%, and 60%, respectively, in the TB cases. The ELISA test shows very good specificity, but low level of sensitivity and negative predict value. It was thought that it might be used in combination with other methods to increase diagnostic accuracy, especially for culture-negative TB pediatric cases, which are difficult to diagnose. Pediatr Pulmonol. 2009; 44:839,844. © 2009 Wiley-Liss, Inc. [source] Anti,Heat Shock Protein 70 Antibodies in Meniere's Disease ,THE LARYNGOSCOPE, Issue 9 2000Steven D. Rauch MD Abstract Objectives To determine the prevalence of anti,heat shock protein 70 (anti-HSP70) antibodies in patients with Meniere's disease and healthy subjects and to probe the relationship between antibody status and clinical features of Meniere's disease. Study Design Prospective cohort study of consecutive consenting patients with Meniere's disease. Methods Serum samples were obtained prospectively from 134 patients with Meniere's disease and 124 blood donors. Serial samples were taken at 3-month intervals in 38 of 134 patients with Meniere's disease. Demographic data and clinical characterization of vestibular and auditory status were acquired with each sample. Serum was assayed for anti-HSP70 antibodies by Western blot using bovine renal extract, recombinant bovine HSP70, and recombinant human HSP70 antigens. Results Immunoreactivity against bovine renal extract HSP70 was found in 38% of patients with Meniere's disease, compared with 25% of blood donors (P < .04). Reactivity with recombinant antigens was not significantly different between patients with Meniere's disease and healthy control subjects. Patients with Meniere's disease who reacted with all three antigens were more likely to have simultaneously active hearing and balance symptoms (P = .03). Neither univariate nor multivariate statistical analysis established any other association between serological findings and clinical features of Meniere's disease. Tests performed on serial samples of patients with Meniere's disease also showed no association of positive or negative test results with changes in clinical course. Conclusions Because of the high prevalence of anti-HSP70 antibodies in healthy subjects and the very limited association of anti-HSP70 antibody status with clinical features or course of Meniere's disease, we conclude that, at present, the detection of anti-HSP70 antibodies by Western blotting offers little clinically useful information in Meniere's disease. [source] Anti-La/SSB antibodies transported across the placenta bind apoptotic cells in fetal organs targeted in neonatal lupusARTHRITIS & RHEUMATISM, Issue 6 2002Hai B. Tran Objective To determine whether La and/or Ro epitopes on apoptotic cells in fetal organs that are targeted in neonatal lupus syndrome (NLS) are accessible for binding by autoantibodies in vivo, we traced the fate of transplacental autoantibodies in a murine passive transfer model. Methods Pregnant mice at day 15 of gestation (E15) were injected intraperitoneally with human anti-Ro/La,positive sera or control sera, and transplacental transfer of human autoantibodies was tested by enzyme-linked immunosorbent assay with recombinant antigens. Multiple cryostat sections at the level of the heart of E17 fetuses were visualized simultaneously for human IgG binding and apoptosis (TUNEL) under confocal microscopy. Serial paraffin sections of E17 and E19 fetuses were examined for histologic evidence of inflammation. Results Human IgG anti,52-kd Ro, anti,60-kd Ro, and anti-La autoantibodies were transported efficiently into the fetal circulation. Human IgG,apoptotic cell complexes were detected in the heart (atrial trabeculae and atrioventricular node), skin, liver, and newly forming bone of fetuses from mothers injected with anti-Ro/La sera but not control sera. The IgG binding was fetal-specific and organ-specific; transplacental autoantibodies did not bind to apoptotic cells in the fetal thymus, lung, brain, or gut. The complexes were not associated with an inflammatory reaction. Injection of mothers with affinity-purified anti-La autoantibodies (but not anti-Ro/La Ig depleted of anti-La) revealed an identical location of IgG binding to apoptotic cells in the fetuses. Conclusion This is the first study to demonstrate that transplacental anti-La autoantibodies bind specifically to apoptotic cells in selected fetal organs in vivo, similar to the organ involvement in NLS. We hypothesize that additional factors are required to promote proinflammatory clearance of IgG,apoptotic cell complexes and subsequent tissue damage. [source] | |||||||||||||