scholarly journals Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs

2014 ◽  
Vol 19 (7) ◽  
pp. 1124-1130 ◽  
Author(s):  
David J. Green ◽  
Edwin A. Rudd ◽  
James A. Laugharn
Keyword(s):  
High Throughput Screening ◽  
Microtiter Plate ◽  
Antibody Binding ◽  
Binding Assays ◽  
Coefficients Of Variation ◽  
Assay Performance ◽  
Antigen Antibody ◽  
Lower Limits ◽  
Hiv 1

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8–10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z′ factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z′ factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.

scholarly journals Research fronts of Chemical Biology

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Shanshan Lv
Keyword(s):  
High Throughput Screening ◽  
Drug Targets ◽  
Chemical Biology ◽  
Rational Design ◽  
Future Research ◽  
Design Strategies ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Diagnostic Technologies

Abstract Over the past decades, researchers have witnessed substantially increasing and ever-growing interests and efforts in Chemical Biology studies, thanks to the development of genome and epi-genome sequencing (revealing potential drug targets), synthetic chemistry (producing new medicines), bioorthogonal chemistry (chemistry in living systems) and high-throughput screening technologies (in vitro cell systems, protein binding assays and phenotypic assays). This report presents literature search results for current research in Chemical Biology, to explore basic principles, summarize recent advances, identify key challenges, and provide suggestions for future research (with a focus on Chemical Biology in the context of human health and diseases). Chemical Biology research can positively contribute to delivering a better understanding of the molecular and cellular mechanisms that accompany pathology underlying diseases, as well as developing improved methods for diagnosis, drug discovery, and therapeutic delivery. While much progress has been made, as shown in this report, there are still further needs and opportunities. For instance, pressing challenges still exist in selecting appropriate targets in biological systems and adopting more rational design strategies for the development of innovative and sustainable diagnostic technologies and medical treatments. Therefore, more than ever, researchers from different disciplines need to collaborate to address the challenges in Chemical Biology.

scholarly journals HSF1 inhibition attenuates HIV-1 latency reversal mediated by several candidate LRAs In Vitro and Ex Vivo

2020 ◽  
Vol 117 (27) ◽  
pp. 15763-15771 ◽  
Author(s):  
Andrew Timmons ◽  
Emily Fray ◽  
Mithra Kumar ◽  
Fengting Wu ◽  
Weiwei Dai ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Ex Vivo ◽  
In Vitro Models ◽  
Model Systems ◽  
Heat Shock Factor 1 ◽  
Primary Cells ◽  
Major Barrier ◽  
Hiv 1 ◽  
In Cells

HIV-1 latency is a major barrier to cure. Identification of small molecules that destabilize latency and allow immune clearance of infected cells could lead to treatment-free remission. In vitro models of HIV-1 latency involving cell lines or primary cells have been developed for characterization of HIV-1 latency and high-throughput screening for latency-reversing agents (LRAs). We have shown that the majority of LRAs identified to date are relatively ineffective in cells from infected individuals despite activity in model systems. We show here that, for diverse LRAs, latency reversal observed in model systems involves a heat shock factor 1 (HSF1)-mediated stress pathway. Small-molecule inhibition of HSF1 attenuated HIV-1 latency reversal by histone deactylase inhibitors, protein kinase C agonists, and proteasome inhibitors without interfering with the known mechanism of action of these LRAs. However, latency reversal by second mitochondria-derived activator of caspase (SMAC) mimetics was not affected by inhibition of HSF1. In cells from infected individuals, inhibition of HSF1 attenuated latency reversal by phorbol ester+ionomycin but not by anti-CD3+anti-CD28. HSF1 promotes elongation of HIV-1 RNA by recruiting P-TEFb to the HIV-1 long terminal repeat (LTR), and we show that inhibition of HSF1 attenuates the formation of elongated HIV-1 transcripts. We demonstrate that in vitro models of latency have higher levels of the P-TEFb subunit cyclin T1 than primary cells, which may explain why many LRAs are functional in model systems but relatively ineffective in primary cells. Together, these studies provide insights into why particular LRA combinations are effective in reversing latency in cells from infected individuals.

scholarly journals Identification of an APOBEC3G Binding Site in Human Immunodeficiency Virus Type 1 Vif and Inhibitors of Vif-APOBEC3G Binding

2007 ◽  
Vol 81 (23) ◽  
pp. 13235-13241 ◽  
Author(s):  
Andrew Mehle ◽  
Heather Wilson ◽  
Chengsheng Zhang ◽  
Andrew Jay Brazier ◽  
Mark McPike ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Assays ◽  
Immunodeficiency Virus ◽  
N Terminus ◽  
Hiv 1

ABSTRACT The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOBEC3 interface as a potential drug target.

scholarly journals Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology

2016 ◽  
Vol 22 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Jing Wang ◽  
Pengfei Fang ◽  
Peter Chase ◽  
Sagi Tshori ◽  
Ehud Razin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Throughput Screening ◽  
Treatment Resistance ◽  
Dna Interaction ◽  
Pigment Formation ◽  
Assay Performance ◽  
Microphthalmia Transcription Factor ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z′ factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

scholarly journals Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1425 ◽  
Author(s):  
Elizabeth M. Beck ◽  
Euan Parnell ◽  
Angela Cowley ◽  
Alison Porter ◽  
Jonathan Gillespie ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
High Throughput Screening ◽  
Partial Agonist ◽  
Fold Increase ◽  
Binding Assays ◽  
Camp Analogue ◽  
Competition Binding ◽  
Multiple Cell ◽  
Gtpase Activation

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

Determination of ruthenium and chromium by X-ray fluorescence spectrometry and the use of inert ruthenium (II) phenanthroline as a solid phase marker in sheep digestion studies

1977 ◽  
Vol 89 (1) ◽  
pp. 17-22 ◽  
Author(s):  
C. C. Evans ◽  
J. C. Macrae ◽  
S. Wilson
Keyword(s):  
Solid Phase ◽  
Quantitative Estimation ◽  
Fluorescence Spectrometry ◽  
Fatty Acid Production ◽  
X Ray ◽  
Coefficients Of Variation ◽  
Lower Limits

SummaryThe quantitative estimation of ruthenium and chromium in faeces and digesta from sheep by X-ray fluorescence spectrometry is described. Estimates of calibration parameters, established by regression analysis, were applicable to a range of matrices. Lower limits of detection of 2·4 mg Ru/kg (solid), 1·8 mg Ru/1 (liquid), 1·6 mg Cr/kg (solid) and 1·1 mg Cr/1 (liquid) and precisions, expressed as coefficients of variation, of 5·4% at 17·7 mg Ru/kg (solid), 5·1% at 182·7 mg Cr/kg (solid) and 1·1% at 152 mg Cr/1 (liquid) were acceptable for the routine analysis of Ru and Cr.The use of inert ruthenium phenanthroline (Ru-P) and inert chromium ethylenediamine tetra-acetic acid (Cr-EDTA) as solid:liquid dual phase markers in digestive physiological studies was evaluated. The specificity of Ru-P for the particulate phase of digesta, established by both in vitro and in vivo experiments, was found to be 92–98%, and in vivo recovery of intraruminally administered Ru-P in faeces was 97–101%. In vitro incubation of rumen liquor showed that concentrations of Ru-P up to 10−4 M had little effect on volatile fatty acid production rates but at 10−3 M there was virtually complete inhibition of acetate and propionate production.Advantages of using inert Ru-P as a solid phase marker are discussed.

scholarly journals Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

2005 ◽  
Vol 10 (6) ◽  
pp. 606-614 ◽  
Author(s):  
Sinu John ◽  
Thomas M. Fletcher ◽  
Colleen B. Jonsson
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Strand Transfer ◽  
Hiv Integrase ◽  
Chromosomal Dna ◽  
Screening Assay ◽  
Viral Dna ◽  
High Throughput Screening Assay ◽  
Hiv 1

Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS™), for HIV-1 IN activity using 5′ biotin-labeled DNA (5′ BIO donor) and 3′ digoxygenin-labeled DNA (3′ DIG target). Following 3′ processing of the 5′ BIO donor, strand transfer proceeds with integration of the 5′ BIO donor into the 3′ DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.

scholarly journals A Multispecies Biofilm In Vitro Screening Model of Dental Caries for High-Throughput Susceptibility Testing

2019 ◽  
Vol 8 (2) ◽  
pp. 14 ◽  
Author(s):  
Lara A. Heersema ◽  
Hugh D. C. Smyth
Keyword(s):  
Dental Caries ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Studies ◽  
Microtiter Plate ◽  
Multispecies Biofilm ◽  
Caries Model ◽  
Multispecies Model ◽  
Multispecies Biofilms

There is a current need to develop and optimize new therapeutics for the treatment of dental caries, but these efforts are limited by the relatively low throughput of relevant in vitro models. The aim of this work was to bridge the 96-well microtiter plate system with a relevant multispecies dental caries model that could be reproducibly grown to allow for the high-throughput screening of anti-biofilm therapies. Various media and inoculum concentrations were assessed using metabolic activity, biomass, viability, and acidity assays to determine the optimal laboratory-controlled conditions for a multispecies biofilm composed of Streptococcus gordonii, Streptococcus mutans, and Candida albicans. The selected model encompasses several of the known fundamental characteristics of dental caries-associated biofilms. The 1:1 RPMI:TSBYE 0.6% media supported the viability and biomass production of mono- and multispecies biofilms best. Kinetic studies over 48 h in 1:1 RPMI:TSBYE 0.6% demonstrated a stable biofilm phase between 10 and 48 h for all mono- and multispecies biofilms. The 1:1:0.1 S. gordonii: S. mutans: C. albicans multispecies biofilm in 1:1 RPMI:TSBYE 0.6% is an excellent choice for a high-throughput multispecies model of dental caries. This high-throughput multispecies model can be used for screening novel therapies and for better understanding the treatment effects on biofilm interactions and stability.

scholarly journals m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

2019 ◽  
Author(s):  
Chia-Chieh Chu ◽  
Bei Liu ◽  
Raphael Plangger ◽  
Christoph Kreutz ◽  
Hashim M. Al-Hashimi
Keyword(s):  
Binding Assays ◽  
Sequence Context ◽  
Rev Response Element ◽  
Stability Structure ◽  
Rna Export ◽  
The Stability ◽  
The Impact ◽  
Melting Experiments ◽  
Hiv 1

AbstractN6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM). Our results indicate that if present in stem IIB, m6A is unlikely to modulate RRE-Rev interaction by altering the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.

scholarly journals Novel Chimpanzee/Human Monoclonal Antibodies That Neutralize Anthrax Lethal Factor, and Evidence for Possible Synergy with Anti-Protective Antigen Antibody

2009 ◽  
Vol 77 (9) ◽  
pp. 3902-3908 ◽  
Author(s):  
Zhaochun Chen ◽  
Mahtab Moayeri ◽  
Devorah Crown ◽  
Suzanne Emerson ◽  
Inna Gorshkova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Dissociation Constant ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Binding Assays ◽  
Anthrax Lethal Factor ◽  
Antigen Antibody ◽  
Domain I

ABSTRACT Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC50) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC50 of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

Sign in / Sign up

Export Citation Format

Share Document