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Detection of the cyanobacterial hepatotoxins microcystins.

Concern regarding the presence of microcystins in drinking water and their possible contamination in food (e.g., salad vegetables, fish, shellfish) has resulted in the need for reliable methods for the detection and accurate quantification of this class of toxins. Currently, routine analysis of microcystins is most commonly carried out using high-performance liquid chromatography with photodiode array detection (HPLC-PDA), although more sensitive biological assays such as antibody-based ELISAs and protein phosphatase inhibition assays have also proven useful. However, many of these methods have been hindered by the availability of a wide range of purified microcystins. Although over 60 variants have now been reported, only a very small number are commercially available and calibrated standards are not yet obtainable. This has led to the common practice of reporting microcystin-LR equivalence regardless of which variant is present. The increased availability of HPLC with online mass spectral analysis (HPLC-MS) may facilitate more accurate detection of toxin variants but as several microcystins share the same molecular mass, definitive identification can be difficult. A further difficulty in analyzing microcystins is the requirement for sample processing before analysis. Solid phase extraction (SPE) is typically used to enrich environmental concentrations of microcystins, or to eliminate contaminants from complex samples such as animal and plant tissues. Recently, new technologies employing recombinant antibodies and molecularly imprinted polymers have been exploited to develop assays and biosensors for microcystins. These novel detection systems are highly sensitive, often do not require sample processing, and offer a simpler, less expensive alternative to analytical techniques. They have also been successfully employed in solid phase extraction formats for the concentration and clean up of environmental samples before HPLC analysis.

Animals↗

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates.

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.

Catalysis↗

Cardiac troponin I in cats with hypertrophic cardiomyopathy.

The molecular structure of cardiac troponin I (cTnI) is highly conserved across mammalian species and assays developed for its measurement in human patients have been validated in a number of veterinary species. A raised concentration of circulating cTnI is a sensitive and specific marker of cardiac myocyte injury. Raised levels have been documented in a variety of cardiac diseases in both human and veterinary patients. This study compared serum cTnI concentrations between 16 cats diagnosed with hypertrophic cardiomyopathy (HCM) using echocardiography and 18 control cats. The results show that cats with HCM have significantly higher concentration of serum cTnI (median 0.95 ng/ml, range 0.2-4.1 ng/ml) than control cats (median <0.2 ng/ml, range <0.2-0.25 ng/ml) [P<0.0001]. Furthermore in cats with cardiomyopathy a weak correlation was found between the thickness of the left ventricular freewall in diastole measured by ultrasound and serum cTnI concentration (r(2)=0.28;P=0.036). These results suggest that measurement of serum cTnI concentration may enable cats with cardiomyopathy to be distinguished from normal cats using the assay described here.

Animals↗

Epitope mapping and use of anti-idiotypic antibodies to the L6 monoclonal anticarcinoma antibody.

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were raised against L6, a murine IgG2a monoclonal antibody specific for a cell surface antigen expressed by many human carcinomas. Ten distinct anti-ids were generated. Eight anti-ids were shown to inhibit the binding of L6 to its target antigen and were characterized in detail. The heavy and light chain variable region gene segments of the monoclonal antibody L6 linked to human constant regions (chimeric L6) were expressed separately or together, to map the epitopes recognized by the anti-ids. Individual anti-ids were shown to recognize heavy chain, light chain, or combinatorial variable region determinants. Defining these specificities enabled us to select particular anti-ids for assays to monitor the pharmacokinetics of either murine or chimeric L6 antibodies in the circulation of human patients. A quantitative enzyme-linked immunosorbent assay developed with two anti-ids accurately detects less than 5 ng/ml. Anti-ids specific for light chain variable region-encoded determinants were capable of recognizing L6 antigen-binding fragments bound to the surface of human carcinoma cells. These anti-ids can be used to study the binding of chimeric L6 antibody at the surface of tumor cells in histological sections of tumor biopsies.

Animals↗

Evaluation of an in vitro assay aimed at measuring protective antibodies against sporozoites.

We have evaluated the in vitro biological activities of antibodies directed against sporozoites and compared them with their capacity to protect against challenge with both human and rodent malaria. The anti-Plasmodium falciparum antibodies evaluated with the test included monoclonal antibodies (MAbs) NFS1 and NFS2 as well as polyclonal antibodies contained in human hyperimmune sera directed against sporozoites of P. falciparum. The inhibitory effect of these antibodies was dependent on their concentration. However, total inhibition was not observed except occasionally with highly concentrated MAbs (10-100 micrograms/ml). Strong but also incomplete inhibition was observed with sera from humans living in hyperendemic areas. In the P. yoelii rodent system, we tested sera from mice immunized with subunit vaccines. None of these mice were protected in vivo against challenge with 40-200 sporozoites. In vitro only a sub-total inhibition was achieved (maximum 91% at 1:10 serum dilution). In contrast, we tested sera from mice that received NYS1, an IgG3 MAb, in passive transfer and were protected against challenge with 5000 sporozoites. At 1:10 dilution, 100% inhibition was achieved in vitro while IFA titres from these mice were similar to those of vaccinated mice. These data show a close correlation between in vivo and in vitro findings and thus suggest that the inhibition of liver-stage development assay (ILSDA) appears appropriate to evaluate the potential of antibodies.

Adult↗

Low prevalence of IgA deficiency in north Indian population.

BACKGROUND AND OBJECTIVES: IgA deficient patients are at risk of severe anaphylactic reaction on being transfused blood and blood products, and its prevalence varies in different parts of the world. No data are available from India. We did a blood donor survey to look for prevalence of IgA deficiency in north India. METHODS: A sensitive enzyme linked immunosorbent assay developed in-house was used to detect IgA deficiency in a total of 3818 blood donors. Complete IgA deficiency was defined as value less than 5 mg/dl whereas partial IgA deficiency was defined as value between 5-30 mg/dl. RESULTS: Of the 3818 blood donors screened, 3640 (95.3%) were males with a mean age of 31.2 yr. No donor was found to have complete IgA deficiency; however, 257 (6.7%) had partial IgA deficiency. INTERPRETATION AND CONCLUSION: Our study shows that IgA deficiency is rare in north India.

Adolescent↗

Resistance to human immunodeficiency virus type 1 in vitro as a surrogate of vaccine-induced protective immunity.

An in vitro assay developed as a correlate of vaccine-induced protection from human immunodeficiency virus (HIV) was validated in populations with relative resistance to HIV-1 as well as in HIV vaccine recipients. Cultures of peripheral blood mononuclear cells (PBMC) were challenged with 10 TCID50 of HIV-1MN or HIV-1BaL, titered in PBMC from normal controls (n=57). PBMC from HIV-1-infected persons with low viremia (n=17), exposed uninfected persons (n=23), and HIV-2-infected Senegalese prostitutes (n=9) were significantly resistant to HIV-1BaL and/or HIV-1MN (P<.001). Among 34 HIV vaccine recipients of live canarypox vector expressing multiple HIV-1 gene products with or without rgp120 booster, PBMC from postvaccination samples were significantly resistant to both strains (P<.001), and cytotoxic T lymphocyte precursor-positive samples were significantly more resistant than were precursor-negative samples (P<.03). This is the first evidence of the induction by vaccination of a validated correlate of protection. This assay should serve as a useful criterion for assessing experimental HIV vaccines before phase III efficacy trials.

AIDS Vaccines↗

Biological effects of a neurotoxic pesticide at low concentrations on sea urchin early development. A terathogenic assay.

Dose-dependent terathogenic effects of an organophosphate insecticide were found during early sea urchin development. This biological assay is low cost, easy to measure, and allows to detect the effects of the exposure of organisms to the active principle at concentrations lower than the acceptable daily intake (ADI) for man. Effects were found independently from the stage of exposure, and were major as earlier exposure occurred. The stronger effects were exerted on the elongation of the skeletal rods, that was easily measured by following the migration of primary mesenchyme cells, labelled by WGA (wheat germ agglutinin).

Animals↗

Rapid enzyme-linked immunosorbent assay for the diagnosis of human brucellosis in surveillance and clinical settings in Egypt.

OBJECTIVE: To optimize and standardize an enzyme-linked immunosorbent assay (ELISA) for rapid diagnosis of human brucellosis in clinical cases identified during a surveillance study for acute febrile illness (AFI). METHODS: Serum samples from patients presenting with AFI at 13 fever hospitals across Egypt between 1999 and 2003 were kept frozen at NAMRU-3 and used in this study. The assay was evaluated in 5 subject groups: brucellosis cases confirmed by blood culture (group I, n=202) 87% positive by standard tube agglutination test (TA), brucellosis cases exclusively confirmed by TA (group II, n=218), blood cultures from AFI cases positive for bacterial species other than Brucella (group III, n=103), AFI cases with unexplained etiologies (group IV, n=654), and healthy volunteers (group V, n=50). All members of groups III-V were negative for brucellosis by TA. RESULTS: Sensitivity and specificity of ELISA for total specific antibodies were >=96% versus 87% for TA as compared to microbial culture, the current gold standard method for Brucella identification. Assessment of Brucella antibody classes by ELISA in random subsets of the 5 groups showed significantly high (p>0.001) levels of anti Brucella IgG (>=81%) and IgM (>=90%) in groups I and II only. CONCLUSION: The obtained sensitivity and specificity results indicate that our ELISA is more suitable for AFI surveillance and clinical settings than blood culture and TA. The developed assay is also cost-effective, easier to use, faster, and the coated plates can be stocked for at least 8 months, providing a potential for field use and automation.

Adolescent↗

The assay of urinary growth hormone in normal and acromegalic adults.

OBJECTIVES: To establish a normal range for urinary growth hormone in adults and to investigate the urinary growth hormone levels in patients with acromegaly, comparing these with the serum growth hormone results of a glucose tolerance test. We also studied the molecular identity of the growth hormone recognized by our assay method. DESIGN: Overnight urine samples and, in some cases, timed urine samples taken during the day were obtained from healthy volunteers and acromegalic patients. A standard glucose tolerance test with serum growth hormone measurements was performed on the acromegalic patients. PATIENTS: One hundred and thirty-five normal adults and 33 acromegalic patients were studied. MEASUREMENTS: Urinary growth hormone was measured using a sensitive and precise assay developed previously. RESULTS: In healthy volunteers overnight urinary growth hormone values fell gradually with increasing age, but there was no significant difference between men and women in any decade or between smokers and non-smokers. Sexual intercourse had no detectable effect on the values, but there was a large increase following strenuous exercise. Studies of the diurnal patterns in normal and abnormal adults suggested that it might be possible to diagnose acromegaly on a random urine sample. Gel filtration studies on a urine sample from an acromegalic patient showed a single peak of molecular weight 22,000. Using overnight collections there was clear discrimination between the values given by the normal adults and the acromegalic patients and an excellent correlation between urinary growth hormone levels in acromegalic patients and the mean serum growth hormone in a glucose tolerance test. CONCLUSIONS: In contrast to some other groups we conclude that urinary growth hormone provides a useful, non-invasive screening test for acromegaly, but this conclusion depends crucially on the assay being sensitive and precise at low values.

Acromegaly↗

Development of a triplex PCR assay for the specific detection of Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7.

A triplex PCR assay was developed and evaluated for efficacy in detecting Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7 in a variety of raw and ready-to-eat food products. Following a short enrichment period, artificially contaminated food samples were subjected to a triplex PCR assay, which incorporated published primers for each food pathogen, a protocol for sample collection, and a PCR procedure designed specifically for the assay. The selected primers amplified fragment sizes of 159 bp, 252 bp, and 360 bp for C. jejuni, E. coli O157:H7, and Salmonella spp., respectively. This assay provides specific and reliable results and allows for the cost-effective detection of all three bacterial pathogens in one reaction tube.

Bacteriological Techniques↗

Immunoglobulin levels in Bull terriers suffering from lethal acrodermatitis.

An enzyme-linked immunosorbent assay, developed by one the authors (Huang, 1993) was used to measure immunoglobulin levels in previously stored plasma samples from eight dogs suffering from lethal acrodermatitis (LAD) and 10 normal Bull terriers. The aim of the study was to document plasma immunoglobulin levels and to determine if dogs suffering from LAD had low IgA levels. The results showed that dogs suffering from LAD had normal IgG and IgM levels but significantly lower IgA levels than the control group. This finding may in part explain the frequent occurrence of microbial infections recorded in dogs suffering from LAD.

Acrodermatitis↗

Measurement of circulating parathyroid hormone-related protein in rats with humoral hypercalcemia of malignancy using a two-site immunoradiometric assay.

The Rice H500 rat Leydig cell tumor is a well characterized model of humoral hypercalcemia of malignancy (HHM). Circulating concentrations of PTH-related protein (PTHRP) have not been reported in this or any other animal model of HHM. Taking advantage of the marked N-terminal amino acid homology between rodent and human PTHRPs, we have adapted a sensitive two-site immunoradiometric assay developed for measurement of human PTHRP for use in measuring rat PTHRP. Circulating calcium and PTHRP concentrations were serially measured after sc passage of the Leydig cell tumor in rats. Significant hypercalcemia and elevation of PTHRP occurred on day 9 after tumor inoculation. When grouped by tumor size, both plasma calcium and PTHRP levels were significantly elevated in animals with tumor burdens greater than 10 cc. The PTHRP concentration was strongly correlated with both serum calcium (r = 0.88) and tumor size (r = 0.80). Circulating rat PTHRP averaged 12.8 pM on day 9 and 27.5 pM on day 10 or 11. PTHRP was undetectable in the plasma of 19 control rats. In 3 rats, plasma calcium returned to normal, and PTHRP became undetectable within 24 h after tumor excision. Rat milk displayed a PTHRP concentration of 2000 pM, while acid-urea extract of the rat tumor contained 0.32 pmol/mg protein. Dilutions of rat plasma, milk, and tumor extract displayed response curves that were parallel to the human PTHRP-(1-74) standard in the assay. This two-site immunoradiometric assay is a sensitive and easily performed means of measuring rat PTHRP. It should be useful in studying this animal model of HHM and the function of PTHRP in normal tissues.

Animals↗

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

Adenoviridae↗

Helper-free foamy virus vectors.

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

Base Sequence↗

Urinary biomarker profiling in transitional cell carcinoma.

Urinary biomarkers or profiles that allow noninvasive detection of recurrent transitional cell carcinoma (TCC) of the bladder are urgently needed. We obtained duplicate proteomic (SELDI) profiles from 227 subjects (118 TCC, 77 healthy controls and 32 controls with benign urological conditions) and used linear mixed effects models to identify peaks that are differentially expressed between TCC and controls and within TCC subgroups. A Random Forest classifier was trained on 130 profiles to develop an algorithm to predict the presence of TCC in a randomly selected initial test set (n = 54) and an independent validation set (n = 43) several months later. Twenty two peaks were differentially expressed between all TCC and controls (p < 10(-7)). However potential confounding effects of age, sex and analytical run were identified. In an age-matched sub-set, 23 peaks were differentially expressed between TCC and combined benign and healthy controls at the 0.005 significance level. Using the Random Forest classifier, TCC was predicted with 71.7% sensitivity and 62.5% specificity in the initial set and with 78.3% sensitivity and 65.0% specificity in the validation set after 6 months, compared with controls. Several peaks of importance were also identified in the linear mixed effects model. We conclude that SELDI profiling of urine samples can identify patients with TCC with comparable sensitivities and specificities to current tumor marker tests. This is the first time that reproducibility has been demonstrated on an independent test set analyzed several months later. Identification of the relevant peaks may facilitate multiplex marker assay development for detection of recurrent disease.

Adult↗

Oxidative Stress and Aging - Second International Conference. Technologies for assessment and intervention strategies. 2-5 April 2001, Maui, USA.

The study of oxidative contributions to aging has reached sufficient maturity to support the development of interventional strategies designed to forestall or reverse protein cross-linking, oxidation of DNA and lipids, and mitochondrial senescence associated with chronic pathology and aging. Catalytic antioxidants, including combined superoxide dismutase (SOD) and catalase mimics, extend the lifespan of oxidatively compromised animals such as Mn-SOD knockout mice, and will be entering the clinic for radiation-induced dermatitis. Substituted phenacylthiozolium compounds that slow the formation and break extant protein cross-linkages formed via Maillard reactions, restoring vascular compliance in aged animals, and are showing efficacy in clinical trials. Non-feminizing estrogen analogs (eg, 17- alpha estradiol) block cytotoxicity in a host of oxidative stress models and moderate neuronal loss in MCAO stroke models. Efficacy of the polycyclic phenols is synergistically amplified by glutathione supplementation, suggesting that the two function as a redox couple analogous to vitamin E and ascorbate. Finally, discovery of novel small molecules designed to stabilize mitochondrial function during Ca(2+)-induced oxidative stress, such as that occurring during stroke or myocardial ischemia reperfusion, will be accelerated by a proprietary fluorescence resonance energy transfer assay developed at MitoKor. Maintaining mitochondrial function under these circumstances will improve cellular bioenergetic and oxidative status, and hence moderate secondary necrosis and apoptosis.

Journal Article↗

[Detection of platelet antibodies in idiopathic thrombocytopenic purpura (author's transl)].

The amount of IgG present on the surface of platelets (platelet-bound IgG, or PBIgG) was measured using a quantitative antiglobulin consumption assay developed by Dixon et al. (1975). Normal platelets or platelets from patients with thrombocytopenia due to a decreased production had less than 10 X 10(-15) g of IgG/platelet. Studied were 364 blood samples drawn from 196 patients; 173 patients had idiopathic thrombocytopenic purpura; 65 (93 %) of 70 in the acute phase of their disease had elevated PBIgG (values up to 100 times greater than the normal); 98 (76.5 %) of 128 blood samples from patients during a chronic phase had abnormally high PBIgG but the levels of IgG were generally lower than those observed in the acute phase; 75 (75 %) of 100 samples from patients in remission did not have pathologically increased PBIgG. Usually when the platelet count improved (after prednisone therapy or after splenectomy), PBIgG decreased. The PBIgG determination was not useful for predicting the response to prednisone therapy or the further evolution of the disease, except when PGIgG remained elevated after high-dose prednisone therapy; this generally implied a poor prognosis. PBIgG have also been found elevated in other diseases such as lupus erythematosus and in one case of chronic lymphocytic leukemia. This assay may be of a great clinical interest, especially for the diagnosis of ITP.

Antibodies↗