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.

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.

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.

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.

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.

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.

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.

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.

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.
CONCLUSIONS: 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.

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.