Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte–macrophage colony-stimulating factor (GM-CSF), and β₂-microglobulin (β₂M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20 ng mL⁻¹ with a detection limit of 0.099 ng mL⁻¹. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-β super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples.

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An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants

The safety of our food is an essential requirement of society. One well-recognised threat is that of chemical contamination of our food, where low-molecular-weight compounds such as biotoxins, drug residues and pesticides are present. Low-cost, rapid screening procedures are sought to discriminate the suspect samples from the population, thus selecting only these to be forwarded for confirmatory analysis. Many biosensor assays have been developed as screening tools in food contaminant analysis, but these tend to be electrochemical, fluorescence or surface plasmon resonance based. An alternative approach is the use of biolayer interferometry, which has become established in drug discovery and life science studies but is only now emerging as a potential tool in the analysis of food contaminants. A biolayer interferometry biosensor was assessed using domoic acid as a model compound. Instrument repeatability was tested by simultaneously producing six calibration curves showing replicate repeatability (n?=?2) ranging from 0.1 to 6.5 % CV with individual concentration measurements (n?=?12) ranging from 4.3 to 9.3 % CV, giving a calibration curve midpoint of 7.5 ng/ml (2.3 % CV (n?=?6)). Reproducibility was assessed by producing three calibration curves on different days, giving a midpoint of 7.5 ng/ml (3.4 %CV (n?=?3)). It was further shown, using assay development techniques, that the calibration curve midpoint could be adjusted from 10.4 to 1.9 ng/ml by varying assay parameters before the simultaneous construction of three calibration curves in matrix and buffer. Sensitivity of the assay compared favourably with previously published biosensor data for domoic acid.     

Discovery of a potent small molecule IL-2 inhibitor through fragment assembly

Using a site-directed fragment discovery method called tethering, we have identified a 60 nM small molecule antagonist of a cytokine/receptor interaction (IL-2/IL2Ralpha) with cell-based activity. Starting with a low micromolar hit, we employed a combination of tethering, structural biology, and computational analysis to design a focused set of 20 compounds. Eight of these compounds were at least 5-fold more active than the original hit. One of these compounds showed a 50-fold enhancement and represents the highest affinity inhibitor reported against this protein-protein target class. This method of coupling selected fragments with a low micromolar hit shows great potential for generating high-affinity lead compounds.     

Back to basics: label-free technologies for small molecule screening

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.     

Phenotypic screening of small molecule libraries by high throughput cell imaging

We have developed high throughput fluorescence cell imaging methods to screen chemical libraries for compounds with effects on diverse aspects of cell physiology. We describe screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway. Each of these screens yielded specific inhibitors for research use, and the mitosis screen identified Eg5 as a potential target protein for cancer chemotherapy. Cell imaging provides a large amount of information from primary screening data that can be used to distinguish compounds with different effects on cells, and together with automated analysis, to quantitate compound effects.     

基于药效团的BRD4靶向小分子抑制剂虚拟筛选

BRD4靶点和多种肿瘤密切相关,是具有良好成药性的热门靶点.本文选取活性较好且结构差异较大的BRD4小分子抑制剂作为训练集分子,基于配体小分子共同特征(HipHop)方法使用Discovery Studio 3.0分子模拟软件构建了药效团.药效团通过测试集验证、ROC曲线验证(SE(sensitivity)=0.937...     

Condensed tetracyclic systems with an isatin fragment in the molecule

A new method for the synthesis of the dioxodihydro-1H-benzo[b]furoindole heterocyclic systems from the corresponding isomeric amino acids with the amino groups at positions 2 and 3 is described. By this method it is possible not only to obtain the indicated tetracyclic systems in the form of one isomer but also to interconvert them; from the tetracyclic systems with angular structure it is possible to obtain the corresponding isomers with linear structure and vice versa. The classical Sandmeyer reaction served a model for such transformations. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1314–1321, September, 2007.     

Electrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity

Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.     

WWW small molecule modeling

Computational chemistry and molecular modeling sites have proliferated on the Internet's world wide web. This paper provides present links to some of the more most useful ones for small organic molecule modeling, and offering free resources.     

WWW small molecule modeling

Computational chemistry and molecular modeling sites have proliferated on the Internet's world wide web. This paper provides present links to some of the more most useful ones for small organic molecule modeling, and offering free resources.     

Micro-X-ray fluorescence as a general high-throughput screening method for catalyst discovery and small molecule recognition

A powerful high-throughput screening technique is described for the rapid screening of bead-based libraries for catalyst discovery and molecular recognition. Micro-X-ray fluorescence (MXRF) screens materials for elemental composition with mesoscale analysis. This method is nondestructive and requires minimal sample preparation and no special tags for analysis, and the screening time is dependent on the desired sensitivity. The speed, sensitivity, and simplicity of MXRF as a high-throughput screening technique were applied to screen bead-based libraries of oligopeptides for phosphate hydrolysis catalysts and molecular recognition of selective receptors for the degradation products and analogues of chemical warfare agents. This paper demonstrates the analytical or HTS capability of MXRF for combinatorial screening. It is meant only to show the capabilities of MXRF and is not meant as an exhaustive study of the catalyst and molecular recognition systems presented.     

Fluorous tagged small molecule microarrays

The affinity fluorous interaction between fluorous tagged small molecules and a fluoroalkyl modified glass surface was shown to facilitate the detection of protein-ligand binding interactions in the fabrication and screening of small molecule microarrays.     

Exploring the active site of human factor Xa protein by NMR screening of small molecule probes

A collection of small molecules (MW < 350 Da) was screened for binding to human factor Xa using saturation transfer difference NMR spectroscopy to detect binding. The NMR screening experiments identified four hits. Binding isotherms constructed from NMR linewidth data showed that the binding affinities of the hits were all in the 30-210 microM range. Competition binding experiments showed that three of the ligands were displaced by a known microM inhibitor of factor Xa. The success of the method for identifying new ligands and the relevance of this information to the design of new factor Xa inhibitors are discussed.     

Design of compound libraries for fragment screening

Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.     

A small neutral molecule with two noble-gas atoms: HXeOXeH

A novel noble-gas compound, HXeOXeH, is identified using IR spectroscopy, and it seems to be the smallest known neutral molecule with two noble-gas atoms. HXeOXeH is prepared using, for example, UV photolysis of water in solid xenon and subsequent annealing at 40-45 K. The experimental observations are fully supported by extensive quantum chemical calculations. A large energy release of 8.3 eV is computationally predicted for the decomposition of HXeOXeH into the 2Xe + H2O global energy minimum. HXeOXeH may represent a first step toward the possible preparation of (Xe-O)n chains and it may be relevant to the terrestrial "missing xenon" problem.