Biological assays: their role in the development and quality control of recombinant biological medicinal products.
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Animal Testing Alternatives↗
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Results for “Biological Assay”
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Biological assays for Ras transformation.
The rodent fibroblast systems described above have provided sensitive and rapid biological assays to characterize the properties of normal and mutated Ras proteins. Furthermore, these assays have provided in vitro systems to measure the ability of other cellular components to modulate Ras signal transduction and transformation. However, while these assays provide an excellent measure of Ras-transforming activity, the fact that these cells are of fibroblastic origin, and can be transformed by a single hit, indicates that caution should be used in extrapolating observations from NIH 3T3 transformation assays to the situation in human tumors. Therefore, using human epithelial cell-based assays that more closely approximate the cell types where mutated ras alleles are most frequently detected may provide more realistic assays for examining the biochemical and biological consequences of aberrant Ras function in human tumors. Nevertheless, despite these cautions, these rodent transformation assays will continue to be the best and most widely applied assays for Ras biological activity.
3T3 Cells↗
Design and evaluation of biological assays.
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Biological Assay↗
Statistical method in the field of biological assay.
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Biological Assay↗
A transgenic mouse model provides a novel biological assay of topical glucocorticosteroid potency.
BACKGROUND AND DESIGN: A homozygous line of transgenic mice that expresses the human elastin promoter/CAT (chloramphenicol acetyltransferase) reporter gene construct in a tissue-specific and developmentally regulated manner is presented. Previous studies have shown that subcutaneous injections of various glucocorticosteroids up-regulate the human transgene in the mouse skin potentially through their interaction with three putative glucocorticosteroid-responsive elements contained within the human elastin promoter. In this study, we propose the use of these transgenic mice as a model system for assaying the potency of various topical glucocorticosteroid preparations. RESULTS: In the first set of experiments, three different commercially available topical glucocorticosteroid creams, 2.5% Hytone (2.5% hydrocortisone) (Dermik Laboratories, Fort Washington, Pa), Cutivate (0.05% fluticasone propionate) (Glaxo Inc, Research Triangle Park, NC), and Temovate (0.05% clobetasol propionate) (Glaxo Inc) (being classified into class VII, V, and I steroids, respectively) were applied to the skin of transgenic mice, with Eucerin (Beiersdorf Inc, Lindenhurst, NY) as the control cream. In a series of six experiments, Hytone 2.5% cream caused a 3.1-fold increase on the average, with Cutivate and Temovate creams resulting in 2.2-fold and 12.4-fold increases in CAT activity over control, respectively. Next, two different preparations of diflorasone diacetate 0.05% cream (Florone [class III] and Psorcon [class II], both from Dermik Laboratories), formulated with different vehicles, were compared. Psorcon caused a 22.8-fold increase in CAT activity over the control compared with a 4.4-fold increase for Florone. However, an assay comparing Psorcon ointment (class I) and Psorcon cream (class II) showed no statistically significant difference in their potencies. CONCLUSIONS: These preliminary findings suggest the usefulness of these transgenic mice as a model system for assaying the potency of topical glucocorticosteroid preparations. Discrepancies between our data and the published classification of some topical steroids may result from anatomic differences between human and murine skin, with mouse skin much thinner, Alternatively, the discrepancies may reflect the fact that our assay measures the biological activity of these steroids on gene transcription, while previous ranking is based on their vasoconstrictive activity.
Administration, Topical↗
Neurokinin receptor subtypes characterized by biological assays.
Neurokinin receptors have been characterized by biological assays using naturally occurring and selective agonists as well as peptide and non peptide antagonists. Six preparations have been used: the rabbit vena cava and the rat urinary bladder, treated with a NK-2 receptor antagonist for the NK-1 receptor, the rabbit pulmonary artery and the hamster urinary bladder for the NK-2, the rat portal vein and the guinea pig ileum, treated with a NK-1 receptor antagonist, for the NK-3. Treatment with antagonists was required because of the presence (in some preparations) of two functional sites contributing to the biological effect. Differences in the order of potency of agonists between each couple of receptors have been demonstrated, especially with tachykinins and the selective agonists. Such differences are even more evident with antagonists, some of which show apparent affinity (pA2) values 1.5 to 3 log units higher in one than in the other member of each couple. Based on data obtained in pharmacological experiments, it is concluded that NK-1, NK-2 and NK-3 receptors show differences strong enough to justify the assumption that their coding and/or expression diverge among species.
Animals↗
[A study of standardization to the rapid detection of Mycobacterium tuberculosis based on phage amplified biologically assay].
OBJECTIVE: To evaluate the phage amplified biologically (PhaB) assay in the rapid detection of Mycobacteria tuberculosis in samples. METHODS: The conditions of the PhaB assay, including various infection times prior to addition of virucide and the effect of the inactivation agents which could inactive the extracellular phages, were investigated and compared. The sensitivity, specificity and accuracy of PhaB assay were tested when it was used in rapid detection of Mycobacterium tuberculosis. Some agents of the method, including Mycobacteriophage, virucide and help cells (Mycobacterium smegmatis) were investigated at different times when they were preserved at 4 degrees C. RESULTS: (1) The optimal infection time prior to addition of virucide was between 3 and 4 hours. Four percent FAS (ferrous ammonium sulphate) could inactive 1 x 10(9) PFU (plaque-forming unit) in five minutes. (2) The samples were positive when 80 - 200 CFU of Mycobacterium tuberculosis were present. However, the positive rate of non-Tuberculosis Mycobateria (NTM) was varied. All bacteria lived in the respiratory tract were negative. (3) The important agents used in this test showed optical effect when they were preserved at 4 degrees C. CONCLUSIONS: The method based on mycobacteriophage-amplified biologically assay could rapidly detect Mycobacterium tuberculosis, and it was effective, accurate, and simple to perform. It was appropriate for using in developing countries, compared with a variety of molecular techniques.
Antitubercular Agents↗
Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin.
The 75,000-dalton cell-binding domain of fibronectin (f75k) and synthetic peptides derived from its sequence have been used to examine the cell-surface fibronectin receptor. The spreading of baby hamster kidney fibrolasts on f75k-coated substrates and the direct binding of [3H]f75k to these cells were both competitively inhibited by a synthetic peptide of fibronectin with the sequence Gly-Arg-Gly-Asp-Ser, indicating that the peptide and f75k interact with the same cell-surface sites. Related peptides, including the inverted sequence Ser-Asp-Gly-Arg, also competed for f75k binding. Our results provide the first evidence that fibroblastic baby hamster kidney cells recognize the same fibronectin amino acid sequence in direct binding of soluble ligand and in indirect inhibitory biological assays, thus correlating our direct fibronectin-receptor binding assay with biological functional assays for the fibronectin receptor in fibroblasts.
Animals↗
Biological assay of luteinizing hormone-releasing hormone (gonadorelin).
The abilities of luteinizing hormone releasing hormone (LHRH) to stimulate the release of luteinizing hormone (LH) in vitro from dispersed pituitary cells or from segments of adenohypophysial tissue have both been exploited in an attempt to develop biological assays for its determination. Columns of dispersed adenohypophysial cells were highly sensitive to LHRH. The responses appeared to be specific, but the marked continual variation in their magnitude rendered the system nonprecise. Pituitary segments were less sensitive to LHRH than the dispersed cells. However, their responsiveness was increased by pretreatment ('priming') with the releasing hormone. Over a wide range of doses, there was a linear relationship between the LH released by 'primed' segments and the logarithm of the concentration of LHRH. The dose-response lines of hypothalamic extracts were parallel with those of the synthetic peptide. The method using segments is accurate, specific, and precise and fulfils the essential requirements of a biological assay.
Animals↗
BALANCED INCOMPLETE BLOCK DESIGNS IN BIOLOGICAL ASSAY ILLUSTRATED BY THE ASSAY OF GASTRIN USING A YOUDEN SQUARE.
General equations are given for computing the results of any (j+j) dose parallel line biological assay based on an incomplete block design. Only the information from comparisons within blocks is used. The computation is illustrated numerically by means of a (2+2) assay of gastrin devised and performed by Lai (1962), in which only three of the four doses could be given to each animal.
Animals↗
Biological assays for interferons.
Interferons (IFN) are potent biologically active proteins synthesised and secreted by somatic cells of all mammalian species. They have been well characterised, especially those of human origin, with respect to structure, biological activities, and clinical therapeutic effects. While structural differences are known to exist among the IFN species that constitute the "IFN family" and despite the existence of different receptors for type I and type II IFN, all species have been shown to exert a similar spectrum of in vitro biological activities in responsive cells. Principal among the biological activities induced by IFN is antiviral activity, the activity used to originally define IFN. Antiviral activity of IFN is mediated via cell receptors and is dependent on the activation of signalling pathways, the expression of specific gene products, and the development of antiviral mechanisms. Sensitivity of cells to IFN-mediated antiviral activity is variable, and depends on a number of factors including cell type, expression of IFN receptors and downstream effector response elements, effectiveness of antiviral mechanisms, and the type of virus used to infect cells. Nevertheless, by the judicious use of sensitive cell lines in combination with appropriate cytopathic viruses, effective assays to measure the antiviral activity have been developed. Historically, "antiviral assays" (AVA) were the first type of biological assays that were developed to measure the relative activity or potency of IFN preparations. However, the subsequent discoveries of several other biological activities of IFN has opened the way to the development of assays based on one or other of these activities. The latter include inhibition of cell proliferation, regulation of functional cellular activities, regulation of cellular differentiation and immunomodulation. More recently, the cloning of IFN responsive genes has led to the development of "reporter gene assays". In this case, the promoter region of IFN responsive genes is linked with a heterologous reporter gene, for example, firefly luciferase or alkaline phosphatase, and transfected into an IFN-sensitive cell line. Stably transfected cell lines exposed to IFN increase expression of the reporter gene product in direct relation to the dose of IFN, the readout being a measure of this product's enzymic action. The current review aims to give a critical overview of the development, specificity, standardisation and present use of the various biological assay methods now available for the quantification of IFN activity.
Animals↗
Relevance of different biological assays in assessing initiating and promoting properties of polycyclic aromatic hydrocarbons with respect to carcinogenic potency.
The results from assays that describe biological effects of polycyclic aromatic hydrocarbons (PAH) were explored using multivariate methods. Based on the availability of data, 29 PAH were included in the study. Five variables described the carcinogenic potency in rodents of the PAH. Biological effects were assayed using 14 variables. These included bacterial mutagenicity, enhancement and inhibition of bacterial mutagenicity, Ah receptor (AhR) affinity, and induction of enzymes that bioactivate many PAH to proximal bacterial mutagens. A principal components analysis (PCA) showed that the highest correlations with the cancer data were observed for variables describing AhR affinity, whereas bacterial mutagenicity data were poorly correlated with cancer data. When a partial least squares (PLS) regression analysis was applied, only one bacterial mutagenicity variable, but all AhR affinity variables were statistically relevant to describe carcinogenic potency. The latter variables were also correlated with the inhibition of bacterial mutagenicity of benzo[a]pyrene. It was concluded that structural requirements for AhR affinity are the same as those required for metabolism by enzymes that bioactivate benzo(a)pyrene. Negative correlations between mutagenicity and induction of enzymes were observed. The roles of cancer initiation and cancer promotion are discussed regarding the biological properties studied. It is proposed that bacterial mutagenicity reflects the cancer initiation potency, whereas the AhR affinity reflects the promotive effect of some PAH at the high doses applied in rodent carcinogenicity tests. It is thus indicated that initiation and promotion are provoked by different chemical species: reactive metabolites and the parent hydrocarbons, respectively. At doses reflecting a normal human exposure situation the effects of initiation may be more important in the course of chemical carcinogenesis. The mechanisms of cancer initiation and cancer promotion should therefore be studied in more detail for reliable quantitative risk assessments.
Animals↗
Biological assay for tissue kallikrein: comparison with the synthetic substrate S2266.
A highly sensitive biological assay for tissue kallikrein is described, using human kininogen as substrate; and quantitation, by radioimmunoassay, of generated kinins. Using purified human urinary kallikrein as a reference standard we have correlated the kininogenase activity of kallikrein with amidase activity as measured by cleavage of the synthetic substrate S2266.
Animals↗
[Biological assay, reliability test, error analysis and pooled calculation for assessing leucomycin yield].
A biological assay for determining leucomycin yield was established by using a curillinear protocol (Sarcina lutea) and (2.2) dosage or (3.3) dosage protocol (Bacillus subtillis 63501). At the same time, according to the principles of biostatistics and the methods of variance analysis, reliability tests, error analysis and pooled calculations were carried out, so as to hold the confidence limit below the internationally accepted level of 5%.
Biological Assay↗
[The characteristics of the sensitivity of Mycobacterium tuberculosis to rifampicin and isoniazid through determination of mutations in the genes rpoB, katG, inhA, oxyR, and kasA by different molecular biological assays].
Two hundred and two patients with different forms of pulmonary tuberculosis were examined to study the characteristics of sensitivity with the signs of multidrug resistance to rifampicin and isoniazid, by using a microbiological assay of the absolute concentrations and determining mutations in the genes rpoB, katG, inhA, oxyR, and kasA, by employing different molecular biological assays. Mycobacterium tuberculosis (MBT) DNA was isolated from both a diagnostic material (such as sputum, bronchial secretion), and clinical MBT isolates. By showing a higher sensitivity and a higher specificity, as cultural techniques, molecular biological assays of MBT drug sensitivity in patients with tuberculosis were ascertained to accelerate its diagnosis until the patient was admitted to a clinic.
Antibiotics, Antitubercular↗
[Short-term screening of anticarcinogenic ingredients of tea by cell biology assays].
By using a panel of short term cell biology assays, several ingredients of tea (tea pigments, caffeine, tea polysaccharide, tea polyphenols tablet and mixed tea) were screened in order to investigate their anticarcinogenic effects. The cytokinesis block micronuclei test in V79 cells induced by mitomycin, the test of metabolic cooperation between V79 and M cells and the test of growth ability of Hela cells in soft agar were used in the screening. The results showed that the six kinds of tea ingredients tested were effective in the test involved in different stages of carcinogenesis, i.e. initiation, promotion and progression. The effects of mixed tea and tea pigments were the strongest among the ingredients tested.
Animals↗
CMV infection of liver transplant recipients: comparison of antigenemia and molecular biology assays.
BACKGROUND: CMV is a major clinical problem in transplant recipients. Thus, it is important to use sensitive and specific diagnostic techniques to rapidly and accurately detect CMV infection and identify patients at risk of developing CMV disease. In the present study, CMV infection after liver transplantation was monitored retrospectively by two molecular biology assays - a quantitative PCR assay and a qualitative NASBA assay. The results were compared with those obtained by prospective pp65 antigenemia determinations. MATERIALS AND METHODS: 87 consecutive samples from 10 liver transplanted patients were tested for CMV by pp65 antigenemia, and CMV monitor and NASBA pp67 mRNA assay. RESULTS: CMV infection was detected in all patients by antigenemia and CMV monitor, whereas NASBA assay identified only 8/10 patients with viremia. Furthermore, CMV infection was never detected earlier by molecular biology assays than by antigenemia. Only 5/10 patients with CMV infection developed CMV disease. Using a cut off value of 8 cells/50,000, antigenemia was found to be the assay that better identified patients at risk of developing CMV disease. However, the kinetics of the onset of infection detected by NASBA and CMV monitor seemed to have better identified patients at risk of developing CMV disease. Furthermore, before onset of disease, CMV pp67 mRNA was found to have similar or better negative and positive predictive values for the development of CMV disease. CONCLUSIONS: The present data, suggests that the concomitant use of antigenemia and pp67 mRNA assay gives the best identification of patients at risk of developing CMV disease.
Adult↗
Validation of biological assays.
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Biological Assay↗