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PubMed1987

Physiological studies of human chorionic gonadotropin (hCG), alpha hCG, and beta hCG as measured by specific monoclonal immunoradiometric assays.

Several libraries of monoclonal antibodies have been produced against epitopes that reside on hCG, alpha hCG, and beta hCG. Having characterized them physically, we explored their use in the construction of highly specific and sensitive immunoradiometric assays. There were several important immunochemical considerations with respect to developing assays that accurately detect low levels of free subunits in serum in the presence of high concentrations of the native hormone. These include physical properties and specificities of the monoclonal antibodies, choice of capture antibody on the solid phase support, assay design, and purity of hormone standards. Using such assays, we found early pregnancy (in vitro fertilization) to be characterized by the sequential appearance of hCG, followed by beta hCG and then alpha hCG. Molar ratios of beta hCG to alpha hCG and beta hCG to hCG were highest in early gestation. However, there was a reversal of the beta hCG to alpha hCG ratio at 12-13 weeks gestation, and an excess of free alpha hCG was observed thereafter. Except for values obtained in very early pregnancy, the beta hCG to hCG ratio remained remarkably constant at approximately 0.5% throughout gestation. In contrast, choriocarcinoma was distinguished by absolute serum beta hCG concentrations 3-100 times greater than the maximum values observed during pregnancy and, more importantly, by exceedingly high beta hCG to hCG ratios. For comparison, we studied hCG, alpha hCG, and beta hCG levels in an additional 178 patients with nontrophoblastic tumors. Ectopic production of alpha hCG and beta hCG was rare (3%), and thus far, we have been unable to demonstrate the presence of hCG in such patients. Therefore, hCG and the free subunits appear not to be useful as serological markers for nontrophoblastic tumors.

Adolescent↗

PubMed1997

Micro-assay method for evaluating the allergenicity of the major soybean allergen, Gly m Bd 30K, with mouse antiserum and RBL-2H3 cells.

A micro-assay method for evaluating the allergenicity of soybean allergen was developed by using the mouse antiserum against Gly m Bd 30K, a major soybean allergen, and RBL-2H3, a rat mucosal mast cell line. The antiserum against Gly m Bd 30K was prepared by subcutaneously immunizing BALB/c mice with the allergen. The behavior by affinity-chromatography and the properties against heat treatment show that the reaginic activity of the antiserum resided in the IgE antibody specific for Gly m Bd 30K. The developed assay method is shown to be useful for simulating IgE mediated type-I allergy and to be highly sensitive for detecting the allergen.

Allergens↗

PubMed1999

Reverse-transcription polymerase chain reaction detection of the enteroviruses.

OBJECTIVE: This review focuses on commercial and in-house-developed reverse-transcription polymerase chain reaction (RT-PCR) assays used for the detection of enteroviral infections. In addition to providing details on the performance of RT-PCR, its specificity, and sensitivity, the clinical utility of this diagnostic method with specific reference to its impact on hospitalization and cost savings is addressed. DATA SOURCES: MEDLINE was searched for reports relating to RT-PCR detection of the enteroviruses in adults and children. The search was restricted to studies reported in English language journals. STUDY SELECTION: Reports documenting detailed information regarding the RT-PCR conditions, primers, sensitivity, specificity and, if relevant, clinical impact were selected for analysis. DATA EXTRACTION: Details regarding method of extraction of the enteroviral genome, the primers used, RT-PCR conditions, and sensitivity and specificity of the assay were extracted from the literature. For reports detailing the use of RT-PCR in the clinical management of enteroviral infections in children, the reduction in duration of hospitalization and health care cost savings were recorded. DATA SYNTHESIS: Reverse-transcription PCR can increase the yield of detection of enteroviruses from cerebrospinal fluid by a mean of approximately 20% over tissue culture. Reverse-transcription PCR of cerebrospinal fluid has been shown to exhibit sensitivity and specificity values of 86% to 100% and 92% to 100%, respectively. Reductions of 1 to 3 days of hospitalization per patient are predicted if RT-PCR is used to diagnose enteroviral meningitis in children. CONCLUSIONS: Reverse-transcription PCR detection of enteroviral infections is an extremely rapid, sensitive, and specific diagnostic modality. Both commercial assays and assays developed in-house appear to be equivalent with regard to sensitivity and specificity. Reverse-transcription PCR diagnosis of enteroviral infections in children could reduce the length of hospitalization and result in significant health care cost savings.

Enterovirus↗

PubMed1999

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

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

Analysis of Variance↗

PubMedDate not supplied

ELISA using enzyme penicillinase for the detection of IgG antibodies to Trypanosoma evansi in sera of experimentally infected rabbits.

An Enzyme Linked Immunosorbent Assay (ELISA) using penicillinase as an enzyme marker has been developed for the detection of IgG antibodies in the sera of Trypanosoma evansi infected rabbits. Anti-T. evansi IgG antibodies were detected on 10th day after the infection and thereafter antibody titre rose progressively for 18 days. The developed assay is simple (end result assessed visually) and reproducible (4.4 and 14.0 percentage of intra and inter coefficient of variation respectively).

Animals↗

PubMed2005

PCR method for the detection of potential ochratoxin-producing Aspergillus species in coffee beans.

Ochratoxin A (OA) is a nephrotoxic and carcinogenic mycotoxin that has been found in cereal and food commodities. Currently, Aspergillus carbonarius, A. niger and A. ochraceus have been recognized as the species responsible for OA in coffee beans. With the aim of developing multiplex-PCR for detection of these species in coffee bean samples, we first developed specific primers for A. niger detection. The primer designed (OPX7(372)) provided an amplicon of 372 pb in all A. niger stricto sensu isolates. The PCR assay developed for A. niger identification in pure culture was also successfully used for detecting this species in coffee beans. No cross-reaction was observed using DNA from coffee beans inoculated with closely related black aspergilli species. A multiplex-PCR method for detection of A. carbonarius, A. niger and A. ochraceus species in coffee beans was developed. From a single assay this method detected the amplicons of 809, 372, and 260 pb that represent the three most ochratoxigenic species found in coffee bean samples, i.e., A. carbonarius, A. niger and A. ochraceus, respectively.

Aspergillus↗

PubMed2002

Clues from wildlife to create an assay for thyroid system disruption.

In 1996 the U.S. Congress charged the U.S. Environmental Protection Agency to develop a screening program to test chemicals for their possible estrogenic and other endocrine effects. Shortly thereafter, the Chemical Guidelines Program of the Organisation for Economic Co-operation and Development's (OECD) Environmental Directorate organized a Task Force on Endocrine Disruption Testing and Assessment to coordinate development of internationally harmonized screening and testing protocols. Most of the research devoted to this effort has focused on detecting impaired estrogenicity, androgenicity, and/or steroidogenesis, with little progress toward developing assays to detect chemicals that might interfere with thyroid function. Despite the fact that wildlife biologists have been reporting abnormal thyroid gland development and unusual thyroid hormone (TH) and retinoid ratios in fish and birds since the early 1960s, few studies have demonstrated an association between an environmental contaminant and a particular health end point other than reduced reproductive success at the population level. This article is a review of the literature that specifically examines THs and their role in normal behavior and development in wildlife. It presents several studies that associated changes in the thyroid gland, TH concentrations, and behavior with contaminant exposure. The goal of this article is to provide fodder for the creation of simple screens to detect possible thyroid system agonists and antagonists.

Amphibians↗

PubMed2002

Viral replication and host gene expression in alveolar macrophages infected with Ebola virus (Zaire strain).

In order to characterize the cellular response to and identify potential diagnostic markers for the early detection of Ebola virus, an in vitro culture system involving nonhuman primate alveolar macrophages was developed. Ebola virus replication in the alveolar macrophages was characterized by plaque assay, immunohistochemical analysis, and in situ hybridization. Fluorogenic 5'-nuclease assays specific for nonhuman primate proinflammatory cytokines and chemokines were designed and used to evaluate mRNA transcription in macrophages infected with Ebola virus. Transient increases in cytokine and chemokine mRNA levels were observed immediately following exposure to Ebola virus. At 2 h postexposure, levels of cytokine and chemokine mRNAs were markedly reduced. Although Ebola virus infection of alveolar macrophages failed to induce a sustained increase in proinflammatory cytokine and chemokine mRNA transcription (potentially reducing the use of these markers as diagnostic tools), the fluorogenic 5'-nuclease assays developed may have prognostic value for individuals infected with Ebola virus. Recently published data have indicated that persons who remain asymptomatic after exposure to Ebola virus are capable of mounting an early proinflammatory cytokine response and that those who become clinically ill are not. If implemented immediately after exposure, these assays could be used to predict which individuals will be more likely to remain asymptomatic as opposed to those who will be more likely to develop clinical signs and eventually succumb to the virus.

Animals↗

PubMed2005

Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase.

We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.

Animals↗

PubMed1998

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry.

BACKGROUND: The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.

Adult↗

PubMed1991

Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes.

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.

Animals↗

PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗

PubMed1996

Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella.

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.

Electrochemistry↗

PubMed2004

Development of a respiratory burst assay using zebrafish kidneys and embryos.

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.

Animals↗

PubMed1987

Induction of prophage lambda by chlorinated pesticides.

Chlorinated organics represent an important class of environmental carcinogens. However, only a small percentage of the carcinogens of this chemical class are genotoxic in prokaryotic bioassays such as the Salmonella assay. In an effort to identify a short-term assay sensitive to chlorinated carcinogens, we have tested a group of chlorinated pesticides, most of which are carcinogenic in rodents, in a prophage-induction assay developed by Rossman et al. (1984). The Microscreen phage-induction assay is a rapid, inexpensive, miniaturized system that uses the induction of prophage lambda in Escherichia coli as an indicator of genetic damage. It has been used successfully to screen complex environmental samples for genotoxicants and has detected carcinogenic metals that are refractory in the Salmonella assay. The pesticides tested were malathion, monuron, p,p'-DDT, mirex, lindane, nitrofen, chlordane, toxaphene, captan, and dichlorvos. All but the first 4 induced prophage. The remaining pesticides were ranked as follows according to induction potency in the presence of S9: captan greater than dichlorvos greater than toxaphene greater than lindane greater than nitrofen greater than chlordane. Rankings were similar in the absence of S9. Of these 6 pesticides, only nitrofen required S9 to induce prophage. Comparisons with mutagenesis data in Salmonella indicated that the Microscreen assay detected as genotoxic each of the pesticides that were mutagenic in Salmonella; moreover, it detected 2 additional carcinogens (chlordane and lindane) that were not mutagenic in the Salmonella assay. The possible use of the Microscreen phage-induction assay to detect chlorinated organics is discussed.

Animals↗

PubMed2000

Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man.

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30-50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra-sensitive kinetic method was developed to identify specific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild-type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.

Actins↗

PubMed2006

Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid.

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

Animals↗

PubMed1990

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus.

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.

Adult↗