SEARCH · PubMed Health

Results for “In vitro assay”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed1988

Evaluation of two in vitro assays to screen for potential developmental toxicants.

To evaluate two in vitro assays for their ability to detect known developmental toxicants and nontoxicants, a series of 44 coded compounds were assayed by two independent laboratories using standardized protocols. The two test systems were (1) the human embryonic palatal mesenchymal cell growth inhibition assay and (2) the mouse ovarian tumor cell attachment inhibition assay. After all compounds were tested, they were decoded and ranked according to the minimum IC50 value (the millimolar concentration of compound which inhibits growth or attachment by 50% compared to the solvent control) from either test. The in vitro test result concordance with established in vivo animal and human test results was examined over a wide range of concentration levels (above which the in vitro results were called positive and below which they were considered negative). A positive response from either test was defined as a positive in vitro response. Concordance was defined as the number of correct responses divided by the number of chemicals tested. At the 1 mM level, the concordance of data from the combined in vitro assays with the in vivo data was 66% in one laboratory and 58% in the other. The maximum agreement between the combined in vitro and in vivo data was reached at the 20 mM level, where there was a 73 and 74% concordance of results in the two laboratories. At that level there was a 16 and 10% incidence of false negative results, and a 54 and 77% incidence of false positive results. A portion of these false negative compounds may require metabolic activation. The use of either assay alone was not as accurate as using a positive result from either test. Agreement of the in vitro data at the 10 mM level with available human data was 71 and 75% for each laboratory. The data indicate that the two assays are complimentary and as such the combination of these assays could be useful as a preliminary screen to establish priorities for in vivo developmental toxicity testing.

Animals↗

PubMed2003

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood.

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.

Animals↗

PubMed2006

In vitro assay for sex pheromone biosynthesis by the female yellow mealworm beetle and identification of a regulated step.

Several pheromones are known to be involved in mediating the mating behavior of the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). The only sex pheromone that has been identified to date in this species is the female-produced male attractant, 4-methylnonanol (MNol). The objective of this study was to develop an in vitro assay for the last biosynthetic step involved in MNol biosynthesis, the reduction of 4-methylnonanoic acid (MNA) to the corresponding pheromone product. In vitro assays were conducted by adding appropriate cofactors and radioactively labelled precursor. Radioactive pheromone product was separated from the radioactive precursor by column chromatography, and quantified through scintillation counting. The conversion of MNA to MNol was observed, and was comparable to that observed in vivo using the same radiolabelled substrate. In both the in vivo and in vitro assays, the (3)H-MNA was also converted to a nonpolar product, tentatively identified as a triacylglyerol (TAG) derivative. The reduction of (3)H-MNA to pheromone proceeded through the coenzyme A intermediate. Mature females reduced significantly more precursor to pheromone than the immature females and the males, both in vivo and in vitro, indicating that the reduction of MNA to MNol is a regulated step involved in pheromone production. Using the in vitro assay reported herein, studies are ongoing in our lab to purify and characterize the enzyme catalyzing the reduction.

Allosteric Regulation↗

PubMed1977

Comparison of antiplaque agents using an in vitro assay reflecting oral conditions.

An in vitro assay is described using saliva-treated bovine enamel slabs for determining the potential of chemotherapeutic agents to adsorb to tooth surfaces and act against plaque-forming bacteria. Chlorhexidine was found to inhibit the formation of in vitro plaque by Actinomyces viscosus, A naeslundii, Streptococcus mutans and S sanguis. Actinobolin was found to have marked antibacterial properties but limited adsorptive qualities.

Actinomyces↗

PubMed1995

Classification of contaminants by mode of action based on in vitro assays.

A concept for toxicity assessments based on in vitro assays of variant levels (i.e., from whole cells to isolated enzymes) is presented. Due to the complexity of organisms of different species, it is evident that no single in vitro test can represent the entire spectrum of toxic potency of chemicals, rather a carefully designed battery of tests has to be employed to account for the various targets attacked in organisms yielding the different modes of action. Cytotoxicity tests like the Neutral-Red Assay predominantly reflect non-specific toxicity, which can be modelled according to a log Pow dependent baseline QSAR. Specific toxicants (e.g., decouplers, acetylcholinesterase inhibitors or photosystem II inhibitors) may be identified based on according in vitro tests and eventually modelled by the respective mode of action related QSARs employing also, e.g., steric or polarizability descriptors to account for specific interactions. The complementation and partial replacement of in vivo (eco)toxicological testing by in vitro assays depends on two criteria: (a) the sensitivity of the tests to reliably detect environmentally relevant concentrations of toxicants and (b) the specificity of the assays to provide an unambiguous classification of toxicants by modes of action: the pattern of interaction with the various targets allows to recognize those compounds of specific effects that frequently occur as outliers in QSAR analyses.

Acetylcholine↗

PubMed1980

Analysis of tumour responses by excision and in vitro assay of cellular clonogenic capacity.

A review is presented of the advantages and problems associated with the use of in vitro assays of cellular clonogenic capacity when used to analyse responses of tumours treated in vivo, either with radiation or chemotherapeutic agents. Three questions are considered: (1) Does the cell suspension obtained by the dispersion technique from various types of solid tumours provide an adequately random sample of the cells initially present in the tumour? (2) Are the properties of cells in suspensions obtained from solid tumours assayed in optimal conditions in vitro? (3) Are the properties expressed and analysed by in vitro techniques equivalent to the properties that the same cells would have expressed if they had been left in vivo, either in untreated or in treated tumours? It is concluded that the in vitro assay provides a valuable tool to analyse tumour responses but that the data obtained must be complemented by information on cell kinetics and other factors to obtain a complete description of tumour responses and to correlate them with tumour curability and growth delay.

Cell Survival↗

PubMed1999

Validation of in vitro assays for botulinum toxin: a case study.

Ensuring the reliability and precision of assay results requires careful attention to assay design. In this case study we describe validation studies of an in vitro assay for botulinum neurotoxin type A based on its endopeptidase activity towards immobilised synthetic substrate. This assay, in common with many in vitro assays, is sensitive to changes in reagents and assay conditions and is time dependent. In addition, the toxin is not stable in solution. Differences in estimates of potency, resulting from positional factors, which are not significant in individual assays, are shown to be consistent and statistically significant over a longer series of assays. This study emphasizes that assay validation should not be viewed as a single step in assay development but must be considered as a continuing process if assay results are to be reliable and reproducible.

Analysis of Variance↗

PubMed1990

Qualitative analysis of antibody binding. An in vitro assay for the evaluation and development of vaccines.

We describe a rapid in vitro assay for the evaluation of in vivo properties of conjugate vaccines. Using human and murine monoclonal antibodies (mAb) specific for lipopolysaccharides (LPS), isolated from Pseudomonas aeruginosa, we determined in a competitive binding assay the amount of LPS or conjugate vaccine which was required to inhibit the antibody binding to LPS by 50% (I50 values). Furthermore, utilizing a murine burn wound sepsis model, we determined the potential of the same conjugates to induce protection in vivo against infection with the corresponding bacteria. Protective mAb have approximately 100-fold lower I50 values for preparations which are highly effective in inducing protection than for preparations which are ineffective. Furthermore, in the case of potent preparations it was noted that protective mAb exhibit similar I50 values for the conjugates and for the corresponding LPS. These results suggest that the fast and easily interpretable in vitro assay described may significantly facilitate the development and optimization of vaccines.

Animals↗

PubMed1976

Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings.

The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.The in vivo NR assay effectively distinguished between two wheat varieties. The variety known to have the higher percentage of seed protein also had the higher amounts of NR activity.With seedling wheat leaves, the addition of NADH plus a surfactant increased in vivo NR activity approximately 2-fold over comparable controls. Because the tissue contained high levels of nitrate and enzyme, we concluded that reducing potential was the rate-limiting factor in nitrate reduction in situ in these growth-chamber-grown plants.

Journal Article↗

PubMedDate not supplied

Selection of a high activity ribozyme against cytostatic drug resistance-associated glypican-3 using an in vitro assay containing total tumor RNA.

We tested 11 hammerhead ribozymes for their ability to bind and cleave RNA transcripts of the cytostatic drug resistance-associated glypican-3-encoding gene (GPC3, MXR7, OCI-5). To select the optimum target sequence, the activity of each hammerhead ribozyme was tested in a short in vitro assay using truncated RNA substrates without time-consuming cloning procedures. Glypican-3-derived RNA was cleaved effectively by 3 of 11 hammerhead ribozymes. One of these, the hammerhead ribozyme Rz967, recognized the GUC sequence at nucleotides +965 to +967 of the open reading frame of the glypican-3-encoding mRNA. Rz967 cleaved in vitro-transcribed fragments derived from glypican-3 mRNA (nucleotides +803 to +1036) most efficiently. Cleavage efficiency was confirmed by a rapid in vitro assay using full-length total tumor cell RNA. We were able to demonstrate that this new in vitro assay is suitable for the selection of hammerhead ribozymes that have the capability to cleave glypican-3-encoding mRNA in human tumors.

Base Sequence↗

PubMed1998

1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but is in mouse liver micronucleus assay.

1,4-Dioxane, an animal carcinogen, was not previously genotoxic in in vitro assays. We reevaluated the compound's genotoxic potential in five in vitro genotoxicity tests in the presence and absence of S9 mix using recommended new protocols. We used the bacterial reverse mutation assay with Salmonella TA and E. coli WP2 strains, including the plate and preincubation methods, the CHO chromosomal aberration assay, including examination of polyploid induction and extended sampling time, the CHO sister-chromatid exchange assay with short and long treatment time, the mouse lymphoma tk assay (microtiter method), including longer treatment time (24 hr), and the CHO micronucleus assay with short and long treatment times. The highest concentration we used was five mg/ml or plate. We also evaluated the genotoxic effect of 1,4-dioxane in vivo by conducting peripheral blood and liver micronucleus assays in the same mice after single oral administration of up to 3,000 mg/kg. All in vitro assays and the peripheral blood micronucleus assay were negative. The mouse liver micronucleus assay, on the other hand, was positive, indicating that 1,4-dioxane might be genotoxic. It is also conceivable that the positive result in mouse liver micronucleus assay was due to a nongenotoxic mechanism, i.e., errors in genetic repair following enhancement of hepatocyte proliferation.

Animals↗

PubMed1988

Phosphorylation of the neuronal protein kinase C substrate B-50: in vitro assay conditions alter sensitivity to ACTH.

We have explored the hypothesis that changes in the in vitro assay conditions alter both the extent of endogenous phosphorylation of B-50 protein in synaptosomal plasma membrane (SPM) and also the ability of the neuropeptide, ACTH-(1-24) to inhibit the phosphorylation of this protein. B-50 phosphorylation is influenced by preincubation, pH and ionic strength. ACTH-(1-24)-induced inhibition of B-50 phosphorylation varies with ionic strength and SPM protein concentration. Reduction of the buffer ionic strength and the SPM protein concentration enhances the ability of ACTH-(1-24) to inhibit B-50 phosphorylation. Furthermore, loss of ACTH-(1-24) by adsorption to plastic pipettes and test tubes reduces the peptide concentration in the assay. Addition of a low concentration of bovine serum albumin (BSA) essentially eliminates this loss without affecting the extent of phosphate incorporation into B-50. These data provide an explanation for the relatively high (and variable) IC50 values for ACTH-(1-24)-induced inhibition of B-50 phosphorylation reported in the literature. Further, these data suggest that in vitro assay conditions must be carefully investigated before modulation of protein phosphorylation can adequately be studied.

Animals↗

PubMed1988

A rapid in vitro assay for predicting thermochemosensitivity of human cancer: comparison with clonogenic assay.

We previously reported a rapid in vitro assay based on morphological changes in the nucleus in order to predict response to thermochemotherapy. It was strongly suggested that this simple method may be clinically useful. In the present study, a comparison with the clonogenic assay was carried out on eight different human tumors (three malignant melanomas, two lung carcinomas, two colon carcinomas and one leukemia). Melphalan, mitomycin C and vincristine were tested. Correlation between the two test systems was dependent upon the criteria for each test system. At the level of less than 50% survival of colony as compared with normothermic dishes in clonogenic assay, there was a high correlation between the two test systems for sensitive tumor to 43 degrees C. In respect of response to thermochemotherapy, when only karyorrhexis changes in the nucleus were selected as an activity criteria in our cytotoxic test, parallel data between the two test systems were obtained.

Antineoplastic Agents↗

PubMed2002

An in vitro assay to evaluate competitive exclusion products for poultry.

An in vitro assay was developed to measure the ability of competitive exclusion (CE) bacteria to protect Caco-2 and CRL-2117 epithelial cells from invasion by Salmonella Typhimurium. The proposed assay is needed to expedite the development of defined-flora CE products. The average significantly protective concentration of the commercial poultry-specific CE product Preempt was 4.05 log CFU/6.41 log human Caco-2 cells and 3.71 log CFU/6.89 log CFU chicken CRL-2117 cells, Enterococcus faecalis isolated from Preempt protected CRL-2117 cells, Escherichia coli isolates protected Caco-2 cells, Lactococcus lactis and Bacteroides distasonis isolates protected both cell lines, and three species of Lactobacillus isolates failed to protect either cell line. A defined mixture of 29 strains of bacteria similar to the constituents of Preempt protected both cell lines from Salmonella invasion at a concentration of 7.83 log CFU. The constituents of the defined CE culture were separated into mixtures of obligate (8.42 log CFU) and facultative (8.49 log CFU) anaerobes, which both protected the cell lines, suggesting that both types of bacteria were equally protective. Although not a substitute for in vivo testing, the in vitro CE assay is a rapid technique for the evaluation of bacterial mixtures for potential CE products.

Animals↗

PubMed2003

An assessment of the phototoxic hazard of a personal product ingredient using in vitro assays.

Where substances are intended for use in personal products applied to the skin an assessment of potential phototoxic hazard is required. This report describes a tiered testing strategy involving in vitro assays used for the phototoxic hazard assessment of a personal product ingredient (Ingredient X). The initial assay was measurement of a UV/visible absorption spectrum to identify absorption at relevant wavelengths. This was followed by in vitro assays for phototoxicity (3T3 cell neutral red uptake phototoxicity test) and photoallergy (photobinding to human serum albumin). These in vitro screens gave equivocal results for Ingredient X which appeared to suggest a weak phototoxic reaction. To further evaluate the phototoxic hazard of Ingredient X to human skin, a phototoxicity assay using a 3-D human skin model was conducted. Ingredient X did not cause phototoxicity in this assay. Overall conclusions from these studies were that although Ingredient X showed slight intrinsic potential for photoactivation, it was unlikely to present a hazard to human skin. This report illustrates the value in a step-wise strategy of the use of human skin models to help interpret the results of other in vitro phototoxicity assays.

3T3 Cells↗

PubMed1988

A novel in vitro assay for murine haematopoietic stem cells.

Study of the biology of haematopoietic stem cells is crucially dependent on the availability of suitable in vitro assays. Existing assays have suffered from the fact that they detect small subcompartments of the total stem cell compartment. This limits experiments where it is required to assay a high proportion of stem cells, e.g. the enumeration of stem cell numbers under varying conditions or the identification and purification of stem cell regulators. We describe an in vitro assay which shows macroscopic colony formation and limited self-renewal capacity in vitro. The detected cell (CFU-A) has a low cycling status in normal bone marrow (NBM) and responds to known stem cell regulators. The incidence (100-200 per 10(5) in NBM), the proliferative characteristics under stress and some of the physical properties are similar to stem cells detected by colony formation after transplantation into lethally irradiated recipients (CFU-S). These data indicate that our assay detects a high proportion of haematopoietic stem cells in vitro. This will facilitate experiments on stem cell behaviour which have previously been difficult to conduct.

Animals↗

PubMed2002

Detection of oestrogenic activity of steroids present during mammalian gestation using oestrogen receptor alpha- and oestrogen receptor beta-specific in vitro assays.

Numerous steroid hormones are present in the foetus but their potential to activate oestrogen receptor (ER) alpha and/or beta is largely unknown. In this study, in vitro assays were developed to rapidly and specifically detect ERalpha or ERbeta activation by these steroid hormones. Our results showed that several oestrogen precursors and androgens are able to activate both ERalpha and ERbeta. Of special interest is that some of these precursors are able to activate ERalpha and ERbeta at concentrations that are present during human gestation. Moreover, some precursors (dehydroepiandrosterone (DHEA) and 17-hydroxylated pregnenolone sulphate) and androgens (5-androsten-3beta,16alpha,17beta-triol and testosterone) showed a more than 100-fold relative preference for ERbeta transactivation over ERalpha transactivation when compared with 17beta-oestradiol. Due to their relatively high levels, the precursor steroids DHEA and pregnenolone may be of particular importance in the regulation of ERbeta activity in vivo. To obtain information about the oestrogenic activity of the total pool of steroid hormones present during mammalian gestation, steroids were extracted from mouse embryos at different prenatal stages and assayed for oestrogenic activity in the established in vitro assays. Oestrogenic activity was detected in steroid extracts from all stages tested. This study has demonstrated that oestrogen receptor agonists are present in the murine embryo and that oestrogen precursors may contribute to the total pool of agonists during foetal life.

Androgens↗

PubMed1983

Development of an in vitro assay for the survival of cells suspended from BA1112 rat sarcomas.

A method has been developed for the in vitro assay of the survival of BA1112 rat sarcoma cells following treatment in vivo. The BA1112 tumor was not adapted for growth in culture and was maintained by serial transplantation in vivo. The assay used a feeder layer of heavily irradiated cells to eliminate any changes in plating efficiency caused by the large number of dying cells which must be plated when assaying cell survival after intensive treatments. The plating efficiency of the tumor cells was not affected significantly by the age of the feeder layer (between 1 and 4 days), the density at which the experimental cells were plated or the interval between preparation of the cell suspension and plating (up to 4 hr). Using the in vitro assay a survival curve was determined for cells from tumors irradiated in air-breathing animals. This curve was similar to that determined previously for BA1112 tumor cells using an in vivo assay to measure cell survival.

Animals↗