A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh.

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.     

Comparison of bioassays and laboratory assays for apple stem grooving virus

The standard field double-budding assay with the indicator Virginia Crab and the glasshouse test with the indicator Malus micromalus, were compared with ELISA and immunocapture PCR for the detection of Apple stem grooving virus (ASGV) in 102 apple trees and three oriental pear. Twenty-two trees were positive for ASGV by both ELISA and IC-PCR but three of these trees were negative by Virginia Crab, three were negative by M. micromalus and one was negative by both these bioassays. The infected trees were re-tested by IC-PCR and ELISA in a second year; the IC-PCR results were confirmed but two of the 22 infected trees were negative by ELISA. On this evidence, IC-PCR is a more reliable assay for ASGV than the slow and expensive bioassays.     

Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin.

The effectiveness of a latex agglutination assay kit for the detection of Escherichia coli heat-labile toxin and cholera toxin was determined for the identification of natural isolates of the corresponding enteric pathogens in Southeast Asia. By selection of the appropriate culture media, the sensitivity of the assay was improved from 90.6% (for the detection of heat-labile toxin) and 75% (for the detection of cholera toxin) to 100%, and the results were confirmed with bioassays and DNA hybridization assays for both clinical and environmental isolates.     

High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.     

Parlin, a general microcomputer program for parallel-line analysis of bioassays

Commonly used manual and calculator methods for analysis of clinically important parallel-line bioassays are subject to operator bias and provide neither confidence limits for the results nor any indication of their validity. To remedy this, the authors have written a general program for statistical analysis of these bioassays for the IBM Personal Computer and its compatibles. The program has been used for analysis of bioassays for specific coagulation factors and inflammatory lymphokines and for radioimmunoassays for prostaglandins. The program offers a choice of no transform, logarithmic, or logit transformation of data, which are fitted to parallel lines for standard and unknown. It analyzes the fit for parallelism and linearity with an F test, and calculates the best estimate of the result and its 95% confidence limits. Comparison of results calculated by PARLIN with those previously obtained manually shows excellent correlation (r greater than 0.99). Results obtained using PARLIN are quickly available with current assay technics and provide a complete evaluation of the bioassay at no increase in cost.     

Recent progress in the development of assays suited for histone deacetylase inhibitor screening

Histone deacetylase (HDAC) inhibitors have an unprecedented potential to occupy a major position in the future market of anticancer agents. However, progress in the development of these new chemotherapeutics is largely dependent on the existence of bioassays well-suited for inhibitor screening. Herein, we summarize recent developments in HDAC assay technology and, particularly, discuss different assay types with respect to their suitability for high-throughput screening programs.     

Short-term bioassays of fractionated emission samples from wood combustion

Extracts of an emission sample from wood burning, consisting of particles and volatiles, have been fractionated on an HPLC silica gel column into five fractions of increasing polarity. Nonfractionated samples and the individual fractions have been tested in three different short-term bioassays: the Ames Salmonella assay, the sister chromatid exchange (SCE) induction-test in Chinese hamster ovary cells (CHO), and the cell transformation test on Syrian hamster embryo (SHE) cells. Most of the total activity was found in the volatile part of the sample with all three bioassays, whereas the particle extract had the highest activity per unit mass extracted. The second most polar fraction contained most of the mass and was also highly active in all assays. The most polar fraction was very potent in the Salmonella assay, but showed only a weak response in the eukaryotic bioassays. Storage of the samples for several months at 0 degrees C revealed that the bacterial mutagens present in the most polar fraction were labile; the mutagenicity was almost totally lost after 1 year's storage.     

Bioassay of hormones

Bioassay of hormones has a small but important role in defining the physiologic status of selected patients in whom immunoassay results do not correlate with clinical signs and symptoms. This article reviews three sensitive bioassays for hormones, which are based on 1) stimulation of adenylate cyclase in membrane preparations, 2) cytochemical changes in fresh tissue segments or sections, and 3) stimulation of cell replication in the Nb2 lymphoma cell culture. The adenylate cyclase assay using kidney membranes can measure parathyroid hormone (PTH), whereas assays using thyroid membranes can measure both thyrotropin (TSH) and thyroid-stimulating immunoglobulins (TSI). The cytochemical assay methods can measure numerous hormones, but the two most promising assays for clinical use are the PTH assay using guinea pig distal convoluted renal tubules and TSH/TSI assay using thyroid follicles. The Nb2 lactogenic assay is sensitive to biologic concentrations of growth hormone, prolactin, and human placental lactogen.     

Assessment of the genotoxicity of trichloroethylene and its metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), in the comet assay in rat kidney

Trichloroethylene (TCE) has been reported to give a small, but significant, increase in renal tumours in the rat. These tumours were always associated with nephrotoxicity which is most likely caused by the metabolism of TCE to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) which accumulates in the proximal tubules. The genotoxicity of TCE and DCVC have been evaluated in vivo using the comet assay to assess DNA breakage in the proximal tubules of rat kidneys. Rats were exposed to TCE by inhalation or to DCVC by oral gavage at dose levels in excess of those which produced effects in long-term bioassays. Cell suspensions were produced from proximal tubules isolated from the kidneys of treated rats and the level of DNA damage assessed in these cells using the pH >13 comet assay. In vitro and in vivo positive controls were included and demonstrated the sensitivity of the assay. TCE gave a clearly negative response in the assay at all dose levels as did DCVC at the 16-h sampling time and at the 2-h sampling time with the lower dose level. At the 2-h sampling time following administration of DCVC at the higher dose level (10 mg/kg), there was limited evidence of DNA damage in a small number of animals, but this was considered insufficient to indicate a positive response in this assay. These data support an overall conclusion, based on these and other published data, that the renal tumours seen in bioassays are non-genotoxic in origin.     

Micronucleus assays with Tradescantia pollen tetrads: an update

Micronucleus (MN) assays with early pollen tetrad cells of Tradescantia (Trad-MN assays) are at present the most widely used bioassays with plants for the detection of genotoxins in the environment. So far, ～ 160 chemicals have been tested and ～ 100 articles that concern complex environmental mixtures were published. This article summarises the results of Trad-MN studies, which have been carried out during the last 15 years with individual compounds and investigations concerning the pollution of environmental compartments (soil, water and air). The evaluation shows that the effects of certain genotoxins such as heavy metals, radionuclides, pesticides and air pollutants can be easily detected with this test. Comparisons with results obtained in MN studies with mitotic (root tip) cells indicate that meiotic tetrad cells are in general more sensitive. Important issues for future research concern the evaluation of the suitability of wildlife Tradescantia species that are sometimes used instead of specific clones (such as #4430 for which standardised protocols have been developed) as well as the assessment of the predictive value of Trad-MN results in regard to the prediction of cancer hazards in humans and adverse effects at the ecosystem level. The fact that the genotoxic effects of certain compound such as metals, which can be detected with plant bioassays, in particular with the Trad-MN assay but not in other commonly used bioassays (e.g. in bacterial tests) makes them an essential element in the batteries for environmental monitoring.     

Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates

Potato virus Y (PVY) has a worldwide distribution and infects several economically important crops from the Solanaceae family. The emergence and spread of the PVYNTN strain, which is the causative agent of potato tuber necrotic ringspot disease (PTNRD), has lead to large economic losses and highlighted the need for accurate discrimination of the different PVY strains. Detection and differentiation of PVY isolates is mainly based on a combination of ELISA, RT-PCR and bioassays; however, PVYNTN isolates are particularly difficult to differentiate from standard PVYN without the use of time-consuming bioassays. A strong correlation has been identified previously between the ability to induce PTNRD and the presence of a recombination point in the virus coat protein. An RT real-time PCR assay has been developed to enable detection of isolates with the recombination point, therefore, enabling rapid differentiation between potentially tuber necrotic PVYNTN isolates and standard PVYN isolates. The assay is also able to detect the presence of PVYO isolates. To aid with routine testing, immuno-capture and post-ELISA virus release were introduced; when coupled with RT real-time PCR the sensitivity of the assays were up to seven orders of magnitude higher than ELISA. The assay was shown to be a suitable method for rapid large-scale diagnostic testing of PVY in different types of plant material including tubers, and specific screening for potentially tuber necrotic recombinant isolates.     

Review and evaluation of the NCI/NTP carcinogenesis bioassays

A comparison of the carcinogenesis bioassay results obtained by the National Cancer Institute (NCI) and the National Toxicology Program (NTP) indicates that approximately one-half of the bioassays directed by both institutions were positive for carcinogenicity. The more recent 85 bioassays completed by NTP reveal a higher proportion of studies interpreted as demonstrating no evidence of carcinogenicity than represented in the initial 198 bioassays conducted by NCI. Of the 100 NCI bioassays that were not positive for carcinogenicity 3 (3%) were classified in the category of "no evidence for carcinogenicity in two animal species." Of the 43 NTP bioassays that were not positive for carcinogenicity 36 (84%) were placed in the category of "no carcinogenic effects." The reason for this shift from a 33:1 positive to negative ratio in the NCI bioassays to an approximately 1:1 ratio in the NTP bioassays appears to be a difference in interpretation of the adequacy of the testing. For example, 6 of the 36 NTP negative bioassays involved testing in only one species. Uniform criteria for concluding that a bioassay is negative must be developed and the results of all existing and future carcinogenesis bioassays must be interpreted with these exclusive criteria. Other bioassay problems are explored, including the incomplete validation of the carcinogenesis bioassay protocol by confirmatory results with positive and negative reference agents, the apparent lack of bioavailability data for some orally administered negative compounds, the continued use of mouse hepatic neoplasia as a single discriminating parameter, the variability in the inter- and intrastudy incidence of spontaneous tumors, and the continued reliance on the maximum tolerated dose.     

The Tg rasH2 mouse in cancer hazard identification

The Tg rasH2 transgenic mouse has been developed as an altemative to the lifetime mouse bioassay to predict the carcinogenic potential of chemicals. Unlike the p53+/- mouse, the Tg rasH2 mouse is sensitive to both genotoxic and nongenotoxic carcinogens. The Tg rasH2 mouse, officially designated CB6F1-TgN (RasH2), contains multiple copies of the human c-Ha-ras oncogene and promoter within its genome. These mice develop spontaneous andchemically inducedneoplasms earlierin life and in greaternumbersthan wild-type mice, reflectingtheirenhanced sensitivity to neoplasia. The most common spontaneous neoplasms in control Tg rasH2 mice 8 to 9 months of age are lung adenomas and carcinomas (7.4% incidence), splenic hemangiomas and hemangiosarcomas (5.4%), forestomach squamous cell papillomas and carcinomas (2.4%), and skin neoplasms (1.2%). Simulations have demonstrated that 20 to 25 mice/sex/treatment group are required to provide the assay with adequate statistical power. Four of 6 known or suspected human carcinogens tested in Tg rasH2 mice were positive in this assay. For 19 nonmutagenic agents testing positive in conventional rodent bioassays, 7 chemicals were positive, 10 chemicals were negative, and 2 were equivocal. None of the 10 nonmutagenic rodent carcinogens that were negative in the Tg rasH2 mouse model are considered to be human carcinogens. All nonmutagenic chemicals that were negative in the conventional rodent bioassays were also negative in the Tg rasH2 model. Results for 15 of 18 mutagenic chemicals tested in Tg rasH2 mice agreed with the results of conventional rodent bioassays, and 3 results were equivocal. The Tg rasH2 mouse model appears to predict known or suspected human carcinogens as well as the traditional mouse bioassay, but with fewer positive results for nongenotoxic compounds that are not considered human carcinogens. The Tg rasH2 mouse model is the most thoroughly tested in vivo altemative to the lifetime mouse bioassay for nongenotoxic compounds administered by oral or parenteral routes. The U.S. FDA Carcinogenicity Assessment Committee has determined that the Tg rasH2 model has been adequately evaluated for consideration for carcinogenicity testing of pharmaceutical candidates and its use could contribute to the weight of evidence for carcinogenicity assessment. The FDA will consider proposals to replace lifetime mouse carcinogenicity studies with 6-month Tg rasH2 mouse studies to support pharmaceutical registration on a case-by-case basis.     

Development and application of a pig IL-8 ELISA detection system

Interleukin 8 (IL-8) is a chemotactic and activating chemokine, especially for neutrophils, which plays an important role in inflammatory process. A pig IL-8 specific enzyme-linked immunosorbent assay (ELISA) was developed to measure IL-8 concentrations in cell culture supernatants and biological fluids. A streptavidin-biotin amplified sandwich method uses mouse capture mAb IZ8.03 and detection biotinylated mouse mAb IZ8.04 against recombinant pig IL-8. The assay specifically and reproducibly recognizes both recombinant and natural pig IL-8. A working range of the assay is 16-1000 pg/mL and takes a mere 3.5 h of incubation time. This pig IL-8 ELISA is a suitable alternative way of measurement of IL-8 concentrations to time consuming and laborious IL-8 bioassays.     

Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein

Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.     

Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.     

Economic comparison of enzyme immunoassay and virus isolation procedures for surveillance of arboviruses in mosquito populations.

Cost-effectiveness analysis of an enzyme immunoassay (EIA) for the surveillance of arboviruses was conducted. The EIA was compared with conventional virus isolation and serologic identification procedures (virus isolation procedures; VIP). Under most circumstances, EIA was more cost-effective than VIP. Costs for processing mosquito pools by VIP increased with the number of viruses included in the surveillance program and with the prevalence rate of each virus. In contrast to VIP, the prevalence rate did not affect costs for processing pools by EIA. In general, EIA was the most cost-effective procedure, followed by cell culture and mouse bioassays. In a 5-year cost-effectiveness analysis of a model surveillance program in which EIA and cell culture bioassays were used, the EIA again proved to be the most cost-effective assay procedure under most circumstances.     

Development and Application of a Pig IL-8 ELISA Detection System

Abstract

Interleukin 8 (IL-8) is a chemotactic and activating chemokine, especially for neutrophils, which plays an important role in inflammatory process. A pig IL-8 specific enzyme-linked immunosorbent assay (ELISA) was developed to measure IL-8 concentrations in cell culture supernatants and biological fluids. A streptavidin-biotin amplified sandwich method uses mouse capture mAb IZ8.03 and detection biotinylated mouse mAb IZ8.04 against recombinant pig IL-8. The assay specifically and reproducibly recognizes both recombinant and natural pig IL-8. A working range of the assay is 16–1000 pg/mL and takes a mere 3.5 h of incubation time. This pig IL-8 ELISA is a suitable alternative way of measurement of IL-8 concentrations to time consuming and laborious IL-8 bioassays.     

Biological assays for interleukin 1 detection

Various biological assays are used for qualitative and quantitative measurements of interleukin 1 (IL-1) in supernatants from cell cultures. The purpose of the present study was to compare the specificity and variability of three cellular IL-1 bioassays: the PHA co-stimulatory human T lymphocyte proliferation assay, the PHA co-stimulatory murine thymocyte (THY) proliferation assay, and the 2-step NOB-1 conversion assay. Three different ways of IL-1 unit calculation, based on a semi-logarithmic plot, a double-logarithmic plot, or a probit-analysis plot were also compared. The T lymphocyte assay can be used only to demonstrate qualitative differences in IL-1-like activity, whereas the THY assay is excellent as a semi-quantitative assay, with a low intra-assay variability, but also with a low specificity. The NOB-1 assay is probably more specific with respect to IL-1 measurement, although, with a high intra-assay variance. The THY and the NOB-1 assays both have a high inter-assay variability, and measurement of samples from longitudinal clinical studies must be done in one and the same analysis if quantitative differences are to be illustrated. Probit analysis for unit calculation is recommended. To generate a consensus view as to assay performance, collaborative laboratory studies are needed.     

Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments

An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.