Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for (125)I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-alpha-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.     

Comparison of four different assays for determination of serum S-100B

BACKGROUND: S-100B determination has been shown to be clinically useful in the management of melanoma patients. After the development of a test for determination of the isoforms S100-A1B and S100-BB in serum (S-100B), several sensitive assays for the detection of serum S-100B have become available. We compared four S-100B assays, two automated (LIAISON Sangtec 100 and Elecsys S100) and two manual ones (Sangtec 100 ELISA and CanAg S100 EIA), with respect to clinical data, reference values and correlation. METHODS: In a total of 280 samples from 155 melanoma patients and 98 healthy individuals S-100B values were measured simultaneously with the different assays. RESULTS: The inter and intra coefficients of variation were best for the automated assays. The functional sensitivity of both manual assays was 0.15 microg/L. Method comparison revealed satisfactory correlation coefficients of 0.9 or higher, but the slopes ranged from 0.29 to 3.36. Except for the Sangtec 100 ELISA, the linearity between the assays was acceptable. The overall sensitivity for melanoma ranged from 37% (Elecsys S100) to 47% (LIAISON Sangtec 100) and the sensitivity increased with stage. ROC curves showed the best accuracy for the LIAISON Sangtec 100 assay. CONCLUSIONS: All assays gave satisfactory results, but it is advisable to improve the performance of the manual assays for better sensitivity. Agreement about an international reference standard is needed.     

Sensitive and accurate quantification of human leukocyte migration using high-content Discovery-1 imaging system and ATPlite assay

Migration is a fundamental aspect of leukocyte behavior and represents a significant therapeutic target clinically. However, most migration assays used in research are relatively low throughput and not easily compatible with rapid analysis or high-throughput screening (HTS) protocols required for drug screening assays. We therefore investigated the quantification of the migration of human leukocytes using the Molecular Devices high-content Discovery-1 platform or PerkinElmer ATPlite assay compared to manual counting. We have conducted extensive assay validation, investigating the detection limits, sensitivity, and precision of each method to count human leukocytes. Leukocyte migration assays were conducted using 96-well HTS-Transwell plates and the potent chemokine stromal cell-derived factor-1 (SDF-1). We reveal that the Discovery-1 and ATPlite methods developed here provide useful approaches to quantify leukocyte migration in an HTS manner with high levels of detection, sensitivity, and precision.     

High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR

The peach-potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.     

Improved donor/acceptor BRET couples for monitoring β-arrestin recruitment to G protein-coupled receptors

We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced β-arrestin (βARR) intramolecular rearrangements and recruitment to G protein-coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing βARR assays and offer new options for the development of future BRET assays.     

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

The ability of plant genotoxicity assays to predict carcinogenicity

A number of assays have been developed which use higher plants for measuring mutagenic or cytogenetic effects of chemicals, as an indication of carcinogenicity. Plant assays require less extensive equipment, materials and personnel than most other genotoxicity tests, which is a potential advantage, particularly in less developed parts of the world. We have analyzed data on 9 plant genotoxicity assays evaluated by the Gene-Tox program of the U.S. Environmental Protection Agency, using methodologies we have recently developed to assess the capability of assays to predict carcinogenicity and carcinogenic potency. All 9 of the plant assays appear to have high sensitivity (few false negatives). Specificity (rate of true negatives) was more difficult to evaluate because of limited testing on non-carcinogens; however, available data indicate that only the Arabidopsis mutagenicity (ArM) test appears to have high specificity. Based upon their high sensitivity, plant genotoxicity tests are most appropriate for a risk-averse testing program, because although many false positives will be generated, the relatively few negative results will be quite reliable.     

An Optimized Immunohistochemistry Technique Improves NMO-IgG Detection: Study Comparison with Cell-Based Assays

Cell-based assays (CBA) have increased the sensitivity of the neuromyelitis optica (NMO)-IgG/aquaporin-4-antibody detection compared to classical tissue-based indirect assays. We describe the sensitivity of an optimized immunohistochemistry (IHC-o) to detect NMO-IgG/aquaporin-4-antibody in comparison with that of two CBA: an in-house (CBA-ih) and a commercial (CBA-c) assay (Euroimmun, Germany). Coded serum from 103 patients with definite NMO and 122 inflammatory controls were studied by IHC-o, CBA-ih, and CBA-c. IHC-o used the same protocol described to detect antibodies against cell surface antigens. CBA-ih used live cells transfected with the aquaporin-4-M23-isoform. The sensitivity of the IHC-o was 74.8% (95% confidence interval [CI] 65-83) and was similar to that of the CBA-ih 75.7% (95% CI 66-84) and the CBA-c 73.8% (95% CI 64-82). The specificity of the three assays was 100% (95% CI 97-100). Interassay concordance was high, 100 of 103 samples were coincident in all techniques. The optimized immunohistochemistry proves to be as sensitive and specific as the cell-based assays. This assay extends the available tools for NMO-IgG/aquaporin-4-antibody detection.     

Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates

beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.     

Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

We present an active oligonucleotide microarray platform for time-resolved F?rster-resonance-energy-transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors.     

Latent Class Modeling Approaches for Assessing Diagnostic Error without a Gold Standard: With Applications to p53 Immunohistochemical Assays in Bladder Tumors

Improved characterization of tumors for purposes of guiding treatment decisions for cancer patients will require that accurate and reproducible assays be developed for a variety of tumor markers. No gold standards exist for most tumor marker assays. Therefore, estimates of assay sensitivity and specificity cannot be obtained unless a latent class model-based approach is used. Our goal in this article is to estimate sensitivity and specificity for p53 immunohistochemical assays of bladder tumors using data from a reproducibility study conducted by the National Cancer Institute Bladder Tumor Marker Network. We review latent class modeling approaches proposed by previous authors, and we find that many of these approaches impose assumptions about specimen heterogeneity that are not consistent with the biology of bladder tumors. We present flexible mixture model alternatives that are biologically plausible for our example, and we use them to estimate sensitivity and specificity for our p53 assay example. These mixture models are shown to offer an improvement over other methods in a variety of settings, but we caution that, in general, care must be taken in applying latent class models.     

Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.     

The coupling of NAD(P)+-producing reactions to a semiautomated bioluminescent reaction

Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P)+. Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests that NAD(P)+ bioluminescence has a wide potential for application.     

轮虫生态毒理学测试指标的敏感性研究进展

随着工业化的发展,人类活动对环境造成了严重污染.轮虫是常用于污染物毒性检测的模式生物之一.针对不同类型的污染物,轮虫各生态毒理学指标的敏感性不尽相同.因此,本文归纳了现有的轮虫生态毒理学测试指标,综述了各生态毒理学检测指标对不同污染物的敏感性:轮虫的生命表统计学参数和生理生化指标对重金属污染通常更敏感;在检测持久性有机...     

不同严重急性呼吸综合征冠状病毒2抗体检测试剂盒的诊断应用评价

本文评估了6个品牌的严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS­CoV­2)特异性抗体检测试剂盒的性能,以指导临床合理选用。收集2020年1月30日至2020年5月11日期间上海市(复旦大学附属)公共卫生临床中心的住院患者样本资料共245例,其中包括SARS­CoV­2感染确诊患者122例,排除SARS­CoV­2感染的其他疾病患者123例。选用6个品牌的抗体检测试剂盒(3种为胶体金法,3种为化学发光法)检测所有患者的血清样本,同步采用聚合酶链反应(polymerase chain reaction,PCR)检测SARS­CoV­2核酸,统计临床灵敏度、特异性、阴性预测值和阳性预测值等指标,比较各检测方法间的差异。 结果显示,各检测试剂盒在临床特异性方面表现相近,但临床灵敏度差异明显,IgG的灵敏度高于IgM。化学发光试剂灵敏度为72.1%～85.2%,整体优于胶体金试剂的47.5%～84.4%。所有试剂检测结果与SARS­CoV­2核酸诊断结果相比均有统计学差异(P<0.05)。SARS­CoV­2抗体的特异性检出率随时间而上升,核酸确诊患者≥16 d抗体检出率最高可达96%。 结果表明,SARS­CoV­2抗体检测可作为核酸诊断的辅助手段,IgG和IgM 联合诊断可提高检测的灵敏度。但是不同试剂盒性能表现有差异,应根据不同临床需求和应用场景选择合适的试剂盒。     

Ligand binding and activation in a prokaryotic cyclic nucleotide-modulated channel

We designed a technique that directly determines binding of cyclic nucleotides to the prokaryotic cyclic nucleotide modulated ion channel MloK1. The ability to purify large quantities of MloK1 facilitated equilibrium binding assays, which avoided the inherent problem of relatively low affinity binding which hindered the use of eukaryotic channels. We found that MloK1 specifically binds cAMP and cGMP with affinity values in the range of those observed for activity assays for eukaryotic channels. Notably, the concentration of ligand that elicited 50% of maximum response in (86)Rb flux assays (K1/2), also referred to as ligand sensitivity, was smaller than the corresponding value obtained from binding assays (Kd) potentially indicating significant channel activity in partially liganded states. To gain further insight into the mechanism of binding and activation of these channels, we mutated several amino acids in the ligand-binding pocket of MloK1, known from electrophysiological studies of homologous eukaryotic channels to affect ligand selectivity and binding efficacy. The S308V MloK1 mutant (a mutation which decreases cGMP selectivity in eukaryotic channels) decreased both the observed cGMP binding affinity and the sensitivity to cGMP relative to the wild-type (WT) channel, leaving those for cAMP unchanged. Conversely, the A352D MloK1 mutant (a mutation which increases cGMP selectivity in eukaryotic channels) increased both the affinity and the sensitivity for cGMP relative to the WT channel, again leaving those for cAMP unchanged. Mutations at R307 in MloK1, the most conserved residue in the binding pocket of cyclic nucleotide-binding proteins, were not tolerated as these mutants do not form functional channels. Furthermore, for each mutation, changes in binding affinities were mirrored by equivalent changes in ligand sensitivity. These data, together with the evidence that partially liganded channels open significantly, suggested strong coupling between cyclic nucleotide binding and MloK1 channel opening.     

Sensitive enzyme-linked immunosorbent assays for the detection of bacterial superantigens and antibodies against them in human plasma

Enzyme-linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic-shock syndrome toxin-1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3 pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150 min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme-linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Accuracy and clinical correlates of two different methods for chromogranin A assay in neuroendocrine tumors

Measurement of chromogranin A (CgA) plays a major role in the management of neuroendocrine tumors (NET); however, reliable assaying of CgA is made difficult by the rapid hydrolysis following its release into the bloodstream. This study was aimed at the assessment of two assays for CgA in NET patients. CgA was measured in 93 patients by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoradiometric assay (IRMA). The specificity and sensitivity of CgA were evaluated in relation to tumor histology. The clinical accuracy of the two assays was evaluated by receiver-operating characteristic (ROC) curve analysis. Regression analysis demonstrated different immunoreactivity for CgA of the antibodies used in the two kits (r = 0.61). The two assays had different accuracy also in classifying patients according to their clinical condition (91% vs 64% specificity and 79% vs 79% sensitivity for the ELISA and IRMA assay, respectively) and tumor histology (81% vs 85% sensitivity for the ELISA and IRMA assays, respectively, in carcinoids; 92% vs 67% sensitivity for the ELISA and IRMA assays, respectively, in pancreatic islet cell tumors). The different clinical accuracy of the two assays was confirmed by the ROC analysis (AUC = 0.90 vs AUC = 0.87 for the ELISA and IRMA assays, respectively). In conclusion, because of the poor standardization of the commercially available measurement tools the clinical accuracy of CgA measurement depends on the assay used. This makes it difficult to compare CgA values measured with different kits and affects the clinical accuracy of the different assays for CgA.