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Duplicate analysis has been a conventional practice in the industry for ligand-binding assays (LBA), particularly for plate-based platforms like Enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) assays. Recent whitepapers and guidance have opened a door to exploring the implementation of single-well (singlicate) analysis approach for LBAs. Although the bioanalytical industry has actively investigated the suitability of singlicate analysis, applications in supporting regulated LBA bioanalysis are limited. The primary reason for this limitation is the absence of appropriate strategy to facilitate the transition from duplicate to singlicate analysis. In this paper we present the first case study with our data-driven approach to implement singlicate analysis in a clinical pharmacokinetics (PK) plate based LBA assay with ISR data. The central aspect of this strategy is a head-to-head comparison with Precision and Accuracy assessment in both duplicate and singlicate formats as the initial stage of assay validation. Subsequently, statistical analysis is conducted to evaluate method variability in both precision and accuracy. The results of our study indicated that there was no impactful difference between duplicate vs singlicate, affirming the suitability of singlicate analysis for the remaining steps of PK assay validation. The validation results obtained through singlicate analysis demonstrated acceptable assay performance characteristics across all validation parameters, aligning with regulatory guidance. The validated PK assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by initial Incurred Sample Reanalysis (ISR) data in which 90.1% of ISR samples fall within the acceptable criteria. [ABSTRACT FROM AUTHOR]

• Dystrophin expression is designed to replace dystrophin in patients with DMD. • Existing platforms, such as western blotting, are not sensitive and lack specificity. • Highly sensitive immunoassay using Single Molecule Counting Technology was developed. • Successfully measured muscle dystrophin levels in patients with DMD. Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle loss caused by mutations in dystrophin, resulting in decreased dystrophin levels. Dystrophin protein expression is a biomarker used to evaluate treatments that restore patient dystrophin levels. Currently, a semiquantitative assay using western blotting, which normalizes dystrophin expression to that of a control population, is used for regulatory filing. However, the current methods are limited in terms of sensitivity, quantification, and reproducibility. To address this, a highly sensitive and quantitative sandwich immune assay using Single Molecule Counting technology was established, with recombinant dystrophin protein as the calibrator. Capture and detection antibodies were selected to detect full-length dystrophin. Using this optimized assay, dystrophin levels in muscle samples from Myotonic Dystrophy (n = 9) and DMD (n = 8) subjects were 93.2 ± 31.9 (range: 49.4–145.3) and 14.5 ± 6.8 (range: 6.18–22.6) fmol/total protein mg, respectively. The lowest concentration of dystrophin measured in the DMD samples was 5 times higher than that in the lower limit of quantitation, a level not detected by western blotting. These data indicate that this assay accurately and sensitively measured dystrophin protein and may be useful in clinical trials assessing dystrophin restoration therapies. [ABSTRACT FROM AUTHOR]

BACKGROUND: General odor‐binding proteins (GOBPs) play critical roles in insect olfactory recognition of sex pheromones and plant volatiles. Therefore, the identification of GOBPs in Hyphantria cunea (Drury) based on their characterization to pheromone components and plant volatiles is remain unknown. RESULTS: In this study, two H. cunea (HcunGOBPs) genes were cloned, and their expression profiles and odorant binding characteristics were systematically analyzed. Firstly, the tissue expression study showed that both HcunGOBP1 and HcunGOBP2 were highly expressed in the antennae of both sexes, indicating their potential involvement in the perception of sex pheromones. Secondly, these two HcunGOBPs genes were expressed in Escherichia coli and ligand binding assays were used to assess the binding affinities to its sex pheromone components including two aldehydes and two epoxides, and some plant volatiles. HcunGOBP2 showed high binding affinities to two aldehyde components (Z9, Z12, Z15‐18Ald and Z9, Z12‐18Ald), and showed low binding affinities to two epoxide components (1, Z3, Z6‐9S, 10R‐epoxy‐21Hy and Z3, Z6‐9S, 10R‐epoxy‐21Hy), whereas HcunGOBP1 showed weak but significant binding to all four sex pheromone components. Furthermore, both HcunGOBPs demonstrated variable binding affinities to the plant volatiles tested. Thirdly, in silico studies of HcunGOBPs utilized homology, structure modeling, and molecular docking revealed critical hydrophobic residues might be involved in the binding of HcunGOBPs to their sex pheromone components and plant volatiles. CONCLUSION: Our study suggests that these two HcunGOBPs may serve as potential targets for future studies of HcunGOBPs ligand binding, providing insight in the mechanism of olfaction in H. cunea. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Biologics, especially monoclonal antibodies (mAbs), are an increasingly important part of the drug discovery and development portfolio across the pharmaceutical industry. To enable robust demonstration of pillars 1 and 2 [1] for mAbs, specialised assays are required to measure the complex interactions between mAb and target. This is especially important for the interpretation of soluble target interactions. In some instances, multiple assays with overlapping purposes (e.g., developing both complex and total assays) have been developed. In retrospect, these efforts may have led to excessive time and resources spent in assay development and the generation of data that is contradictory or misleading. Our recommendation is to invest resources early into the development of total assays for both mAb and target. Free target assay data may be inaccurate and report higher levels of free target than are present in the sample at collection due to re-equilibrium during measurement. Total assay formats are inherently less sensitive to the effects of sample preparation, assay conditions, and re-equilibration than free or complex assays. It is acknowledged that pathology/pharmacology is ultimately driven by the free target and knowledge of its dynamics are critical. However, generation of appropriate total target data and using model-based estimation of free target concentrations is a more robust approach than utilisation of direct assay derived estimates. Where free data are utilised, the potential biases should be prospectively considered when developing the assay and utilising the data for quantitative analyses. [ABSTRACT FROM AUTHOR]

Abstract Anti-drug antibodies (ADAs) can interfere with ligand binding assays (LBAs) by binding to epitopes recognized by the assay antibodies or by preventing assay antibody binding through steric hindrance. This can lead to underestimation of total drug concentration in pharmacokinetic (PK) samples which can confound decisions during drug development. We hypothesized that ADA interference in LBAs can be removed by sample heat pre-treatment. We heat pre-treated ADA-spiked samples by incubating them in a shallow water bath at 56–100 °C for 5–30 min prior to measuring the samples by a traditional electrochemiluminescence (ECL) assay. Heat pre-treatment at minimum 85 °C for 5 min completely removed the ADA interference. We then compared the analyte concentrations measured with and without heat pre-treatment of blood samples from toxicology studies performed for two different analytes in 60 cynomolgus monkeys and 29 minipigs, respectively. The overall difference in measured concentration of ADA-positive samples was significantly different from the overall difference in measured concentration of ADA-negative samples. For the cynomolgus monkey study samples, the ADA titer was determined, and the difference in measured concentration, when comparing heat pre-treatment to no heat pre-treatment, was significantly correlated to the ADA titer. Additionally, heat pre-treatment removed parallelism issues observed in a subset of study samples. Our data suggest that sample heat pre-treatment can abolish ADA interference in an LBA and could serve as a tool to assess the degree of ADA interference and the total drug concentration in a PK sample. Of note, before utilizing this strategy on a new analyte, it is necessary to assess whether heat pre-treatment negatively affects the detection of the analyte by the assay antibodies.

Biological networks are characterized by diverse interactions and dynamics in time and space. Many regulatory modules operate in parallel and are interconnected with each other. Some pathways are functionally known and annotated accordingly, e.g., endocytosis, migration, or cytoskeletal rearrangement. However, many interactions are not so well characterized. For reconstructing the biological complexity in cellular networks, we combine here existing experimentally confirmed and analyzed interactions with a protein-interaction inference framework using as basis experimentally confirmed interactions from other organisms. Prediction scoring includes sequence similarity, evolutionary conservation of interactions, the coexistence of interactions in the same pathway, orthology as well as structure similarity to rank and compare inferred interactions. We exemplify our inference method by studying host-pathogen interactions during infection of Mus musculus (phagolysosomes in alveolar macrophages) with Aspergillus fumigatus (conidia, airborne, asexual spores). Three of nine predicted critical host-pathogen interactions could even be confirmed by direct experiments. Moreover, we suggest drugs that manipulate the host-pathogen interaction.

A stepwise, data-driven approach for Anti-Drug Antibodies (ADA) singlicate assays has been developed to evaluate the feasibility of singlicate ligand binding assay (LBA) method development, qualification and validation to support our clinical programs. With initial precision runs in method validation and subsequent statistical analysis to directly compare duplicate and singlicate formats, our results indicated no meaningful difference in assay precision between duplicate and singlicate. Consequently, the rest of the ADA method validation proceeded with singlicate analysis yielding acceptable validation results. The validated ADA assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by Signal-to-Noise (S/N) data from the three tiers of the confirmatory positive samples, which showed strong correlation in S/N values., Competing Interests: Declaration Conflict of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

The ICH M10 guideline establishes global standards for bioanalytical method validation for pharmacokinetic assays, focusing on data reliability and accuracy across studies. A significant component is cross-validation, which should be performed to ensure data comparability when multiple methods or laboratories are involved in a single study or across studies where comparison will be performed. However, ICH M10 does not specify acceptance criteria for cross-validation, creating challenges for the industry because traditionally many laboratories have always utilized acceptance criteria to "pass" or "fail" the study. This editorial discusses how bioanalytical labs should conduct cross-validation for PK assays post-ICH M10, highlighting the role of statistical methods and the need for close collaboration with clinical pharmacology and biostatistics departments. Proper implementation and strategic focus on relevant studies are essential for effective cross-validation.

E3112 is a recombinant human hepatocyte growth factor currently in development for the treatment of acute liver failure. The assessment of immunogenicity is crucial in the development of biotherapeutics. Consequently, a semi-quantitative assay of anti-drug antibody (ADA) was developed in rat and monkey serum using a ligand binding assay with electrochemiluminescence detection. A standard tiered approach was employed for the immunogenicity assessment, comprising a screening assay and a subsequent confirmatory assay. In the assay validation studies, selectivity, sensitivity, prozone effects, reproducibility, drug tolerance, and stability were evaluated. These assessments were conducted using a surrogate positive control of ADA. The accuracy and precision of the surrogate ADA were within ± 20 % and 20 %, respectively. The stability of ADA was also confirmed under a variety of conditions. The developed assays were successfully employed for the assessment of immunogenicity in rats and monkeys following the administration of a repeated dose of E3112. The administration of E3112 resulted in an increase in ADA levels, with higher levels observed in rats than in monkeys. Systemic exposures of E3112 in rats with higher ADA levels were lower than those with lower ADA, confirming the utility of nonclinical immunogenicity in interpreting pharmacokinetics and its inter-individual variability. • Immunogenicity assays for E3112, a recombinant human HGF, in rats and monkeys were developed. • The assays were successfully validated and were applied to real samples from rats and monkeys to support toxicity studies. • Incidence of ADA in rats and monkeys was 87.5 % and 77.3 % with ADA levels up to 2159.8 and 116.6, respectively. • Decreases in serum E3112 exposures in rats can be explained by high levels of ADA. [ABSTRACT FROM AUTHOR]

Anti-drug antibodies (ADAs) can interfere with ligand binding assays (LBAs) by binding to epitopes recognized by the assay antibodies or by preventing assay antibody binding through steric hindrance. This can lead to underestimation of total drug concentration in pharmacokinetic (PK) samples which can confound decisions during drug development. We hypothesized that ADA interference in LBAs can be removed by sample heat pre-treatment. We heat pre-treated ADA-spiked samples by incubating them in a shallow water bath at 56–100 °C for 5–30 min prior to measuring the samples by a traditional electrochemiluminescence (ECL) assay. Heat pre-treatment at minimum 85 °C for 5 min completely removed the ADA interference. We then compared the analyte concentrations measured with and without heat pre-treatment of blood samples from toxicology studies performed for two different analytes in 60 cynomolgus monkeys and 29 minipigs, respectively. The overall difference in measured concentration of ADA-positive samples was significantly different from the overall difference in measured concentration of ADA-negative samples. For the cynomolgus monkey study samples, the ADA titer was determined, and the difference in measured concentration, when comparing heat pre-treatment to no heat pre-treatment, was significantly correlated to the ADA titer. Additionally, heat pre-treatment removed parallelism issues observed in a subset of study samples. Our data suggest that sample heat pre-treatment can abolish ADA interference in an LBA and could serve as a tool to assess the degree of ADA interference and the total drug concentration in a PK sample. Of note, before utilizing this strategy on a new analyte, it is necessary to assess whether heat pre-treatment negatively affects the detection of the analyte by the assay antibodies. [ABSTRACT FROM AUTHOR]

Abstract. The development of monoclonal antibody (mAb) biosimilars is a complex process. The key to their successful development and commercialization is an in-depth understanding of the key product attributes that impact safety and efficacy and the strategies to control them. Functional assessment of mAb is a crucial part of the comparability of biopharmaceutical drugs. The development of a relevant and robust functional assay requires an interdisciplinary approach and sufficient flexibility to balance regulatory concerns as well as dynamics and variability during the manufacturing process. Although many advanced tools are available to study and compare the potency and bioactivity of the protein, most of these techniques suffer from major shortcomings that limit their routine use. These include the complexity of the task, establishment of the relevance of the chosen method with the mechanism of action (MOA) of the biosimilar, cost and extended time of analysis, and often the ambiguity in interpretation of the resulting data. To overcome or to address these challenges, the use of multiple orthogonal state-of-the-art techniques is a necessary prerequisite. [ABSTRACT FROM AUTHOR]

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at −40 °C prior to distribution and the participants are instructed to store the samples frozen at −20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC–MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays. [ABSTRACT FROM AUTHOR]

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography–tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure. [ABSTRACT FROM AUTHOR]

Apriona germari (Hope) presents a significant threat as a dangerous wood-boring pest, inflicting substantial harm to forest trees. Investigating the olfactory sensory system of A. germari holds substantial theoretical promise for developing eco-friendly control strategies. To date, however, the olfactory perception mechanism in A. germari remains largely unknown. Therefore, we performed transcriptome sequencing of A. germari across four distinct body parts: antennae, foreleg tarsal segments, mouthparts (maxillary and labial palps), and abdomen terminals, pinpointing the odorant binding protein (OBP) genes and analyzing their expression. We found eight AgerOBPs (5, 19, 23, 25, 29, 59, 63, 70) highly expressed in the antennae. In our competitive binding experiments, AgerOBP23 showed strong binding abilities to the pheromone component fuscumol acetate, eight plant volatiles (farnesol, cis -3-hexenal, nerolidol, myristol acetate, cis -3-hexenyl benzoate, (-)-α-cedrene, 3-ethylacetophenone, and decane), and four insecticides (chlorpyrifos, phoxim, indoxacarb, and cypermethrin). However, AgerOBP29 and AgerOBP63 did not show prominent binding activities to these tested chemicals. Through homology modeling and molecular docking, we identified the key amino acid sites involved in the binding process of AgerOBP23 to these ligands, which shed light on the molecular interactions underlying its binding specificity. Our study suggests that AgerOBP23 may serve as a potential target for future investigations of AgerOBP ligand binding. This approach is consistent with the reverse chemical ecology principle, establishing the groundwork for future studies focusing on attractant or repellent development by exploring further the molecular interactions between OBP and various compounds.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays. [ABSTRACT FROM AUTHOR]

Serial blood sampling from one animal is useful to understand relationship between pharmacokinetics (PK) and pharmacological or toxicological events in individual animals. To assess its feasibility in mice, two therapeutic antibodies were used to evaluate impacts by different blood sampling methods, sampling sites, and assay platforms on PK. Denosumab and Panitumumab were intravenously administered to mice and only 0.05 mL of blood sample per point was collected from jugular vein or tail vein. Blood samples were collected serially from a mouse or collected by traditional composite sampling from each mouse. Plasma concentrations of the two drugs were assayed by a generic ligand binding assay using Gyrolab or by a generic ultra-performance liquid chromatography with tandem mass spectrometry. The two assay platforms showed acceptable accuracy and precision and gave comparable PK parameters of the drugs, suggesting that both assays were successfully applied to the PK assessments. Comparable results in the PK profiles were noted between serial and composite blood samplings and differences in the two sampling sites did not impact PK. These findings suggest that microsampling combined with generic assays is useful to assess PK profiles of therapeutic antibodies in mice. • A generic assay of therapeutic antibodies in mouse plasma was developed by LC-MS/MS and ELISA. • The assays were validated and their reproducibility was ensured. • Microsampling coupled with the generic assay was applied to a pharmacokinetic study in mice. • Effects of blood sampling sites and assay platforms on pharmacokinetics were evaluated. [ABSTRACT FROM AUTHOR]

Chemical sensing systems are vital in the growth and development of insects. Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is an important natural enemy of many pests. The molecular mechanism of odorant binding proteins (OBPs) binding with common insecticides is still unknow in O. sauteri. In this study, we expressed in vitro OsauOBP8 and conducted fluorescence competition binding assay to investigate the function of OsauOBP8 to insecticides. The results showed that OsauOBP8 could bind with four common insecticides (phoxim, fenitrothion, chlorpyrifos, deltamethrin). Subsequently, we used molecular docking to predict and obtained candidate six amino acid residues (K4, K6, K13, R31, K49, K55) and then mutated. The result showed that three key residues (K4, K6, R31) play important role in OsauOBP8 bound to insecticides. Our study identified the key binding sites of OsauOBP8 to insecticides and help to better understand the molecular mechanism of OBPs to insecticides in O. sauteri. [Display omitted] • The expression levels of OsauOBP8 were higher in male and female antennae. • OsauOBP8 could bind with four insecticides (phoxim, fenitrothion, chlorpyrifos, deltamethrin). • Three key residues (K4, K6, R31) play important role in OsauOBP8 bound to insecticides. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. Method: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. Results: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). Conclusion: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments. [ABSTRACT FROM AUTHOR]

The chemosensory system plays an important role in the host plants location. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a worldwide leaf-eating forest pest that feeds exclusively on salicaceous trees. There is no function study of odorant binding proteins (OBPs) in P. versicolora . In the current study, we found that PverOBP37 has a high expression in male and female antennae, heads, and legs by quantitative real-time PCR. The binding properties of PverOBP37 to 18 host plant volatiles were determined by fluorescence competition binding assays. The results showed that PverOBP37 could bind to the host plant volatile, o -cymene. Furthermore, four candidate key amino acid residues (F8, Y50, F103, and R107) of PverOBP37 to o -cymene were identified by molecular docking. The functional assay to confirm Y50, F103, and R107 mutations were key amino acid residues of PverOBP37 involved in the binding to o -cymene. Knockdown of PverOBP37 and Y-tube behavioral bioassays of mated females led to a significantly reduced attraction to o -cymene. This study not only revealed the molecular mechanism of PverOBP37 but also suggested that PverOBP37 is essential to detect host plant volatiles as cues to search for egg-laying sites in P. versicolora .

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers. [ABSTRACT FROM AUTHOR]

Spodoptera frugiperda is a kind of polyphagous pest, and can damage a large number different host plants around the worldwide. The molecular mechanisms of two general odorant binding proteins (GOBPs) binding with general volatiles and insecticides are still blank. In this study, we investigated the function of two GOBPs in S. frugiperda , by expressing two SfruGOBPs and tested the binding affinities by the fluorescence competition binding assays. The results exhibited that SfruGOBP1 has binding affinities to 4 of 38 general volatiles and 3 of 7 insecticides. In contrast, SfruGOBP2 showed a broader ligand-binding spectrum to 21 volatiles and 4 insecticides, suggesting SfruGOBP2 may plays a more important role in perceiving host volatiles than SfruGOBP1. Furthermore, we used molecular docking and site-directed mutagenesis assay to explored the key amino acid residues of two SfruGOBP to insecticides ligand. This study provides some valuable information to exploring the olfactory mechanism of two GOBPs bound the host plant volatiles and insecticides in S. frugiperda. [ABSTRACT FROM AUTHOR]

Abstract Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

• A multiplex assay was developed to measure two monoclonal antibodies in one sample. • The assay was fully validated with no significant cross-reactivity. • The assay is used in supporting combination therapy in the clinic. Clinical development of biotherapeutics for combination therapy requires monitoring the concentrations of both drugs in biological samples. Traditionally, two assays are required to measure drug levels one at a time, which poses challenges in sample management, data reporting, and cost. The Meso Scale Discovery (MSD®) U-PLEX™ platform provides a simple and flexible way to create custom multiplex ligand binding assays (LBAs). We developed and fully validated a two-plex assay on the U-PLEX platform where two therapeutic monoclonal antibodies (mAbs) in Merck's pipeline, which we call MK-A and MK-B in this manuscript, can be measured simultaneously in one sample. Our results demonstrated that the multiplexed pharmacokinetic (PK) assay has performances, including accuracy, precision, and cross-reactivity, that meet requirements in regulatory guidance. Furthermore, results of MK-A from the multiplex assay are comparable to results from a previously validated MK-A single-plex assay with 80% of samples tested in both assays having concentration differences < 30% relative to the mean of the two measurements. The multiplex assay was used to support a phase I MK-A/MK-B combination therapy clinical study and generated results consistent with historical MK-A monotherapy PK data. The ability to measure both biotherapeutics in a multiplexed assay is beneficial in that it improves consistency and efficiency while reduces sample volume and cost. With the number of combination therapies increasing in development, multiplexed assays can potentially have wide applications in biologics bioanalysis. [ABSTRACT FROM AUTHOR]

Abstract: Reliable bioanalytical tools for compound selection as well as studies of pharmacokinetics, pharmacodynamics, and toxicity are an important part of preclinical and clinical development. Developing a selective and specific analytical method in today's regulated bioanalysis framework is a formidable challenge for the analyst. The molecular structure and weight of analytes determine the method to be applied. Chromatographic assay (CC), predominantly liquid chromatography coupled with mass spectrometry, is the method of choice for measurement of small molecule concentrations in the biological matrix. For quantification of large molecules such as peptides and proteins, the gold standard is the ligand binding assay (LBA). LBAs differ substantially from CC methods; hence the recommendations for their validation differ too. Recently published (May 2018) United States Food and Drug Administration guidelines for bioanalytical method validation include a number of elements and acceptance criteria for CCs and LBAs individually. The differences in the requirements are based on the principles of analysis. This review summarizes the differences between small and large molecules, CCs, LBAs, and elements for bioanalytical method validation.Graphical abstract: [ABSTRACT FROM AUTHOR]

E6011, a humanized anti-fractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. Therapeutic antibodies may induce production of anti-drug antibodies (ADA) that may deteriorate efficacy and/or enhance immunogenic reaction. It is important to have an ADA assay to understand the characteristics of biotherapeutics under development. A simple and reproducible assay has thus been developed for the determination of ADA against E6011 in monkey and human serum by electrochemiluminescence (ECL) detection. An immune-complex of biotinylated E6011, ADA, and ruthenium-labeled E6011 was attached to avidin-coated wells for ECL signal detection. Screening and confirmatory cutpoints were determined to judge negative or positive ADA. Sensitivity of ADA was 1.61 and 1.34 ng/mL in monkey and human serum, respectively. Accuracy and precision of the assay were within ±20% and 20%, respectively. Drug tolerance of the assay in monkey and human sera was ensured up to 100 and 1000 μg/mL E6011 at the surrogate ADA levels of 1 and 4 μg/mL, respectively. The developed assay was successfully applied to ADA quantification in monkeys and humans in support of immunogenicity assessments. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization. [ABSTRACT FROM AUTHOR]

The accuracy of reported sample results is contingent upon the quality of the assay calibration curve, and as such, calibration curves are critical components of ligand binding and other quantitative methods. Regulatory guidance and lead publications have defined many of the requirements for calibration curves which encompass design, acceptance criteria, and selection of a regression model. However, other important aspects such as preparation and editing guidelines have not been addressed by health authorities. The goal of this publication is to answer many of the commonly asked questions and to present a consensus and the shared views of members of the ligand binding assay (LBA) community on topics related to calibration curves with focus on providing recommendations for the preparation and editing of calibration curves. [ABSTRACT FROM AUTHOR]

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides. [ABSTRACT FROM AUTHOR]