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Improved fermentation and isolation procedures for antitumor antimetabolites U-42,126 and U-43,795 increased drug yields 30-fold. The sensitivity limit of a newly developed assay is 0.03 mug of U-42,126 and 2.0 mug of U-43,795 per ml. The in vitro antimicrobial effect of both drugs was antagonized by histidine.
The utilisation of 31 sole carbon sources by bacterial communities of soil in the presence of increasing concentrations of Hg(II) was measured by a colour development assay. The assay was performed on Biolog microtitre plates (Ecoplates) in the presence of Hg(II) and compared to Hg(II)-free Ecoplates. Furthermore, community tolerance to Hg(II) was measured by colour development in microtitre plates supplemented with LB broth and by enumeration of colony-forming units on LB agar plates. Both microtitre plates supplemented with LB and LB agar plates contained increasing concentrations of Hg(II). The difference in substrate utilisation profile, as shown by growth on 31 different carbon substrates in the Ecoplates, suggested an adaptation of the soil community that correlated with the metal exposure level in the soil. Similarly, growth on microtitre plates supplemented with LB and plate-spreading data showed an increased community tolerance with increasing levels of mercury in the soil. Both the multi-function microtitre plate assay (Ecoplate) and the LB broth microtitre plate assay are suitable for evaluating the adaptation of the bacterial community in soil to a heavy metal pollutant.
Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.
Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.
A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA).
We present a rapid flow cytometric and non-radioactive functional assay developed for the determination of the cytotoxic activity of T lymphocytes, natural killer cells, and lymphokine-activated killer cells. In contrast to indirect evaluation of cytotoxicity using radioactive assays, this assay is based on the quantitative and qualitative flow cytometric analysis of cell damage on a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane, without disturbing its surface marker expression. It, thus, permits the distinction between target and effector cells. After short term in vitro incubation (1.5-3h), AnnexinV-FITC (ann-FITC) staining allows to discriminate between apoptotic and non-apoptotic target cells. Data analysis is performed first by gating on PKH-26 positive target cells, followed by the analysis of the ann-FITC positive subpopulation. The percentage of cytotoxicity in the PKH-26 gated cell population is calculated by subtracting unspecific ann-FITC positive target cells, measured in appropriate controls without effector cells. Using in vitro generated antigen-specific cytotoxic T lymphocytes, we demonstrate that this flow cytometric assay is sensitive, correlates well with the standard 51Cr release assay, and is easy to handle.
Monitoring anthelmintic resistance in strongyle nematodes by the faecal egg count reduction test and a commercial larval development assay on an organized sheep farm in the semi-arid area of Rajasthan revealed the emergence of resistance to benzimidazoles and rafoxanide and a potential risk of the development of levamisole/tetramisole resistance. A benzimidazole/levamisole combination, avermectins and closantel were each found to be efficacious.
To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.
A simple, rapid and highly sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (AFB1)-DNA adducts is reported. Polyclonal antibodies specific to the aflatoxin B1-N7-guanine adduct were produced using a novel synthetic antigen, bovine serum albumin (BSA)-guanine-AFB1. The antibodies were characterized by the Ouchterlony double diffusion technique and by antibody capture assay. The working range of the indirect competitive assay developed was between 0.45 and 330 ng of the analyte [calf thymus (CT)-DNA-AFB1]. A 50% inhibition was attained at 15 ng of the analyte (CT-DNA-AFB1). The antibody capture assay indicated that the antibody produced cross-reacted 100, 92 and 110% with BSA-guanine-AFB1, CT-DNA-AFB1 and CT-DNA-formamidopyrimidine-AFB1, respectively. When free AFB1 and guanine were used as competing analytes, the antibodies showed < or = 5% and zero cross-reactivity at the 50% inhibition level. Spiking studies indicated a recovery in the range 96-97 and 74-78% when standard CT-DNA-AFB1 was added to 10 mM phosphate buffer (pH 7.2) and control rat liver tissue, respectively. Rats exposed to a single oral dose of 1 mg kg-1 body mass of pure AFB1 were used to validate the method. The AFB1-DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB1-DNA adduct ranged between 6.06 and 7.94 micrograms mg-1 DNA. The proposed method may find application in the biological monitoring of aflatoxin B1 in molecular epidemiological studies to assess the dietary exposure of aflatoxins.
Tacrolimus (FK506) is a widely used immunosuppressant for preventing allograft rejection and the treatment of atopic dermatitis. FK506 necessitates therapeutic drug monitoring because of inter- and intrapatient variability and the lack of correlation between the administered dose and the blood concentration. Previous immunoassay-based methods required a relatively long assay time and troublesome liquid-handling procedures. In the present study, we aimed to establish a rapid monitoring method for FK506 determination by using a poly(dimethylsiloxane) (PDMS)-based microfluidic device. Polystyrene beads were coated with mouse anti-FK506 antibody and placed in the flow channel. As a competitive assay, sample solution was allowed to react in the flow channel. After the addition of the fluorogenic substrate, the fluorescent signal was observed under a microscope. As a result, the developed assay allowed a short detection time of approximately 15 min per each sample and a high sensitivity even by using only a single bead. The feasibility of performing a competitive assay using a PDMS-based antibody chip gives promising results over the existing immunoassay-based methods.
Over 500 studies have examined the association of genetic variants of glutathione S-transferases with various malignancies yielding inconsistent results. The genotyping was based on PCR assays that identified the GSTM1 and GSTT1 null (-/-) genotypes but did not distinguish homozygous wild-type +/+ and heterozygous +/- individuals. Complete GSTM1 and GSTT1 genotyping can be accomplished by recently developed assays [Cancer Res. 64 (2004) 1233-1236; Pharmacogenetics 10 (2000) 557-565] that allow the definition of +/+, +/-, and -/- genotypes by separate identification of the respective GSTM1 and GSTT1 wild-type and null alleles. Application of the new GSTM1 assay to a breast cancer case-control study revealed that the relative risk of breast cancer for the +/+ genotype compared to the -/- genotype was 2.83 (95% confidence interval 1.45-5.59; P=0.002), suggesting a protective effect of the GSTM1 deletion [Cancer Res. 64 (2004) 1233-1236]. Regardless of the explanation for the association between the +/+ genotype and increased breast cancer risk, these results warrant application of true GSTM1 and GSTT1 genotyping to additional or previously analyzed groups with breast cancer or other malignancies.
The sensitive/less-sensitive (S/LS) enzyme immunoassay (EIA) testing strategy for discriminating "early" from "longstanding" HIV infection has been widely applied for detecting recent seroconverters and estimating HIV incidence rates. The originally developed assay (3A11-LS EIA; Abbott Laboratories, Abbott Park, IL) involved performance of LS EIAs using a bead-based assay that required specialized equipment and reagents of limited availability. In contrast, 96-microwell-based EIAs are more universally applied for HIV serodiagnosis throughout the world. The authors report development and preliminary validation of an LS protocol using an EIA in a 96-well format: the Vironostika HIV-1 MicroElisa System (Vironostika-LS EIA; Bio Merieux, Raleigh, NC). The results with samples from recent HIV-1 seroconverters, persons with longstanding HIV-1 asymptomatic infection, patients on highly active antiretroviral therapy, and AIDS patients show a high degree of correlation between the Vironostika-LS EIA and 3A11-LS EIA. The authors also demonstrate that the Abbott 3A11-LS EIA and Vironostika-LS EIA performed comparably on HIV-1-positive samples from persons infected with non-B HIV-1 subtypes. These results support the potential use of the Vironostika-LS EIA for detection of recent HIV-1 infections for incidence projections and for other epidemiologic, clinical, and molecular surveillance applications.
We used the nested polymerase chain reaction (PCR) assays developed previously to detect and identify Mycobacterium tuberculosis (M. tuberculosis) in the cerebrospinal fluid (CSF) samples from patients with suspected tuberculous meningitis and non-tuberculous patients. Our nested PCR assays target the multi-copy IS6110 insertion element and the single-copy mtp40 genomic DNA of M. tuberculosis. These assays, when used in combination, allowed us to detect a very low number of M. tuberculosis in the CSF samples, which otherwise would be undetectable by the culture method, and to distinguish M. tuberculosis from M. bovis. We applied these nested PCR assays to analyze eleven CSF samples. Among these, five of them were from patients with suspected tuberculous meningitis but all except one were culture negative. Our results of PCR assays show that three of these five are M. tuberculosis positive, one of which is M. bovis positive, and only one is M. tuberculosis negative. The other six CSF samples were from the clinically diagnosed non-tuberculous patients. Surprisingly, two of these so called non-tuberculous patients, a subarachnoid hemorrhage (SAH) and the syndrome of inappropriate antidiuretic hormone secretion (SIADH), were shown M. tuberculosis positive. This finding supports a long-standing argument that tuberculous meningitis is one of the causes of these neurological diseases. These nested PCR assays thus provide the neurologists with an important adjunct, in addition to the patient's clinical presentation and laboratory data, for the diagnosis of tuberculous meningitis.
Protease inhibitor (PI) monitoring may improve the care of human immunodeficiency virus (HIV)-infected patients; however, pediatric data are limited. A high-performance liquid chromatographic (HPLC) assay developed for the simultaneous determination of indinavir, ritonavir, saquinavir, and nelfinavir in 0.2 mL of plasma was used to quantify PI concentrations in HIV patients. The reliability, sensitivity, and specificity of the assay were first verified in stored adult samples. Later, blood collected prospectively from patients aged 2.9 to 42 years of age (10 adults aged 24 to 42 and 15 children aged 2.9 to 18 years) was tested. Nondetectable (below 25-50 ng/mL) concentrations (ND) were found in 33% of adult samples and 24% of pediatric samples. Four patients taking from 13.7 to 28 mg/kg/d of ritonavir (mean of 21.3) had concentrations ranging from ND to 11.4 microg/mL, quite different from predicted values. Correctable, important clinical problems including drug-drug interactions, drug administration problems, and confirmed noncompliance were identified and corrected using modern therapeutic drug monitoring (TDM) techniques. Routine PI monitoring and interpretation (TDM) could improve the care of adult and pediatric HIV patients; especially in patients who do not respond as expected to treatment, develop viral resistance or toxicity, and have questionable compliance.
Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.
PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.
The higher proportion of repeated DNA sequences in the garden pea (Pisum sativum) than in the mung bean (Vigna radiata), as well as other differences between these legume genomes, are consistent with a higher rate of sequence amplification in the former. This hypothesis leads to a prediction that repeated sequence families in Pisum are mostly heterogeneous, as defined by Bendich and Anderson (1977), while Vigna families are homogeneous. An assay developed by these authors to distinguish between the two types of families, by comparison of reassociation rates at different temperatures, was utilized. The results for Vigna defied the predictions of the assay for either homogeneous or heterogeneous model. Evaluation of the kinetic data in light of the great diversity of repeated family copy numbers in both genomes enabled an interpretation of the results as consistent with heterogeneous families in Pisum and homogeneous families in Vigna. These tentative conclusions were supported by the results of a thermal denaturation (melting) assay described in the accompanying paper.
In vitro (larval development assay) and in vivo studies were conducted to determine possible direct anthelmintic effect of ethanolic and aqueous extracts of Spigelia anthelmia towards different ovine gastrointestinal nematodes. The effect of extracts on development and survival of infective larvae stage (L(3)) was assessed. Best-fit LC(50) values were computed by global model of non-linear regression curve fitting (95% confidence interval). Therapeutic efficacy of the ethanolic extracts administered orally at a dose rate of 125, 250, and 500 mg/kg, relative to a non-medicated control group of sheep harbouring naturally acquired infection of gastrointestinal nematodes, was evaluated in vivo.The presence of S. anthelmia extracts in the cultures decreased the survival of L(3) larvae. The LC(50) of aqueous extract (0.714 mg/ml) differ significantly from the LC(50) of the ethanolic extract (0.628 mg/ml) against the strongyles (p < 0.05, paired t-test). Faecal egg counts on day 12 after treatment showed that the extract is effective, relative to control (one-way analysis of variance [ANOVA], Dunnett's multiple comparison test) at 500 mg/kg against Strongyloides spp. (p < 0.01), 250 mg/kg against Oesophagostomum spp., Trichuris spp. (p < 0.05), and 125 mg/kg against Haemonchus spp. and Trichostrongylus spp. (p < 0.01). The effect of the doses is significant in all cases, the day after treatment is also extremely significant in most cases, whereas interaction between dose and day after treatment is significant (two-way ANOVA). S. anthelmia extract could, therefore, find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.