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Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms.

ABSTRACT: The extraction of targets from biological samples for immunoassays using organic solvents, such as methanol, is often necessary. However, high concentrations of organic solvents in extracts invariably lead to instability of the employed antibody, resulting in poor performance of the immunoassay. Evaluating the tolerance ability and exploring the molecular mechanisms of antibody tolerance in organic solvents are essential for the development of robust immunoassays. In this work, 25 monoclonal antibodies and methanol are utilized as models to address these questions. A novel protocol is initially established to precisely and rapidly determine antibody tolerance in methanol, identifying two distinct methanol effect patterns. Through a detailed investigation of the structural basis, a novel hypothesis regarding methanol effect patterns is proposed, termed "folding-aggregation," which is subsequently validated through molecular dynamics simulations. Furthermore, the investigation of sequence basis reveals significant differences in residue types within the complementarity-determining regions and ligand-binding residues, distinguishing the two antibody methanol effect patterns. Moreover, the methanol effect patterns of the antibodies are defined by germline antibodies. This work represents the first exploration of antibody methanol effect patterns and associated molecular mechanisms, with potential implications for the discovery and engineering of tolerant antibodies for the development of robust immunoassays.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC11336977 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms.

Zhang Yingjie Y Mi Jiafei J Wu Weilin W Fei Jie J Lv Bochen B Yu Xuezhi X Wen Kai K Shen Jianzhong J Wang Zhanhui Z

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20240618 31

The extraction of targets from biological samples for immunoassays using organic solvents, such as methanol, is often necessary. However, high concentrations of organic solvents in extracts invariably lead to instability of the employed antibody, resulting in poor performance of the immunoassay. Evaluating the tolerance ability and exploring the molecular mechanisms of antibody tolerance in organic solvents are essential for the development of robust immunoassays. In this work, 25 monoclonal ant ...[more]

PMID: 38889249

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Project description:Background and aimsHerbivory and plant defence differ markedly among seedlings and juvenile and mature plants in most species. While ontogenetic patterns of chemical resistance have been the focus of much research, comparatively little is known about how tolerance to damage changes across ontogeny. Due to dramatic shifts in plant size, resource acquisition, stored reserves and growth, it was predicted that tolerance and related underlying mechanisms would differ among ontogenetic stages.MethodsOntogenetic patterns in the mechanisms of tolerance were investigated in Plantago lanceolata and P. major (Plantaginaceae) using the genetic sib-ship approach. Pot-grown plants were subjected to 50 % defoliation at the seedling, juvenile and mature stages and either harvested in the short-term to look at plasticity in growth and photosynthesis in response to damage or allowed to grow through seed maturation to measure phenology, shoot compensation and reproductive fitness.Key resultsTolerance to defoliation was high in P. lanceolata, but low in P. major, and did not vary among ontogenetic stages in either species. Mechanisms underlying tolerance did vary across ontogeny. In P. lanceolata, tolerance was significantly related to flowering (juveniles) and pre-damage shoot biomass (mature plants). In P. major, tolerance was significantly related to pre-damage root biomass (seedlings) and induction of non-photochemical quenching, a photosynthetic parameter (juveniles).ConclusionsBiomass partitioning was very plastic in response to damage and showed associations with tolerance in both species, indicating a strong role in plant defence. In contrast, photosynthesis and phenology showed weaker responses to damage and were related to tolerance only in certain ontogenetic stages. This study highlights the pivotal role of ontogeny in plant defence and herbivory. Additional studies in more species are needed to determine how seedlings tolerate herbivory in general and whether mechanisms vary across ontogeny in consistent patterns.

Project description:Pearl millet (Pennisetum glaucum (L.)) is a vital cereal crop renowned for its ability to thrive in challenging environmental conditions; however, the molecular mechanisms governing its salt stress tolerance remain poorly understood. To address this gap, next-generation RNA sequencing was conducted to compare gene expression patterns in pearl millet seedlings exposed to salt stress with those grown under normal conditions. Our RNA sequencing analysis focused on shoots from 13-day-old pearl millet plants subjected to either salinity stress (150 mmol of NaCl for 3 days) or thermal stress (50°C for 60 s). Of 36,041 genes examined, 17,271 genes with fold changes ranging from 2.2 to 19.6 were successfully identified. Specifically, 2388 genes were differentially upregulated in response to heat stress, whereas 4327 genes were downregulated. Under salt stress conditions, 2013 genes were upregulated and 4221 genes were downregulated. Transcriptomic analysis revealed four common abiotic KEGG pathways that play crucial roles in the response of pearl millet to salt and heat stress: phenylpropanoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, and plant hormone signal transduction. These metabolic pathways are necessary for pearl millet to withstand and adapt to abiotic stresses caused by salt and heat. Moreover, the pearl millet shoot heat stress group showed specific transcriptomics related to KEEG metabolic pathways such as cytochrome P450, cutin, suberine, and wax biosynthesis, zeatin biosynthesis, crocin biosynthesis, ginsenoside biosynthesis, saponin biosynthesis, and biosynthesis of various plant secondary metabolites. In contrast, pearl millet shoots exposed to salinity stress exhibited transcriptomic changes associated with KEEG metabolic pathways related to carbon fixation in photosynthetic organisms, mismatch repair, and nitrogen metabolism. Our findings underscore the remarkable cross-tolerance of pearl millet to simultaneous salt and heat stress, elucidated through the activation of shared abiotic KEGG pathways. This study emphasizes the pivotal role of transcriptomics analysis in unraveling the molecular responses of pearl millet under abiotic stress conditions.

Dataset Information

Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms.

Publications

Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms.

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