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Detection of toxin production by Bacteroides fragilis: assay development and screening of extraintestinal clinical isolates.

Strains of Bacteroides fragilis that produce an extracellular 20-kD heat-labile toxin have been epidemiologically associated with diarrheal disease. To standardize detection of the B. fragilis toxin (BFT), a detailed description of the methods for identifying BFT production by B. fragilis strains is reported. To further study the role of toxigenic strains of B. fragilis in clinical disease, extraintestinal clinical isolates that were recovered at an urban east-cost hospital were evaluated. Four (6.2%) of 65 isolates produced BFT. Three cases in which BFT-producing B. fragilis were isolated from extraintestinal sites are reported. In contrast to studies from Japan that have reported BFT production by 23.4% of extraintestinal B. fragilis isolates, we conclude that BFT-producing strains of B. fragilis are infrequently associated with extraintestinal B. fragilis disease at our institution. Additional study will be necessary to better define the importance of BFT production in human disease due to B. fragilis.

Adult↗

A newly developed assay to study the minimum inhibitory concentration of Satureja spinosa essential oil.

AIMS: The minimum inhibitory concentration (MIC) of Satureja spinosa essential oil against Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Salmonella serovar Enteritidis PT4 and Bacillus cereus was comparatively assessed with an established optical density method as well as a novel impedimetric method. METHODS AND RESULTS: The impedimetric analysis takes into account information of microbial growth, such as detection time, maximum conductance, and slope of the conductance curve. For each pathogen two levels of inoculation were studied, a high (10(5) CFU ml(-1)) and a low level (10(2) CFU ml(-1)). Non-linear regression analysis was used to fit the data using a modification of a previously published model, from which a more exact value can be obtained for the MIC. Both methods gave similar MICs as shown by t-test statistical analysis. Salm. Enteritidis seems to be the least sensitive to the action of S. spinosa essential oil followed by L. monocytogenes, E. coli, B.cereus and Staph. aureus. The MICs of low inoculum were lower than that of high inoculum. CONCLUSIONS: The new impedimetric assay of MIC of essential oils can be considered a reliable rapid method for screening antimicrobial effectiveness of natural additives. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the minimum inhibitory concentration of an essential oil with the simple conductance technique and further study of the mode of action of its components is a good combination for obtaining additional knowledge for industrial application of such natural additives.

Anti-Bacterial Agents↗

Effect of bisphosphonates on cartilage turnover assessed with a newly developed assay for collagen type II degradation products.

BACKGROUND: Animal studies of arthritis have suggested that bisphosphonates may have chondroprotective abilities. OBJECTIVE: To evaluate the effect of bisphosphonate treatment on cartilage degradation. METHODS: Type II collagen is almost exclusively localised in cartilage, where it is the major structural component of the tissue. Hence fragments derived from this protein should represent a specific index for cartilage degradation. The urinary concentration of collagen type II C-telopeptide degradation products (CTX-II) was measured by a new immunoassay (enzyme linked immunosorbent assay (ELISA)). The serum concentration of collagen type I C-telopeptide degradation products (CTX-I), a marker of bone degradation, was also measured by ELISA. PARTICIPANTS: Two groups were studied. The alendronate group included 63 healthy postmenopausal women aged 45-54 randomly allocated to receive three years' treatment with 1 mg, 5 mg, 10 mg, or 20 mg alendronate daily or placebo. In the third year the women receiving 20 mg were switched to placebo. The ibandronate group included 119 women at least 10 years after the menopause aged <75 randomly allocated to receive 12 months' treatment with 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5 mg ibandronate daily or placebo followed by 12 months without treatment. RESULTS: 20 mg of alendronate and 2.5 and 5 mg of ibandronate treatment produced significant decreases in urinary CTX-II to about 50% of baseline. The level reached after three months of treatment remained practically constant during the following 12-36 treatment months. When treatment was withdrawn CTX-II values returned towards baseline. Serum CTX-I also decreased rapidly within three months, but to a level of about 30% of baseline. CONCLUSIONS: The urinary excretion of CTX-II, a new marker of cartilage degradation, decreases significantly in response to bisphosphonate. This suggests that bisphosphonates may have chondroprotective effects in humans. By measurement of CTX-II it should be possible to monitor the effects of drugs that potentially inhibit cartilage destruction.

Alendronate↗

Cartilage turnover assessed with a newly developed assay measuring collagen type II degradation products: influence of age, sex, menopause, hormone replacement therapy, and body mass index.

BACKGROUND: Cartilage normally has a slow turnover but in arthritis increased metabolism results in degradation of the tissue. OBJECTIVE: To assess cartilage turnover in a sample of the general population by an assay measuring cartilage derived urinary collagen type II (CTX-II) C-telopeptide degradation products. METHODS: CTX-II concentrations were measured in urine samples from 615 healthy men and women aged 20-87 years, and the influence of age, sex, menopause, hormone replacement therapy (HRT), and body mass index (BMI) was assessed. RESULTS: CTX-II concentrations showed age dependent variations, with notable differences between men and women. Mean (SD) CTX-II concentration in postmenopausal women (220 (118) ng/mmol, n=25) was significantly higher than in an age matched group of premenopausal women (112 (79) ng/mmol, n=26, p<0.001). CTX-II concentration in women using HRT (118 (57) ng/mmol, n=50) was significantly lower than in an age and BMI matched group of women not receiving HRT (215 (99) ng/mmol, n=50, p<0.001). In subjects with a BMI >or=25 kg/m(2), CTX-II concentrations were significantly higher than in those with a BMI <25 kg/m(2) (185 (114) v 148 (91) ng/mmol, p<0.001). CONCLUSIONS: Cartilage turnover, as assessed by measuring urinary degradation products of CTX-II varies considerably with age, and significant differences between CTX-II levels in men and women as well as in pre- and postmenopausal women are found. Further studies are required to validate the marker for assessing cartilage degradation in arthritis.

Adult↗

Measurement of circulating salmon IGF binding protein-1: assay development, response to feeding ration and temperature, and relation to growth parameters.

Fish plasma/serum contains multiple IGF binding proteins (IGFBPs), although their identity and physiological regulation are poorly understood. In salmon plasma, at least three IGFBPs with molecular masses of 22, 28 and 41 kDa are detected by Western ligand blotting. The 22 kDa IGFBP has recently been identified as a homolog of mammalian IGFBP-1. In the present study, an RIA for salmon IGFBP-1 was established and regulation of IGFBP-1 by food intake and temperature, and changes in IGFBP-1 during smoltification, were examined. Purified IGFBP-1 from serum was used for as a standard, for tracer preparation and for antiserum production. Cross-linking (125)I-labelled IGFBP-1 with salmon IGF-I eliminated interference by IGFs. The RIA had little cross-reactivity with salmon 28 and 41 kDa IGFBPs (< 0.5%) and measured IGFBP-1 levels as low as 0.1 ng/ml. Fasted fish had significantly higher IGFBP-1 levels than fed fish (21.6 +/- 4.6 vs 3.0 +/- 2.2 ng/ml). Plasma IGFBP-1 was measured in individually tagged 1-year-old coho salmon reared for 10 weeks under four different feeding regimes as follows: high constant (2% body weight/day), medium constant (1% body weight/day), high variable (2% to 0.5% body weight/day) and medium variable (1% to 0.5% body weight/day). Fish fed with the high ration had lower IGFBP-1 levels than those fed with the medium ration. Circulating IGFBP-1 increased following a drop in feeding ration to 0.5% and returned to the basal levels when feeding ration was increased. Another group of coho salmon were reared for 9 weeks under different water temperatures (11 or 7 degrees C) and feeding rations (1.75, 1 or 0.5% body weight/day). Circulating IGFBP-1 levels were separated by temperature during the first 4 weeks; a combined effect of temperature and feeding ration was seen in week 7; only feeding ration influenced IGFBP-1 level thereafter. These results indicate that IGFBP-1 is responsive to moderate nutritional and temperature changes. There was a clear trend that circulating IGFBP-1 levels were negatively correlated with body weight, condition factor (body weight/body length(3) x 100), growth rates and circulating 41 kDa IGFBP levels but not IGF-I levels. During parr-smolt transformation of coho salmon, IGFBP-1 levels showed a transient peak in late April, which was opposite to the changes in condition factor. Together, these findings suggest that salmon IGFBP-1 is inhibitory to IGF action. In addition, IGFBP-1 responds to moderate changes in dietary ration and temperature, and shows a significant negative relationship to condition factor.

Animals↗

Assay Development of a Serum Marker for Myocardial Injury: Experiences with an Avian anti-Troponin I Antibody.

The diagnosis of acute myocardial infarction is still difficult in certain cases. The measurement of human cardiac Troponin I, a new marker for heart muscle cell necrosis, can help to support current procedures of diagnosis. The detection is usually done with antibodies in a sandwich immunoassay. For development of such a test for human cardiac Troponin I on the automated analyser Technicon Immuno 1 avian egg yolk antibodies were tested. Antibody preparations from two immunised chickens were tested for content of specific anti-Troponin I antibodies. Following affinity purification against a peptide from the Troponin I sequence the antibodies were conjugated with alkaline phosphatase and used as detector antibodies. The combination with different monoclonal antibodies resulted in standard curves for Troponin I, which were in terms of sensitivity inferior to a commercial polyclonal antibody from goat.

Journal Article↗

One-step, labeled-antibody assay for measuring free thyroxin. I. Assay development and validation.

We describe a one-step, labeled-antibody radioassay for measuring free thyroxin (FT4) in serum or plasma, based on a novel principle. FT4 in the sample competes with a gross molar excess (over antibody) of a cross-reactant (L-triiodothyronine, T3), chemically coupled to magnetizable polymer particles, for binding to avid 125l-labeled monoclonal anti-thyroxin antibodies. As in conventional immunoassays, 125I counts bound to the solid phase (T3-magnetizable particles) are inversely proportional to sample FT4 concentration. We demonstrate here the development and technical validity of this new method.

Antibodies, Monoclonal↗

Immunological measurement of transforming growth factor-beta 1 (TGF-beta1) in blood; assay development and comparison.

Development of a new, sensitive immunoassay for measuring transforming growth factor beta 1 (TGF-beta1) is described and compared with four commercially available TGF-beta1 immunoassays. Preanalytical conditions were evaluated. The nonlinearity found in serum or plasma is due to masking of TGF-beta1 by binding proteins in blood. Mixing TGF-beta1 with latency-associated peptide or alpha2-macroglobulin at physiological concentrations suppressed most of the TGF-beta1 signal. Plasma fibronectin showed no effect, even at concentrations exceeding its physiological range. Equilibrium concentrations computed from a model system confirmed the experimental results. Dilutional nonlinearity could be markedly reduced by an appropriately designed activation procedure that minimized the effects of reassociation between TGF-beta1 and its binding partners during restoration of a neutral pH. Plasma should be used for measuring TGF-beta1 in blood. Because serum TGF-beta1 is highly significantly correlated with the platelet count, probably most of the TGF-beta1 is released by platelet degranulation.

Cross Reactions↗

Substrate capacity considerations in developing kinase assays.

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased. In contrast, when the biotin-peptide was captured onto NeutrAvidin-coated plates, the finite peptide binding capacity of the plates limited the amount of substrate that could be incorporated into the assay system and thereby limited the rate of the reaction at a given kinase concentration. Solution-phase kinase reactions can tolerate high substrate concentrations; however, analysis of kinase reaction samples containing biotin-peptide concentrations higher than the binding capacity of NeutrAvidin-coated plates resulted in an inability to detect differences between reactions run at different substrate concentrations. For AlphaScreen detection following solution-phase kinase reactions, limitations in the binding capacity of the donor and acceptor beads caused loss of signal for substrate concentrations above the maximum binding capacity. Overall, the solution-phase assays required significantly more kinase than the in-plate assays (1-4 microg/ml versus <100 ng/ml, respectively). These studies demonstrate that the amount of substrate that can be incorporated into an assay system substantially affects the rate of the kinase reaction and therefore the amount of kinase required for the assay.

Enzyme-Linked Immunosorbent Assay↗

TSH-displacing activity versus TSH-binding inhibiting activity of immunoglobulins from patients with Graves' disease.

Inhibition of binding of TSH to thyroid plasma membranes by immunoglobulins G (IgG) from patients with Graves' disease has been interpreted as evidence of an interaction with the TSH receptor, but other explanations may account for this phenomenon. Displacement of receptor-bound TSH might be a more direct index of such an interaction. The aim of the present study was to assess TSH-displacing activity (TDA) of Graves' IgG by a newly developed assay and compare TDA to TSH-binding inhibiting activity (TBIA) and thyroid-stimulating antibody (TSAb) activity. TDA was assessed by preincubating 125I-TSH with thyroid plasma membranes and by measuring residual bound 125I-TSH after addition of IgG. TBIA was evaluated by determining binding of 125I-TSH to thyroid plasma membranes preincubated with IgG. Results were expressed as % TSH-displacing or binding inhibition activities of test IgG with respect to normal control IgG. The percent of TDA positive Graves' IgG preparations (72.7%) was greater than that of TBIA positive (48.5%). There was no correlation between TDA and TBIA results, since 11 Graves' IgG preparations were TDA positive but TBIA negative, and 6 were TDA negative but TBIA positive. Hashimoto's or idiopathic myxedema IgG preparations were positive in 2/7 in the TDA and in 1/7 in the TBIA assays, respectively. Neither TDA nor TBIA correlated with TSAb determined by cAMP accumulation in thyroid plasma membrane. The TDA assay developed in this study appeared to be more sensitive than the more widely used TBIA assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗

Miniaturized HTS technologies - uHTS.

The transition from slow, manual, low-throughput screening to industrialized robotic ultra-high throughput screening (uHTS) in the past few years has made it possible to screen hundreds of thousands of chemical entities against a biological target in a short time-frame. The need to minimize the cost of screening has been addressed primarily by reducing the volume of sample to be screened. This, in turn, has resulted in the miniaturization of HTS technology as a whole. Miniaturization requires new technologies and strategies for compound handling, assay development, assay adaptation, liquid handling and automation in addition to refinement of the technologies used for detection systems and data management. This review summarizes current trends in the field of uHTS and illustrates the technological developments that are necessary to enable the routine application of miniaturized uHTS systems within an industrial environment.

Journal Article↗

Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.

Human respiratory syncytial virus (RSV) is a major causative agent of lower respiratory tract infections in children and the elderly. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed assay to amplify the genome of RSV subgroups A and B, in order to improve current diagnostic methods for RSV infection. The primer sets for RT-LAMP were designed using highly conserved nucleotide sequences in the matrix protein region of subgroups A and B, and were specific for each subgroup. The RT-LAMP efficiency was compared to virus isolation and a commercially available enzyme immunoassay (EIA) for RSV detection (BD Directigen EZ RSV test), using nasopharyngeal aspirates from 59 children with respiratory tract infections. The RT-LAMP was specific for RSV and could not detect other respiratory pathogens. 61% (36/59) of children were positive by RT-LAMP, 34% (20/59) by viral isolation, and 56% (26/46) by EZ RSV. Of 16 specimens that were negative by both antigen detection and virus isolation, 12.5% (2/16) were RT-LAMP positive. These results suggest that the RT-LAMP is more sensitive than other methods used to detect RSV. The RT-LAMP assay developed in this study may be useful for diagnostic and epidemiological studies of RSV infection.

Base Sequence↗