Enhanced competitive protein exchange at the nano-bio interface enables ultra-deep coverage of the human plasma proteome

We have developed a scalable system that leverages protein nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Introducing proprietary engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle protein interface, driven by the relationship between protein-NP affinity and protein abundance. Here we demonstrate the importance of tuning the protein to NP surface ratio (P/NP), which determines the competition between proteins for binding. We demonstrate how optimized P/NP ratio affects protein corona composition, ultimately enhancing performance of a fully automated NP based deep proteomic workflow (Proteograph). By limiting the available binding surface of NPs and increasing the binding competition, we identify 1.2 to 1.7x more proteins with only 1% false discovery rate on the surface of each NP, and up to 3x compared to a standard neat plasma proteomics workflow. Moreover, increased competition means proteins are more consistently identified and quantified across replicates, yielding precise quantification and improved coverage of the plasma proteome when using multiple physicochemically distinct NPs. In summary, by optimizing NPs and assay conditions, we capture a larger and more diverse set of proteins, enabling deep proteomic studies at scale.

Verification of the Conditions for Determination of Antioxidant Activity by ABTS and DPPH Assays—A Practical Approach

The ABTS and DPPH methods are among the most popular assays of antioxidant activity determination. Attempts to adapt them to different analytes and the search for the highest values of antioxidant activity has resulted in a large variety of assay conditions to be presented in the literature, including the way the measurement is made. This makes it difficult to relate the results to real oxidation systems, and often makes it impossible to compare them. Such a comparison is limited in advance by the use of stable radicals that do not exist in nature and that react differently from those generated in food or in vivo. Therefore, it is important to introduce measures aimed at standardizing the conditions of the activity assay, including reaction time and several reaction environments suitable for testing different groups of compounds. In this study, we used natural antioxidants of various structures: phenolic acids, flavonoids, peptides and corresponding amino acids, ascorbic acid and α-tocopherol, and also synthetic analogues of selected compounds. The curves of dependence of the measured absorbance on the concentration of antioxidants were described, the ranges of linearity were determined, and the value of the error made when reading in various ranges of dependencies was estimated. We also determined and compared the activity values using two popular methods (IC50 and TEAC), taking into account different environments and reaction times. Based on the collected data, recommendations were formulated regarding the reaction conditions adapted to the studies of individual groups of antioxidants, and unified reaction times were proposed. Taking into account the state before reaching the equilibrium of antioxidants reacting in a complex manner, this approach may introduce a simplified reference to the competing reaction that occurs in reality.

Cross-Platform Bayesian Optimization System for Autonomous Biological Assay Development

Current high-throughput screening assay optimization is often a manual and time-consuming process, even when utilizing design-of-experiment approaches. A cross-platform, Cloud-based Bayesian optimization-based algorithm was developed as part of the National Center for Advancing Translational Sciences (NCATS) ASPIRE (A Specialized Platform for Innovative Research Exploration) Initiative to accelerate preclinical drug discovery. A cell-free assay for papain enzymatic activity was used as proof of concept for biological assay development and system operationalization. Compared with a brute-force approach that sequentially tested all 294 assay conditions to find the global optimum, the Bayesian optimization algorithm could find suitable conditions for optimal assay performance by testing 21 assay conditions on average, with up to 20 conditions being tested simultaneously, as confirmed by repeated simulation. The algorithm could achieve a sevenfold reduction in costs for lab supplies and high-throughput experimentation runtime, all while being controlled from a remote site through a secure connection. Based on this proof of concept, this technology is expected to be applied to more complex biological assays and automated chemistry reaction screening at NCATS, and should be transferable to other institutions. Graphical Abstract

Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Development and validation of a novel counter-immunoelectrophoresis assay for the detection of antibodies against extractable nuclear antigens

<p>The identification of autoantibodies is a primary diagnostic marker for the diagnosis of some autoimmune diseases. The sera of patients with connective tissue diseases commonly contain autoantibodies that target nuclear antigens. As such these antibodies are called antinuclear antibodies (ANAs). Furthermore, the clinical identification of ANAs in patient sera to specific nuclear antigens is a primary tool for the diagnosis of connective tissue diseases. For this purpose specific extractable nuclear antigens (ENAs) are used. The most commonly employed laboratory technique for the detection of ENA antibodies is the enzyme linked immunoabsorbant assay (ELISA). ELISA is a simple technique that can be automated and provides high diagnostic sensitivity for the detection of ENA antibodies comparatively to a counter immunoelectrophoresis (CIE) assay. However, compared to CIE ELISA lacks diagnostic specificity. Therefore there is a demand for an assay with high diagnostic specificity as a secondary diagnostic test for the detection of ENA antibodies. The central aim of this project was to develop and validate a novel CIE assay that used fluorescently labelled ENAs to detect ENA antibodies in patient serum. Development of the assay began with comparative testing of fluorescently labelled and unlabelled antigens to confirm that the immunochemical properties of the antigen remained intact. The CIE assay conditions were optimised for the detection of four ANAs SSA, SSB, RNP/Sm, and Sm using their corresponding ENAs. Assay conditions optimised were flurophore type, gel composition, buffer composition, antigen concentration, the running time, and analytical specificity. Evaluation of 281 clinical sera samples known to have previously tested positive test by ANA indirect immunofluorescence were performed using the optimised CIE assay and ELISA. The inter-rater agreement between the CIE assay and ELISA results was determined. Clinical details were used to retrospectively classify the clinical sera samples into clinical categories and case groups. This data was used for the diagnostic comparison of the CIE assay and ELISA results. There was strong inter-rater agreement between the SSA CIE assay and ELISA results. The diagnostic specificity and positive likelihood ratio values of the SSA CIE assay were superior to those of the ELISA, while diagnostic sensitivity and the negative likelihood ratio values were approximately the same. There was strong inter-rater agreement between the RNP/Sm CIE assay and ELISA. However, it was observed that the statistical measures of assay accuracy (diagnostic specificity and sensitivity, positive likelihood ratio, and negative likelihood ratio) for the RNP/Sm ELISA were superior to those of CIE assay. The diagnostic specificity of the SSB and Sm CIE assays were higher than that of their respective ELISAs. This was also true for the positive likelihood ratio values of the SSB and Sm CIE assays when compared to their respect ELISAs. The SSA CIE assay results suggest that this assay maybe a suitable replacement for ELISA as a diagnostic tool for the identification of anti-SSA antibodies and Sjogren’s syndrome. The SSB and Sm CIE assay results suggest that these assays would be suitable as secondary confirmatory diagnostic assays for the identification of their respective ENA antibodies. However, further performance evaluation of the assays within a routine diagnostic laboratory is required.</p>

Development and validation of a novel counter-immunoelectrophoresis assay for the detection of antibodies against extractable nuclear antigens

<p>The identification of autoantibodies is a primary diagnostic marker for the diagnosis of some autoimmune diseases. The sera of patients with connective tissue diseases commonly contain autoantibodies that target nuclear antigens. As such these antibodies are called antinuclear antibodies (ANAs). Furthermore, the clinical identification of ANAs in patient sera to specific nuclear antigens is a primary tool for the diagnosis of connective tissue diseases. For this purpose specific extractable nuclear antigens (ENAs) are used. The most commonly employed laboratory technique for the detection of ENA antibodies is the enzyme linked immunoabsorbant assay (ELISA). ELISA is a simple technique that can be automated and provides high diagnostic sensitivity for the detection of ENA antibodies comparatively to a counter immunoelectrophoresis (CIE) assay. However, compared to CIE ELISA lacks diagnostic specificity. Therefore there is a demand for an assay with high diagnostic specificity as a secondary diagnostic test for the detection of ENA antibodies. The central aim of this project was to develop and validate a novel CIE assay that used fluorescently labelled ENAs to detect ENA antibodies in patient serum. Development of the assay began with comparative testing of fluorescently labelled and unlabelled antigens to confirm that the immunochemical properties of the antigen remained intact. The CIE assay conditions were optimised for the detection of four ANAs SSA, SSB, RNP/Sm, and Sm using their corresponding ENAs. Assay conditions optimised were flurophore type, gel composition, buffer composition, antigen concentration, the running time, and analytical specificity. Evaluation of 281 clinical sera samples known to have previously tested positive test by ANA indirect immunofluorescence were performed using the optimised CIE assay and ELISA. The inter-rater agreement between the CIE assay and ELISA results was determined. Clinical details were used to retrospectively classify the clinical sera samples into clinical categories and case groups. This data was used for the diagnostic comparison of the CIE assay and ELISA results. There was strong inter-rater agreement between the SSA CIE assay and ELISA results. The diagnostic specificity and positive likelihood ratio values of the SSA CIE assay were superior to those of the ELISA, while diagnostic sensitivity and the negative likelihood ratio values were approximately the same. There was strong inter-rater agreement between the RNP/Sm CIE assay and ELISA. However, it was observed that the statistical measures of assay accuracy (diagnostic specificity and sensitivity, positive likelihood ratio, and negative likelihood ratio) for the RNP/Sm ELISA were superior to those of CIE assay. The diagnostic specificity of the SSB and Sm CIE assays were higher than that of their respective ELISAs. This was also true for the positive likelihood ratio values of the SSB and Sm CIE assays when compared to their respect ELISAs. The SSA CIE assay results suggest that this assay maybe a suitable replacement for ELISA as a diagnostic tool for the identification of anti-SSA antibodies and Sjogren’s syndrome. The SSB and Sm CIE assay results suggest that these assays would be suitable as secondary confirmatory diagnostic assays for the identification of their respective ENA antibodies. However, further performance evaluation of the assays within a routine diagnostic laboratory is required.</p>

A comparative analysis of cell surface targeting aptamers

Aptamers represent a potentially important class of ligands for the development of diagnostics and therapeutics. However, it is often difficult to compare the function and specificity of many of these molecules as assay formats and conditions vary greatly. Here, with an interest in developing aptamer targeted therapeutics that could effectively deliver cargoes to cells, we chemically synthesize 15 aptamers that have been reported to target cell surface receptors or cells. Using standardized assay conditions, we assess each aptamer’s binding properties on a panel of 11 different cancer cell lines, correlate aptamer binding to antibody controls and use siRNA transfection to validate each aptamer’s binding to reported target receptors. Using a subset of these molecules known to be expressed on prostate cancers, we use near-infrared in vivo imaging to assess the tumor localization following intravenous injection. Our data demonstrate some surprising differences in the reported specificity and function for many of these molecules and raise concerns regarding their cell targeting capabilities. They also identify an anti-human transferrin aptamer, Waz, as a robust candidate for targeting prostate cancers and for future development of aptamer-based therapeutics.

Kinetics of Crude Peroxidase from the Rind of Watermelon Fruit

Aims: To study the kinetics of crude peroxidase from the rind of watermelon fruit in various assay conditions. Study Design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between October 2015 and January 2016. Methodology: The activity of the crude peroxidase extracted from the rind of watermelon was determined by measuring the rate of oxidation of KI at 25oC in a 3.0 ml reaction mixture which contained 2.3 ml of 25 mM - 400 mM sodium acetate buffer (pH 3.5-6.0), 0.2 ml of 2 mM KI, 0.1ml of the crude peroxidase, and 0.2 ml of varying concentrations of chlorpromazine (0.01 mM - 0.1 mM). In all cases, 0.2 ml of 0.01 mM – 1 mM H2O2 was added last to initiate the reaction. Only one parameter was varied per assay. Assays were done in five replicates. The initial velocity of the crude peroxidase for KI oxidation was determined using the absorbance at 353 nm. Results: The concentration of H2O2 that generated an optimal activity for the crude peroxidase extracted was 0.2 mM, while a pH of 5.5 was optimal for the crude enzyme. The activity of the crude enzyme increased proportionately within a buffer concentration range of 25 mM and 400 mM. Chlorpromazine (0.01 mM - 0.1 mM) proportionately increased the enzyme activity, while promethazine within a range of 0.01 mM and 0.06 mM proportionally increased the enzyme activity. Further increase in promethazine concentration beyond 0.6 mM resulted in a decreased activity of the enzyme. Conclusion: This study suggests that the Rind of watermelon is an alternative source of peroxidase. The activity of this peroxidase can be enhanced by high buffer concentrations in the presence of some redox mediators like promethazine and chlorpromazine at a pH of 5.5.