Gentamicin enzyme-linked immunosorbent assay for microdialysis samples.

The authors investigated the use of a commercially available gentamicin enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of gentamicin concentrations in microdialysis samples. The assay demonstrated good accuracy and precision in the concentration range of 100 to 967 pg/mL. The developed quantitative ELISA is a highly sensitive and valid method for measuring gentamicin concentrations in microdialysis samples. This assay may be a useful alternative to other assay methodologies where analysis may be restricted by sample volume requirements and limited sensitivity.     

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.     

Collaborative study for validation of a serological potency assay for rabies vaccine (inactivated) for veterinary use

European Pharmacopoeia (Ph. Eur.) monograph 0451 on Rabies vaccine (inactivated) for veterinary use describes an in vivo batch potency test that is based on the NIH test. This assay uses a large number of mice and results in a significant degree of suffering. In the interest of replacement, reduction and refinement of animal tests (3R) a serological potency assay for Rabies vaccine (inactivated) for animal use, developed and validated at the Paul-Ehrlich-Institut, has been assessed in a collaborative study organised by the EDQM (European Directorate for the Quality of Medicines & HealthCare). The goal was to demonstrate the wider transferability of the proposed assay and confirm its suitability. The study involved 13 laboratories and assessed 4 different vaccines from the EU market. Results of the study confirm that a limit test using a relatively small number of animals in a serological assay is possible, reproducible and reliable. The optimal number of animals per vaccine is product specific but may roughly be indicated to be between 8 and 10 for the products included in this study. Non-responders should be included in the analysis because they may reflect sub-potent vaccines. However, there may be a need to impose a maximum on the number of non-responders allowed for the reference vaccine as a monitor for assay validity. This assay provides a significant 3R improvement in terms of both the number of animals used and the amount of suffering entailed and provides a more reliable and reproducible assay format than the vaccination challenge assay. It also reduces the time required as compared to the vaccination challenge assay. It has been recommended to the Ph. Eur. group of experts 15V that this assay be included as an alternative to the batch potency assay in the Ph. Eur. monograph 0451.     

Effect of gonadotropin-releasing hormone antagonist on ovarian and uterine weights in immature female rats

The immature rat uterotrophic assay has been proposed as a screening test method for detecting estrogenic and antiestrogenic chemicals. Although the immature rat uterotrophic assay is advantageous because the test animals are not traumatized by the ovariectomizing process, the effect of endogenous estrogen on ovarian and uterine weight in the immature animals that are used in immature rat uterotrophic assay has not received much attention. In this study, 19-day-old rats were treated with antide, a gonadotropin-releasing hormone antagonist, antide, to block gonadal production of endogenous estrogen. Uterine and ovarian weights of the antide-treated animals were markedly lower than those of control animals. This finding suggests that endogenous gonadal estrogen may already be acting on the uterus and ovaries in immature rats. Blocking endogenous estrogen with a gonadotropin-releasing hormone antagonist may enhance the sensitivity of the immature rat uterotrophic assay; however, the possibility that this protocol may interfere with the ability of the immature rat uterotrophic assay to detect centrally-mediated effects can not be discounted.     

Use of the GTP��S ([35S]GTP��S and Eu-GTP��S) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors

In this review I consider assays for G protein-coupled receptor (GPCR) activity based on the binding of labelled analogues of GTPγS ([³⁵S]GTPγS or Eu-GTPγS) to G proteins in tissues (GTPγS binding assays). Such assays provide convenient measures of GPCR activity close to the receptor in the signalling cascade. In order to set up a GTPγS binding assay, the requirements of the assay must be considered. These are tissue source, GTPγS analogue, G protein, GDP, Mg²⁺/Na⁺ ions, saponin, incubation time. The assay, once optimized, can be used to generate concentration/response curves for GPCRs signalling via G_i/o proteins (or to other G proteins with a modified assay) and actions of agonists, inverse agonists and antagonists may, in principle, be assessed. For agonists and inverse agonists, data for the maximal agonist effect, the concentration of ligand giving a half-maximal response and the Hill coefficient may be derived. For antagonists, data for the equilibrium dissociation constant can be obtained. The mechanistic basis of the assay is considered. Although the assay can be used to profile ligands, under the conditions it is used, it may not be measuring the same event that determines GPCR action in cells.     

Design of a flexible cell-based assay for the evaluation of heat shock protein 70 expression modulators

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.     

Genotoxicity testing of a Salacia oblonga extract.

Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal alpha-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.     

Genotoxicity testing of aqueous extracts of Mahwangyounpae-tang, a polyherbal formula

Mahwangyounpae-tang (MT), consisting of 22 types of herbal extracts has been used for thousands of years in Korean traditional medicine for the oral treatment of respiratory diseases including asthma. As part of a safety evaluation of MT extracts for use in asthma, the potential genotoxicity of an aqueous MT extract was evaluated using the standard battery of tests (bacterial reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by Korea Food and Drug Administration (KFDA). The MT extract was determined not to be genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay and mouse micronucleus assay. Use of MT is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, prove to be a useful anti-asthma agent.     

On methods to utilize HIV-RNA data measured by two different PCR assays

The plasma HIV-RNA level has been used as the primary efficacy measurement in clinical trials to evaluate antiretroviral regimens in HIV-infected patients. It is measured by polymerase chain reaction (PCR) assays, which usually have limits of reliable quantification (LoQ). For example, the commercially available Amplicor Standard assay has a reliable range of 400-750,000 copies/mL while the Ultrasensitive assay has a range of 50-75,000 copies/mL. Values below the lower LoQ are usually reported as categorical variables such as " < 400 copies/mL" for the Standard assay and " < 50 copies/mL" for the Ultrasensitive assay. The Standard assay, which has a higher ceiling of 750,000 copies/mL, is typically used as the first tool to measure HIV-RNA levels; if a value of " < 400 copies/mL" is reported by the Standard assay, the plasma sample may be re-tested by the Ultrasensitive assay, which has a lower LoQ of 50 copies/mL, in an effort to quantify the HIV-RNA level. However, for the calculation of change from baseline in log10 HIV-RNA, which is an important efficacy endpoint, the additional data measured by the Ultrasensitive assay are usually ignored due to a lack of simple and appropriate statistical methods. The conventional approach, which only uses the Standard assay data, may result in loss of information; the naive approach, which simply replaces " < 400 copies/mL" reported by the Standard assay with corresponding Ultrasensitive assay results, may lead to a biased estimate because the two assays may have different assay variability; the likelihood-based approach, which can utilize all data from both assays, is computationally intensive and requires a large sample size, which may limit its use in practice. In this paper, we propose a simple imputation approach that, unlike the naive method, accounts for the different variability in the two assays. A simulation study is used to compare these approaches. An example from a clinical trial in HIV-infected patients is used to illustrate the proposed approach.     

氯芬黄敏片中人工牛黄测定方法的改进

将氯芬黄敏片(感冒通片)人工牛黄测定过程中的加氯仿研磨改成加氯仿超声,改进前后测定结果对比表明此方法可行,为以后质量标准的修订提供了有益参考     

On Methods to Utilize HIV-RNA Data Measured by Two Different PCR Assays

The plasma HIV-RNA level has been used as the primary efficacy measurement in clinical trials to evaluate antiretroviral regimens in HIV-infected patients. It is measured by polymerase chain reaction (PCR) assays, which usually have limits of reliable quantification (LoQ). For example, the commercially available Amplicor Standard assay has a reliable range of 400–750,000 copies/mL while the Ultrasensitive assay has a range of 50–75,000 copies/mL. Values below the lower LoQ are usually reported as categorical variables such as “ < 400 copies/mL” for the Standard assay and “ < 50 copies/mL” for the Ultrasensitive assay. The Standard assay, which has a higher ceiling of 750,000 copies/mL, is typically used as the first tool to measure HIV-RNA levels; if a value of “ < 400 copies/mL” is reported by the Standard assay, the plasma sample may be re-tested by the Ultrasensitive assay, which has a lower LoQ of 50 copies/mL, in an effort to quantify the HIV-RNA level. However, for the calculation of change from baseline in log10 HIV-RNA, which is an important efficacy endpoint, the additional data measured by the Ultrasensitive assay are usually ignored due to a lack of simple and appropriate statistical methods. The conventional approach, which only uses the Standard assay data, may result in loss of information; the naïve approach, which simply replaces “ < 400 copies/mL” reported by the Standard assay with corresponding Ultrasensitive assay results, may lead to a biased estimate because the two assays may have different assay variability; the likelihood-based approach, which can utilize all data from both assays, is computationally intensive and requires a large sample size, which may limit its use in practice. In this paper, we propose a simple imputation approach that, unlike the naïve method, accounts for the different variability in the two assays. A simulation study is used to compare these approaches. An example from a clinical trial in HIV-infected patients is used to illustrate the proposed approach.     

Urinary N-acetyl- beta-D-glucosaminidase activities in patients with renal disease.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were assayed in every urine void throughout 24 hours in 17 normal people and in four patients with renal disease. The variation in NAG activity due to changing rates of urine flow was almost eliminated by factoring enzyme activity by the urinary creatinine concentration. Random samples of urine may thus be used for assay. The results of NAG assay in 36 patients with acute and chronic renal diseases showed that NAG was a sensitive indicator of renal damage. This simple test may be valuable in detecting or monitoring renal disease.     

Challenges in developing antidrug antibody screening assays

Robert Dodge is the Laboratory Director for Immunochemistry and Cell Biology at Taylor Technology, a Pharmanet company in Princeton, NJ, USA. Taylor Technology is a contract research laboratory specializing in assay development and sample testing in a good laboratory practice environment. Robert oversees a staff of scientists responsible for the development of assays for use in protein drug quantitation, antidrug antibody screening and biomarker detection. Protein drugs may elicit an immune response in the form of production of antidrug antibodies (ADAs) by a subject. This ADA response may have serious safety implications for subjects or, at a minimum, may affect drug efficacy. Bioanalytical testing for antidrug antibodies has therefore become a required part of safety testing for subjects receiving protein drugs. Regulatory guidance and scientific white papers recommend a tiered approach for bioanalytical ADA testing. The first assay in the tiered testing scheme is a screening assay, which tests all subjects in the study for the presence of ADAs. The screening assay is quasiquantitative, typically lacks a specific positive control and must be designed to detect numerous isotypes and subclasses of antibodies. These characteristics of an ADA screening assay differ from a those of a standard immunoassay designed to quantitate a specific protein in matrix and thus present unique challenges. This article reviews the unique challenges encountered during the development of an ADA assay.     

Equivalence in test assay method comparisons for the repeated-measure, matched-pair design in medical device studies: statistical considerations

In medical device clinical studies, including therapeutic device and in vitro test assay method studies, the investigator is frequently interested in demonstrating the equivalence of clinical response, generally in continuous measurements, between a standard assay method and a new assay method over various occasions or times. The new assay method may be less invasive or more convenient or cheaper to use than the standard assay method. In this paper, several statistical approaches are discussed, including various repeated-measure regression models, the simultaneous 95% confidence interval for paired mean differences derived from Hotelling's multivariate T2 analysis for repeated-measure, paired data, repeatability and reproducibility studies, and concordance correlation coefficient.     

Evaluation of the mutagenic potential of corn (Zea mays L.) grown in untreated and atrazine (AAtrex) treated soil in the field

Bacterial assays using extracts from field corn plants (harvested at one month, silage and mature stages) do not indicate that soil treatment with atrazine, at its maximum use rate, alters the endogenous mutagens present in these extracts, nor that atrazine itself is degraded to mutagenic products. Extracts of corn grown in soil treated with AAtrex were equally mutagenic with those of corn grown in untreated soil when tested in Salmonella typhimurium TA-100 by a reversion assay or in Salmonella typhimurium TM-677 in a forward mutation assay. Higher concentrations of histidine in corn grown in AAtrex treated soil may interfere with the reversion assay, but do not affect the forward mutation assay. The nature of the agent(s) responsible for the positive response was not determined. The mutagenicity may be due to natural plant constituents, an artifact of the sample preparation, or mycotoxins from some unrecognized plant infection. The experimental results in these field studies do not show that atrazine is degraded or metabolized by corn plants to mutagens in this sensitive bacterial assay.     

Evaluation of in vitro models of stem cell-derived cardiomyocytes to screen for potential cardiotoxicity of chemicals

Cardiotoxicity is an important toxicological endpoint for chemical and drug safety assessment. The present study aims to evaluate two stemcell-based in vitro models for cardiotoxicity screening of chemicals. Eleven model compounds were used to evaluate responses of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) using beating arrest as a readout and the analysis of electrophysiological parameters measured with a multi-electrode array (MEA) platform of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Results revealed that the hiPSC-CM MEA assay responded to all compounds. The mESC-CM beating arrest assay was not responsive to potassium channel blockers and showed a lower sensitivity to sodium channel blockers and Na⁺/K⁺ ATPase inhibitors compared to the hiPSC-CM MEA assay. Calcium channel blockers and a β-adrenergic receptor agonist showed comparable potencies in both models. The in vitro response concentrations from hiPSC-CMs were highly concordant with human effective serum concentrations of potassium and sodium channel blockers. It is concluded that both in vitro models enable the cardiotoxicity screening with different applicability domains. The mESC-CM beating arrest assay may be used as a first step in a tiered approach while the hiPSC-CM MEA assay may be the best starting point for quantitative in vitro to in vivo extrapolations.     

Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput screening assay. In the patch-clamp assay, the alpha1 GlyR exhibited the properties expected from a strychnine-sensitive glycine-gated chloride channel. In the FMP assay exposure of the cell line to GlyR agonists elicited a concentration-dependent increase in fluorescent intensity, a signal that could be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC50 and Ki values in good agreement with previously reported values from studies of recombinant alpha1 GlyRs and native alpha1beta GlyRs. The rank orders of potencies was glycine > beta-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the alpha1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn2+ inhibited alpha1 GlyR signalling but in contrast to previous reports the metal ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited alpha1 GlyR function, the potentiation of alpha1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.     

Screening haemostasis--looking for global assays: the Overall Haemostasis Potential (OHP) method--a possible tool for laboratory investigation of global haemostasis in both hypo- and hypercoagulable conditions

A broad spectrum of global haemostatic assays has recently been developed and modified in an attempt to overcome the drawbacks of classical screening tests used for evaluation of coagulation and fibrinolysis. The Overall Haemostasis Potential (OHP) assay is one of such assays. The assay is based on repeated spectrophotometric registration of fibrin-aggregation in citrated plasma, to which small amounts of exogenous thrombin, tissue type plasminogen activator and calcium chloride have been added. The area under the fibrin aggregation curve which then develops is calculated and is the laboratory parameter used for OHP determination. The Overall Coagulation Potential (OCP) and Overall Fibrinolytic Potential (OFP) are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The sensitivity of the assay for detecting hypercoagulation in normal pregnancy, in preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke and vascular surgery has been evaluated. Since the assay can monitor haemostasis balance in the sample, it may serve as a laboratory tool to determine hypocoagulation, especially in patients with haemophilia A or B. Preliminary findings also indicate that the OHP assay may be useful in the monitoring of anticoagulant treatments. Larger controlled clinical studies are, however, mandatory before a definite conclusion about the usefulness of the method can be drawn.     

Human T cell priming assay (hTCPA) for the identification of contact allergens based on naive T cells and DC – IFN-γ and TNF-α readout

Many small protein reactive organic and inorganic chemicals can cause allergic contact dermatitis, a T cell mediated inflammatory skin disease. In vitro alternatives to animal testing are needed for the identification of chemicals that pose such risks to human health. We here publish the standard operation procedure for a human T cell priming assay developed primarily for the identification of contact allergens within the integrated EU project Sens-it-iv. This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. Using sorted naïve T cells and monocyte-derived dendritic cells pulsed with the test chemical or with chemical-protein conjugates, the successful priming of an antigen-specific T cell response is controlled after antigen-specific restimulation by cytokine readout. As the most specific response of the immune system to contact allergens the analysis of the chemical-specific T cell response may be a useful in vitro assay for hazard identification in immunotoxicology. This assay may be a valuable, highly specific element of an integrated testing strategy for the identification of chemicals and drugs that cause T cell mediated respiratory or gastrointestinal tract hypersensitivities or adverse drug reactions.