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PubMed1976

Staphylococcal scalded-skin syndrome: development of a primary binding assay for human antibody to the exfoliative toxin.

Exfoliative toxin (ET) from a phage group II Staphylococcus aureus strain causing staphylococcal scalded-skin syndrome was purified by electrofocusing. Ampholytes and salts were removed from the final product by column chromatography on G-50 Sephadex. Sodium dodecyl sulfate-polyacrylamide gels of the final product yielded a single band upon gel electrophoresis, even when 60 mug of protein was placed in the gels. Radiolabeling of the purified toxin with 125I yielded a product that still caused exfoliation of suckling mice, indicating that the toxin was still biologically active. A radioimmunobinding assay was developed and used to test rabbit and human sera for antibodies to exfoliative toxin. Although the maximum percentage of binding was not as high as expected (approximately 40%), it was postulated that either iodination had not been sufficiently vigorous or the toxin had sustained immunological damage. The assay was reproducible and more sensitive than the existing neutralization method and readily applicable to the testing of human sera for exfoliative toxin antibodies.

Animals↗

PubMed2000

Steroid hormone activity of flavonoids and related compounds.

Soy isoflavones have been studied extensively for estrogenic and antiestrogenic properties. Other flavonoids, found in fruits, vegetables, tea and wine, have been much less tested for steroid hormone activity. We therefore assessed the estrogenic, androgenic and progestational activities of 72 flavonoids and structurally-related compounds. These compounds were tested on BT-474 human breast cancer cells at concentrations of 10(8)-10(-5) M, with estradiol (estrogen), norgestrel (progestin) and dihydrotestosterone (androgen) used as positive controls, and ethanol (solvent) as a negative control. pS2, an estrogen-regulated protein, and prostate-specific antigen (PSA), regulated by androgens and progestins, were quantified in tissue culture supernatants using ELISA-type immunofluorometric assays developed in-house. Of the 72 compounds tested, 18 showed estrogenic activity at 10(-5) M. Of this subset, seven also showed progestational activity at this concentration. The soy isoflavones, biochanin A and genistein, showed the most potent estrogenic activity, with a dose-response effect up to 10(-7) M. Of all other flavonoids, luteolin and naringenin displayed the strongest estrogenicity, while apigenin had a relatively strong progestational activity. Based on our data, we have formulated a set of structure/function relationships between the tested compounds. Flavonoids, therefore, exhibit significant steroid hormone activity, and may have an effect in the modification of cancer risk by diet, or in cancer therapeutics and prevention.

Breast Neoplasms↗

PubMed1984

Effect of Rhodococcus equi on equine polymorphonuclear leukocyte function.

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.

Animals↗

PubMed1994

Development of PCR-based assays for the detection of two human mollicute species, Mycoplasma penetrans and M. hominis.

Recently, a 16S rDNA-based polymerase chain reaction (PCR) assay was developed for the selective and sensitive detection of Mycoplasma pirum. In this study, the same procedure was used in order to selectively detect by PCR two human mycoplasmas, M. hominis and M. penetrans, with a high level of sensitivity even in a context of human DNA. For each assay, the specificity was verified by testing DNA from other mollicute species (including those closely related to the corresponding mycoplasma), from bacteria phylogenetically close to mollicutes, from Escherichia coli and from human peripheral blood mononuclear cells (PBMCs). Each assay proved to be highly sensitive since it reliably detected 10 DNA molecules, even in a context of human DNA. The results of this study demonstrate the suitability of our procedure using primers which were designed for the PCR detection of human mollicutes with a high specificity and a low and reproducible threshold of sensitivity.

Base Sequence↗

PubMed1995

Detection of circulating Trichinella spiralis muscle larva antigens in serum samples of experimentally and naturally infected swine.

A follow up study was carried out to determine the kinetics of appearance of surface/stichosomal (S/S) components, recently included in the TSL-1 group of Trichinella spiralis muscle larva (ML), in serum samples from 13 experimentally infected pigs. Detection of circulating antigens in these animals was done by a sandwich enzyme-linked immunosorbent assay (ELISA) using T. spiralis specific rabbit polyclonal immunoglobulins to capture free antigens and monoclonal antibody NIM-M1 to recognize S/S antigens. The assay developed was able to detect as little as 35 ng ml-1 of S/S components added to normal pig serum. Antigenemia was observed in 54% of the experimentally infected swine with two peaks of appearance, one early at 1-4 weeks post-infection (pi) and one late at 10-14 weeks pi. Specific antibodies against S/S components were demonstrated in serum samples from all experimentally infected pigs starting at 3-4 weeks pi. Free antigen was also detected in serum samples from naturally infected backyard pigs with a sensitivity of 56% compared with 94% when antibody production was determined using purified S/S components in an ELISA.

Animals↗

PubMed1986

Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.

A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.

Animals↗

PubMed1994

Isolation of the 36-kD German (Blattella germanica) cockroach allergen using fast protein liquid chromatography.

Cockroach allergens have been established as important sensitizing agents in the induction of asthma. In the present investigation, we identified a 36-kD allergen from German cockroach (Blattella germanica) using fast protein liquid chromatography that reacted in the Bla g II monoclonal assay developed by Pollart et al. Defatted whole body German cockroaches were extracted in phosphate-buffered saline and fractionated using gel filtration (Superdex 75) and anion exchange (MonoQ) chromatography. The 36-kD allergen was isolated from this enriched allergen preparation by excision from modified preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroelution, dialysis and lyophilization. SDS-PAGE/Western immunoblot analysis using a serum pool of cockroach-sensitive individuals revealed a single band. The lyophilized 36-kD allergen was then analyzed for amino acid composition and digested for peptide amino acid sequencing.

Allergens↗

PubMed1995

Stereospecific high-performance liquid chromatographic assay of lomefloxacin in human plasma.

This report describes an HPLC assay developed for the quantification of the enantiomers of lomefloxacin (LFLX), a quinolone antibiotic, in plasma. Following addition of racemic acebutolol (internal standard, IS), plasma samples were extracted at pH 7 with a mixture of chloroform-isopentyl alcohol-diethyl ether (71.25:3.75:25, v/v/v). The organic layer was evaporated, and LFLX and IS enantiomers in the resulting residue were derivatized with chloroform solutions of 1% triethylamine and 1% (S)-(+)-(1-naphthyl)ethyl isocyanate, followed by 2% ethyl chloroformate (ECF) 1 min later. Ethanolamine was added 30 s after the addition of ECF. The enantiomers were separated as diastereomers on an 8 x 100 mm Radial Pak normal phase column using a mobile phase of hexane-chloroform-methanol (64.5:33:2.5, v/v/v) pumped at 2.0 ml min-1. The IS was detected by fluorescence at 245 and 420 nm (excitation and emission, respectively) during the first 12 min, after which time the wavelengths were 280 and 470 nm for detection of LFLX. The method: (1) was sensitive and showed excellent linearity (10-1000 ng ml-1, r2 > 0.99) between added enantiomer concentrations and peak-area-ratio (LFLX/IS); and (2) separated LFLX and IS enantiomers within 25 min. The assay is suitable for the quantification of LFLX enantiomers in plasma samples.

Anti-Infective Agents↗

PubMed1994

Optimization of 32P-postlabelling assays for the quantitation of O6-methyl and N7-methyldeoxyguanosine-3'-monophosphates in human DNA.

The 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling. Under optimal conditions, the labelling efficiencies of O6-MedGp and N7-MedGp were respectively approximately 100 and approximately 15%, with detection limits of approximately 1.1 and approximately 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [3H]-O6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O6-MedGp, indicating that no inhibitory factors co-eluted with the O6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O6-MedGp was approximately 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 mumol O6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32P-postlabelled. Whereas O6-MedGp was detected at levels between 0.3 and 3.4 mumol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.

Chromatography, Affinity↗

PubMed2005

Towards quantitative biology: integration of biological information to elucidate disease pathways and to guide drug discovery.

Developing a new drug is a tedious and expensive undertaking. The recently developed high-throughput experimental technologies, summarised by the terms genomics, transcriptomics, proteomics and metabolomics provide for the first time ever the means to comprehensively monitor the molecular level of disease processes. The "-omics" technologies facilitate the systematic characterisation of a drug target's physiology, thereby helping to reduce the typically high attrition rates in discovery projects, and improving the overall efficiency of pharmaceutical research processes. Currently, the bottleneck for taking full advantage of the new experimental technologies are the rapidly growing volumes of automatically produced biological data. A lack of scalable database systems and computational tools for target discovery has been recognised as a major hurdle. In this review, an overview will be given on recent progress in computational biology that has an impact on drug discovery applications. The focus will be on novel in silico methods to reconstruct regulatory networks, signalling cascades, and metabolic pathways, with an emphasis on comparative genomics and microarray-based approaches. Promising methods, such as the mathematical simulation of pathway dynamics are discussed in the context of applications in discovery projects. The review concludes by exemplifying concrete data-driven studies in pharmaceutical research that demonstrate the value of integrated computational systems for drug target identification and validation, screening assay development, as well as drug candidate efficacy and toxicity evaluations.

Animals↗

PubMed2004

Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain.

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis ) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14+/-0.013 mM and 0.19+/-0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a K(i) of 2.1+/-0.9 nM. A truncated BC domain that included amino acids 2-560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1+/-0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

Acetyl Coenzyme A↗

PubMed1996

Function of beta 1 integrins on human decidual cells during implantation.

In the present study, the effects of antibodies against specific beta 1 integrin heterodimers on mouse embryo attachment and spreading were tested to identify the role of the beta 1 integrins in early implantation. We developed assays for the attachment of mouse embryos and for trophoblastic spreading on cultured human decidual cells. Blastocysts became attached to the cultured decidual cells in the presence of a purified mouse monoclonal IgG1 antibody (negative control) after the embryos hatched from the zona pellucida. The majority of hatched blastocysts attached within 24 h of culture. Blastocysts that were attached to decidual cells exhibited extensive outgrowth after 48 h. The addition of antibodies directed against the beta 1 and alpha integrin subunits to the cultured decidual cells did not affect the rates of hatching or attachment of the blastocysts. However, the outgrowth of embryos on the decidual cells was inhibited by the addition of a monoclonal antibody against the beta 1 subunit in a dose-dependent manner, implying that blastocyst attachment and outgrowth are mediated by different mechanisms. Although the area of trophoblast outgrowth in the presence of a mouse monoclonal lgG1 antibody was increased during 96 h of culture, anti-beta 1 antibody blocked the outgrowth of trophoblasts in a dose-dependent manner. Both the incidence and extent of trophoblastic outgrowth were also significantly reduced in the presence of antibodies against the alpha 1, alpha 2, alpha 5, and alpha 6 subunits. These observations suggest that beta 1 integrins on decidual cells may be involved in blastocyst development and differentiation following attachment.

Adult↗

PubMed2000

p21WAF1 protein expression determined by quantitative immunoassay in relation to non-small-cell lung cancer aggressiveness.

PURPOSE: p21WAF1, a cyclin-dependent kinase inhibitor, is an important mediator of the cell-cycle arrest and tumor suppression induced by the protein p53. Although alterations of the p53 gene and its overexpression are frequent in most malignancies, including non-small-cell lung cancer (NSCLC), and may be associated with poor patient prognosis, the clinical utility of p21WAF1 expression in NSCLC has not been established. METHODS: We have used a commercial enzyme-linked immunosorbent assay (ELISA) kit for p21WAF1 to test soluble extracts of 54 NSCLC specimens with known clinicopathological properties. RESULTS: There was no correlation between p21WAF1 and p53 concentrations, the latter being determined by a time-resolved immunofluorometric assay developed in-house. Furthermore, p21WAF1 levels were not associated with patient age, tumor/node/metastasis (TNM) stage, lymph node metastasis, histological grade or type, or smoking history, in Mann-Whitney analysis. chi2-tests, based on cutoffs equal to the 25th, 50th, or 75th percentiles of the p21WAF1 distribution, similarly did not reveal any statistically significant associations between p21WAF1 and other clinicopathological variables. Because of the small number of patients and the median follow-up of only 18 months, a meaningful survival analysis could not be performed. CONCLUSION: In summary, this preliminary study suggests that ELISA-quantified p21WAF1 levels in NSCLC extracts are weaker than p53 in terms of prognostic value and do not contribute to the further subclassification of patients.

Aged↗

PubMedDate not supplied

Prevalence of specific IgA and IgM anti-HBc antibodies compared with HBV DNA in the sera of HBsAg chronic carriers.

OBJECTIVE: We evaluated the significance of IgA antibodies directed against the hepatitis B virus core antigen (IgA anti-HBc) as a marker for viral replication. STUDY DESIGN/METHODS: Serum samples of 143 hepatitis B surface antigen (HBsAg) carriers and 189 HBsAg-negative subjects were studied. Hepatitis B virus (HBV) DNA was detected by polymerase chain reaction. IgA anti-HBc was determined by a capture enzyme-linked immunosorbent assay developed in our laboratory. The results were compared with those for IgM anti-HBc, which were determined by a commercially available method. RESULTS: IgA anti-HBc was detected in 57 (40%) and HBV DNA in 38 (27%) of the HBsAg carriers. Among the HBsAg-negative subjects, IgA anti-HBc and HBV DNA were detected simultaneously in four samples. All 42 HBV DNA-positive samples were IgA anti-HBc positive. IgM anti-HBc was detected in 27 (64%) of them. CONCLUSIONS: IgA anti-HBc is a sensitive marker for HBV replication, and its absence may exclude HBV replication. The role of IgA anti-HBc in monitoring response to therapy and predicting clinical course is being evaluated.

Biomarkers↗

PubMed2005

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis.

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

Bacterial Typing Techniques↗

PubMed1997

Initial evaluation of CYFRA 21-1 diagnostic performances as a urinary marker in bladder transitional cell carcinoma.

PURPOSE: CYFRA 21-1, an immunoradiometric assay developed for the detection of a soluble cytokeratin 19 fragment, is evaluated for its diagnostic performance in urine of patients with transitional cell carcinoma. MATERIALS AND METHODS: CYFRA 21-1 was investigated in serum and urine of 128 patients, including 48 with bladder transitional cell carcinoma (group 1), 44 with other urological pathological conditions (group 2) and 36 free of urothelial disease (group 3). Urinary cytopathology was also performed. RESULTS: Mean urinary CYFRA was 123.5 +/- 53, 11.9 +/- 4.8 and 2.3 +/- 0.2 ng./ml. for groups 1 to 3, respectively, and was significantly different. From the receiver operating characteristics curve, the optimal combination of 96% sensitivity and 74% specificity was determined for a threshold value of 4 ng./ml. while overall cytopathology sensitivity was 43%. CONCLUSIONS: Urinary CYFRA 21-1 may be a useful marker for diagnosing transitional cell carcinoma.

Aged↗

PubMed1994

Lack of an immune response to Albunex, a new ultrasound contrast agent based on air-filled albumin microspheres.

Albunex is a new ultrasound contrast agent consisting of air-filled microspheres of heat-aggregated human albumin suspended in a 5% (w/v) solution of human albumin for infusion. The structural alterations induced in the albumin molecules during heat aggregation may cause the formation of new epitopes that may provoke an immunological response in recipients. To assess the possible immunogenicity of the contrast agent, 34 healthy volunteers were randomized to receive four injections of either Albunex or 5% human serum albumin (0.01 ml/kg) at 4-week intervals. Analysis of blood samples taken 3 weeks after each injection did not reveal any formation of IgE or IgG antibodies directed against the Albunex microsphere protein or albumin in any of the recipients. The evaluation of novel enzyme immunosorbent assays developed to detect the presence of these specific IgE and IgG antibodies is described. It was also investigated whether the heat-aggregated microsphere albumin was structurally altered in such a way that it could be recognized by cat albumin specific IgE, present in the sera of individuals allergic to cat albumin. 187 serum samples shown to contain elevated levels of cat albumin specific IgE were used in the study. No cross-reactive binding of the heat-aggregated human albumin to anticat albumin IgE could be detected. There is no evidence from the present studies that the heat-aggregated albumin of the Albunex microspheres will provoke an immunological reaction upon repeated injections or cause an adverse reaction when administered to allergic individuals having cat albumin as one of their allergens.

Adolescent↗

PubMed2002

Development of a microslide agglutination assay with the aid of an inexpensive projection microscope.

A microslide agglutination assay was developed involving the mixing of 2.5 microl each of antiserum and a cell suspension of Listeria monocytogenes. Cell agglutination in the final volume of 5.0 microl was visually observed at a direct magnification of 22 x on the projection screen of an inexpensive 20 US dollar projection microscope. The procedure has the advantage of increasing by a factor of 20 the number of agglutination assays that can be performed with a given volume of antiserum with the use of an inexpensive optical projection system.

Agglutination Tests↗