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PubMed1988

[A six-day subrenal capsule assay for predictive testing of primary human tumors].

We carried out a total of 36 in vivo chemosensitivity tests in 33 cases of human malignant tumor using the subrenal capsule assay, developed by A.E. Bogden et al. Of the 36 assays, 31 were evaluable. The chemosensitivity of each tumor varied individually. UFT, 5-fluorouracil, mitomycin-C and adriamycin were administered to gastrointestinal cancer patients regularly, but our SRC-assay showed a high sensitivity rate for UFT and 5-fluorouracil but a low sensitivity rate for mitomycin-C and adriamycin. Nine patients had clinically evaluable lesions and a correlation between the assay results and clinical response existed in 6 cases. The true positive rate was 50% (3/6), the true negative rate 100% (3/3), and the overall predictive accuracy 66% (6/9). This study suggested that 6-day SRC assay is useful for selecting effective anti-tumor agents for the treatment of cancer patients.

Animals↗

PubMed1986

Improved fluorescence polarization assay for use in evaluating fetal lung maturity. I. Development of the assay procedure.

We describe a fluorescence polarization assay for use in predicting fetal lung maturity, which is suitable for the TDx Analyzer (an automated fluorescence polarimeter). The assay requires 0.5 mL of amniotic fluid and approximately 30 min, and involves the fluorophore 1-palmitoyl-2(6-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]caproyl) phosphatidylcholine. Reproducible polarization measurements depend on proper regulation of incubation time and temperature, but variations in the concentrations of fluorescent probe and amniotic fluid have little effect on measured polarization and therefore little effect on assay precision. Working solutions of the fluorescent probe are stable for at least nine months when stored at -20 degrees C and pH 5. Interferences include erythrocytes, serum, bilirubin, meconium, and lidocaine.

Amniotic Fluid↗

PubMedDate not supplied

An overview of assay methods for D-penicillamine.

The stability and biotransformation of D-penicillamine as it relates to assay development is discussed. A review of published literature on assays is presented with respect to blood/plasma levels of D-penicillamine in man and to statistical evaluation of assays. Also, the paper describes gas chromatographic assay for D-penicillamine disulfide and L-cysteine D-penicillamine disulfide in urine and plasma, as well as a new high performance liquid chromatography assay for D-penicillamine in plasma.

Biological Assay↗

PubMed1976

Roles of individual mgl gene products in the beta-methylgalactoside transport system of Escherichia coli K12.

Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-beta-D-galactopyranoside, while mgl A- and mgl C- mutants are not. When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. In this study, we present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A+,C+ mutants defective in galactose-binding protein accumulate methyl-beta-D-galactopyranoside up to six times the concentration gradient while mgl A- and mgl C- mutants failed to accumulate this substrate. Similar results were obtained using D-glyceryl-beta-D-galactopyranoside, another substrate of the methylgalactoside transport system. In contrast, all sugars tested which are not substrates of this system were transported equally by all mgl- mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-beta-D-galactopyranoside against a 10,000-fold concentration gradient. The apparent Km of methyl-beta-D-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains. In contrast, there was no significant difference between these strains in either the Vmax of substrate influx or the rate of substrate exit. D-Galactose competitively inhibited methyl-beta-D-galactopyranoside influx into both mgl B- and mgl+ strains; the Ki of inhibition in mgl B- cells was 2,000-fold greater than that in mgl+ cells.

Biological Transport, Active↗

PubMed2001

A peptide enzyme linked immunosorbent assay (ELISA) for the detection of human immunodeficiency virus type-2 (HIV-2) antibodies: an evaluation on polymerase chain reaction (PCR) confirmed samples.

BACKGROUND: HIV-1 and HIV-2 infections differ in prognosis, and may also require different prevention and/or treatment approaches. Thus, estimating the true prevalence of HIV-1 and HIV-2 infections, as well as co-infections, is a critical step in controlling the disease. There are a few commercial ELISA and immunoblot kits, which can differentiate between HIV-1 and HIV-2 infections. However, some of these assays overestimate the prevalence of dual infection. Hence, it is necessary to develop assays capable of discriminating between the two infections. OBJECTIVES: To develop a synthetic HIV-2 env based peptide ELISA for the detection of HIV-2 specific antibodies and evaluate its performance on samples from HIV positive individuals previously tested by HIV-1 and HIV-2 PCR and HIV seronegative individuals. STUDY DESIGN: We studied 45 HIV seronegative and 63 HIV infected individuals, including 30 HIV-1 PCR and immunoblot positives, 19 HIV-2 PCR and immunoblot positives, five HIV-1 and two PCR and dual immunoblot positives, two PCR negative but positive for HIV-2 by immunoblot and seven dual immunoblot positives who were only positive for HIV-1 by PCR. RESULTS: All 24 HIV-2 PCR positive samples tested were positive by the peptide assay. Among 30 HIV-1 PCR and immunoblot positive samples, only one (3.3%) showed an absorbance value above the cut off level. The seven dual positive samples by immunoblot (only positive for HIV-1 by PCR) were negative by the HIV-2 peptide ELISA. There was a 100% concordance between HIV-2 PCR and peptide ELISA. The sensitivity, specificity, and the likelihood ratio for the peptide ELISA were 100,94.9, and 19.5, respectively when compared against the PCR findings. CONCLUSIONS: This ELISA, using a specific immunodominant epitope (11 amino acids) from the transmembrane (gp36) portion of the HIV-2 envelope glycoprotein showed a high concordance with PCR findings. This can be considered as a highly sensitive, specific and economically feasible assay for the discrimination of HIV-1 and HIV-2, and may serve as an alternative to HIV-2 PCR in epidemiological studies.

Enzyme-Linked Immunosorbent Assay↗

PubMed1991

Lymphocyte proliferation assays as potential biomarkers for toxicant exposures.

Recently there has been interest in developing assays that can be used as indicators (biomarkers) of exposure to toxic agents. We have been exploring the potential utility of three lymphocyte proliferation assays [the responses of B lymphocytes to the mitogen lipopolysaccharide (LPS), the responses of T lymphocytes to the mitogen concanavalin A (ConA), and the responses of T lymphocytes to antigenic stimuli in a mixed lymphocyte culture (MLC) assay] as biomarkers of toxicant exposure. Studies were initiated to assess the applicability and specificity of these assays and to investigate the mechanisms by which toxicants alter lymphocyte proliferation. All studies were performed using cells isolated from Fischer 344 rats. To assess applicability, mitogen assays were performed using in vitro exposures to eight different toxicants: hydroquinone, benzoquinone, Aroclor 1254, styrene oxide, and the salts of mercury, cadmium, chromate, and nickel. In vitro concentrations spanned five orders of magnitude (100 to 0.01 mg/l). At the lowest concentration tested, all eight compounds induced changes in at least one mitogen assay, indicating that these assays may be applicable to a wide range of toxicants. Variations of the ConA and MLC assays were used to test for specificity. In both assays, splenocytes taken from rats exposed in vivo to either chromate or to cadmium responded differently when the cells were cocultured with exogenously added chromate or cadmium ions, indicating that it may be possible to detect exposure to a specific toxicant by performing modified lymphocyte proliferation assays. In the mechanistic studies, splenocytes from cadmium and chromate-treated rats altered the ConA-induced proliferation of cocultured syngeneic cells. In addition, the antigenicity of splenocytes isolated from cadmium-treated rats was enhanced when these cells were used as stimulators for allogeneic splenocytes. The results of these studies indicate that lymphocyte proliferation assays may be useful for detecting exposure to a wide range of toxicants and that variations of these assays may be useful for implementing immunologically based tests for detecting exposures to specific chemicals.

Animals↗

PubMed2005

Is an assay for simultaneous detection of hepatitis C virus core antigen and antibody a valuable alternative to nucleic acid testing?

BACKGROUND: A new enzyme immunoassay based on the simultaneous detection of nucleocapsid proteins of hepatitis C virus (HCV) and anti-HCV (Monolisa HCV antigen-antibody Ultra, Bio-Rad) was evaluated as an alternative to nucleic acid testing (NAT) for the diagnosis of HCV infection during the window period in blood donations. STUDY DESIGN AND METHODS: The study included 107 sequential samples from 10 HCV seroconversion commercial panels; 81 samples were in the preseroconversion phase, and 26 were collected after seroconversion. All samples were tested with HCV antigen-antibody assay and the two minipool (MP) NAT procedures that are routinely used in France (transcription-mediated amplification in pools of 8 and COBAS AmpliScreen HCV test [Roche Diagnostic] in pools of 24 donations). RESULTS: From the 44 samples collected during window period that were MP-NAT-positive, 31 (70.5%) were also positive with the Monolisa HCV antigen-antibody assay. The mean delay in detecting HCV infection between these two methods was 5.1 days (range, 0-24 days). The Monolisa HCV antigen-antibody assay led to a reduction in the window period of 26.8 days (range, 0-72 days). All samples collected after seroconversion were detected with the HCV antigen-antibody assay. The specificity analyzed in 2503 consecutive blood donations was estimated at 99.88 percent. CONCLUSION: This new developed assay presents an improvement for the detection of HCV infection, especially in the early phase of infection when antibodies are undetectable. Although less sensitive than NAT, this assay could be a suitable solution for blood screening in developing countries where NAT (or HCV core antigen-specific assay) is not affordable or its implementation is not feasible.

Biomarkers↗

PubMed2002

The effect of diet fed to lambs on subsequent development of Trichostrongylus colubriformis larvae in vitro and on pasture.

Contrasting herbage diets were fed to lambs to evaluate their effect on subsequent development of Trichostrongylus colubriformis larvae in faeces and on pasture. The diets had either no condensed tannin (CT), lucerne (Medicago sativa cv. Otaio), white clover (Trifolium repens cv. Tahora), or had moderate to high concentrations of CT, sulla (Hedysarum coronarium cv. Grassland Aokau), Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grassland Maku), Dorycnium pentophyllum, and Dorycnium rectum. Trials were carried out in summer (warm) and in autumn (cool and moist). In summer, egg viability was evaluated in vitro with egg hatch and larval development assays. In both seasons faeces were placed on pasture to compare recovery of eggs and larvae from faeces and larvae from herbage on the high and low fertility farmlets on the AgResearch Ballantrae Hill Country Research Station. D. rectum and D. pentophyllum diets decreased (P<0.01) egg hatching and larval development in laboratory assays relative to other diets. In summer, the number of larvae recovered from faeces placed on pasture was far greater (P<0.001) if the lambs had been fed lucerne than any other diet, whereas recovery was always lowest from faeces of sheep fed D. rectum and D. pentophyllum. Although dietary differences were lower in autumn than in summer, larval recoveries were lower (P<0.05) from faeces of lambs fed D. rectum and L. corniculatus than from white clover, lucerne and sulla diets. This study indicates that the diet of the host can have a significant impact on egg hatching and the subsequent development of T. colubriformis larvae in the laboratory and in the field. In particular, D. rectum consistently reduced T. colubriformis development. Effects measured in vitro generally under-estimated effects measured under field conditions.

Animal Feed↗

PubMed1997

A highly sensitive electrochemiluminescence immunoassay for interferon alfa-2b in human serum.

A highly sensitive immunoassay for the quantitative measurement of recombinant interferon alfa-2b (IFN alfa-2b) in serum was developed using the ORIGEN electrochemiluminescence (ECL) detection system. The ECL assay developed uses a ruthenylated mouse monoclonal anti-IFN-alfa antibody and a sheep polyclonal anti-IFN-alfa antibody that has been biotinylated. An immune complex, formed between the antibodies and the IFN in the serum, is captured by streptavidin coated paramagnetic beads. The assay is sensitive and can accurately measure 4 IU/ml in undiluted serum samples. In addition, this assay requires less labor than classical ELISAs and is not significantly affected by individual serum factors. The total assay variance for both intra- and inter-assay precision is < 10%. The assay is specific for the analyte. Mean percent recoveries in pooled and individual donor serum samples range from 84 to 114%. In addition, results of the ECL assay correlate well with a bioassay and were more accurate that an ELISA for the detection of interferon alfa-2b in individual human serum samples. The assay can be modified to measure other forms of the analyte and/or interferon in other matrices.

Cytopathogenic Effect, Viral↗

PubMed1994

Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation.

Rats carrying the Eker tumor susceptibility mutation are genetically predisposed to renal cell carcinoma. Rats heterozygous for the Eker mutation (Eker carriers) develop multiple bilateral renal cell carcinomas by the age of 1 year. Using an in vitro rat kidney epithelial (RKE) transformation assay developed in our laboratory, proximal tubule cells derived from known Eker rat carriers (+/ek) and non-carriers (+/+) were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to determine if cells derived from Eker carriers were more susceptible to in vitro transformation than cells derived from non-carrier animals. The percent transformation frequency following MNNG treatment was 7.5-fold higher in cells derived from carrier animals when compared to cells from non-carrier animals. This increased susceptibility to transformation due to inheritance of the Eker mutation is consistent with a predisposition resulting from inactivation of a tumor suppressor gene. The increased susceptibility of kidney epithelial cells carrying the Eker mutation may prove useful in the further development of the RKE transformation assay as a sensitive tool to identify potential renal carcinogens. In addition, because transformation frequency in the RKE assay measures a very early step in multistage transformation, these results also suggest that alterations (by Loss of Heterozygosity or mutation) at the Eker tumor susceptibility locus are an early event in the development of renal tumors in the rat.

Animals↗

PubMed1988

The development of fertility assay kits: an overview.

This paper analyzes the importance of developing fertility assays kits, specially in order to shorten the estimate of the fertile period in NFP users. It presents the criteria that an assay kit needs to meet. Urinary hormones, metabolites of progesterone, estrogens, and luteinizing hormones are analyzed as fertile-period markers. Progesterone and estradiol in saliva, as well as other constituents of saliva, the volumetric vaginal aspirator, and the change in the electrical conductivity of the vaginal fluid are also analyzed with the same purposes.

Family Planning Services↗

PubMed1992

Evidence of embryo- and trophoblast-toxic cellular immune response(s) in women with recurrent spontaneous abortion.

OBJECTIVE: The purpose of this study was to determine whether reproductive antigens stimulate lymphocytes and macrophages from women with recurrent abortion to secrete factors that are toxic to preimplantation embryos or trophoblast cells in vitro. STUDY DESIGN: Mononuclear cells were isolated from 30 fertile controls and 300 nonpregnant women being evaluated for recurrent abortion. Supernatants generated from these cells after separate culture with sperm and trophoblast antigen extracts were added to two-cell mouse embryo cultures and trophoblast proliferation assays. Toxicity was assumed when the median percentage of embryos developing to blastocysts or trophoblast proliferation was less than or equal to 50% of control values. Both parametric and nonparametric statistical methods were used where appropriate. RESULTS: Mouse embryo development and/or trophoblast proliferation were significantly inhibited by supernatants from trophoblast and/or sperm antigen-activated peripheral blood leukocyte cultures from a majority of 300 women with recurrent abortion but not from 30 women with normal reproductive histories. The mouse blastocyst development assay was more sensitive than the trophoblast proliferation assay in determining toxic factor production. Embryo-toxic factors were produced by activated leukocyte cultures from 90% of 180 women with a history of recurrent abortion of unexplained etiology, whereas trophoblast-inhibitory factors were detected in 50% of women from the same group. The embryo-toxic factor(s) was heat labile, had a molecular weight(s) between 10 and 30 kd, and was absorbed out by passage through affinity columns containing anti-interferon gamma beads. CONCLUSION: We conclude that recurrent abortion in some women is associated with embryo- and/or trophoblast-toxic factor production in response to stimulation by sperm or trophoblast antigens and that the principal factor may involve the 18 kd, heat-labile, T-lymphocyte cytokine interferon gamma. This study suggests a new cause of recurrent abortion.

Abortion, Habitual↗

PubMed1997

Assays for HIV with improved sensitivity and specificity.

Increased knowledge of the human immunodeficiency virus (HIV) and the infection it causes in humans has resulted in an enormous expansion in the understanding of viral parameters and host changes. HIV is a virus which mutates readily and rapidly, presenting many challenges to assay developers, and monitors of therapy and drug-resistance. Prolific viral replication at all the stages of the disease means that an accurate assessment of viral burden, viral load and changes to immune system markers is essential for effective clinical management and treatment. In the present review we have summarised current opinion on the kinetics of HIV infection and the pathogenesis of the disease it causes, and have provided a background to the evolution of HIV assays. Sensitivities and specificities of assays used for anti-HIV and HIV detection have improved, and new assays have been developed employing novel molecular techniques, which are being applied to meet continually evolving demands for more sensitive measurement of an increasing number of parameters. The future of HIV testing is also considered in the light of new knowledge concerning virus dynamics in vivo, the likelihood of the emergence of new subtypes and the changing approach to therapy. Assays will be, on the whole, used to quantify virus and to measure the host reactions to infection, often in the presence of antivirals. Thus, extreme sensitivity and specificity will be required.

Journal Article↗

PubMed1994

A simple quantitative assay for ristocetin cofactor activity using microtiter plate and ELISA plate reader.

Ristocetin cofactor activity is the test of choice for diagnosing von Willebrand disease. There is however no simple and precise quantitative method for its rapid determination in the clinical laboratory. We describe here a new approach for measuring ristocetin cofactor activity using an ELISA plate reader. Paraformaldehyde-fixed platelets, diluted plasma samples and ristocetin were mixed and incubated in the wells of a microtiter plate. Platelet agglutination was measured by light absorbance at 405 nm. Measurements of platelet agglutination at 1x to 64x dilution of a normal pooled plasma were used to construct a standard curve. Further characterization showed a significant linear correlation between results determined by the new method and by an aggregometer assay (p < 0.0001), and that the intra- and inter-assay variations were 6% and 17%, respectively. Therefore, the newly developed assay provides a simple and rapid way for precise quantitation of plasma ristocetin cofactor activity.

Blood Proteins↗

PubMed1997

A single [3H]thymidine-based limiting dilution analysis to determine HTLp and CTLp frequencies for bone marrow donor selection.

Histocompatibility between recipient and donor is a critical factor in allogeneic BMT which, to a large extent, determines the incidence of GVHD after BMT. Functional histocompatibility assays, such as the helper T lymphocyte precursor frequency assay (HTLp) and the cytotoxic T lymphocyte precursor frequency assay (CTLp), have proved to be helpful tools in facilitating donor selection procedures. However, a major drawback of these assays is that they are laborious and require large numbers of cells. We therefore adapted a [3H]thymidine-based assay, the 'JAM' test, as a read-out for CTLp frequencies, to replace the more cumbersome 51Cr-release assay. Furthermore, we applied an experimental setup that enables the assessment of HTLp and CTLp frequencies from a single limiting dilution assay to reduce the number of cells needed. The newly developed assay is relatively easy to perform and has the advantage that different subsets of T cells can be quantified in a single ongoing alloreaction. When the combined assay was applied in unrelated donor selection it proved to be a sensitive method that enables differentiation in suitability of distinct donors for a single patient. Therefore, the combined HTLp/CTLp assay appears to be a practical and sensitive method for identifying functional histocompatibility in related and unrelated donor/recipient combinations.

Bone Marrow Transplantation↗

PubMed2003

Detection of mutagenic pollution of natural environment using microbiological assays.

One of the most important and serious ecological problems is mutagenic pollution of the natural environment. Therefore, detection of mutagenic compounds in samples taken from natural habitats is of special interest. Microbiological mutagenicity tests seem to be very useful tools for such detection. In this review article, a general view on the tests employing genetically modified bacterial strains designed for detection of low concentrations of mutagenic compounds is presented. Moreover, a comparison of advantages and disadvantages of selected assays, developed early on and more recently, and features of these assays are discussed. It appears that none of the currently available mutagenicity tests is perfect or optimal for all purposes. Thus, a choice for the particular assay must depend on the nature of studies and specific tasks of the experiments to be performed.

Bacteria↗

PubMed1995

Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay.

Chemosensitivity assays are widely used to predict the ability of tumor cell lines to respond to potential or existing cytotoxic drugs. In this study we have compared the cell cloning assay first described by Salmon and Hamburger with a recently developed assay which measures viable cell number by ATP luminescence. Methotrexate (MTX) was chosen as the test agent, since cell lines with varying degrees of sensitivity to this agent were readily available. The results shown good correlation between the two assays, both of which are able to discriminate between the various cell lines used. MTX inhibition of primary breast carcinomas and cell lines shows a steep dose-response curve with a threshold concentration above which increasing dose does not increase sensitivity. In solid tumors, the plateau is usually reached at a level well below 100% inhibition. The ATP luminescence assay allows discrimination of MTX sensitivity between breast carcinomas and has considerable technical advantages over the cloning assay.

Adenocarcinoma↗

PubMed1998

Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures.

BACKGROUND: Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host. METHODS: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice. RESULTS: L. casei induced IFN-gamma, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-gamma. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody. CONCLUSIONS: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.

Allergens↗