L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclo...L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imunohistocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.展开更多
The current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies(mAbs).Despite their increasing potential,mAbs have an intrinsic limitation relate...The current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies(mAbs).Despite their increasing potential,mAbs have an intrinsic limitation related to their inability to cross cell membranes and reach intracellular targets.Nanotechnology offers promising solutions to overcome this limitation,however,formulation challenges remain.These challenges are the limited loading capacity(often insufficient to achieve clinical dosing),the complex formulation methods,and the insufficient characterization of mAb-loaded nanocarriers.Here,we present a new nanocarrier consisting of hyaluronic acid-based nanoassemblies(HANAs)specifically designed to entrap mAbs with a high efficiency and an outstanding loading capacity(50%,w/w).HANAs composed by an mAb,modified HA and phosphatidylcholine(PC)resulted in sizes of~100 nm and neutral surface charge.Computational modeling identified the principal factors governing the high affinity of mAbs with the amphiphilic HA and PC.HANAs composition and structural configuration were analyzed using the orthogonal techniques cryogenic transmission electron microscopy(cryo-TEM),asymmetrical flow field-flow fractionation(AF4),and small-angle X-ray scattering(SAXS).These techniques provided evidence of the formation of core-shell nanostructures comprising an aqueous core surrounded by a bilayer consisting of phospholipids and amphiphilic HA.In vitro experiments in cancer cell lines and macrophages confirmed HANAs’low toxicity and ability to transport mAbs to the intracellular space.The reproducibility of this assembling process at industrial-scale batch sizes and the long-term stability was assessed.In conclusion,these results underscore the suitability of HANAs technology to load and deliver biologicals,which holds promise for future clinical translation.展开更多
Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of et...Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.展开更多
文摘L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imunohistocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.
基金supported by the government of Xunta de Galicia(Competitive Reference Groups,Consellería de Educación e Ordenación Universitaria,Xunta de Galicia,No.ED431C 2021/17)by the ISCⅢ thorough AES 2020,Award No.AC20/00028 and within the framework of EuroNanoMed Ⅲ+3 种基金part of the project Proof of Concept(No.PDC2021-120929-I00)financed by the Spanish Ministry of Science and Innovation-AEI/10.13039/501100011033the European Union NextGenerationEU/PRTRthe Spanish Ministry of Science,Innovation and Universities(No.FPU18/00095).
文摘The current spotlight of cancer therapeutics is shifting towards personalized medicine with the widespread use of monoclonal antibodies(mAbs).Despite their increasing potential,mAbs have an intrinsic limitation related to their inability to cross cell membranes and reach intracellular targets.Nanotechnology offers promising solutions to overcome this limitation,however,formulation challenges remain.These challenges are the limited loading capacity(often insufficient to achieve clinical dosing),the complex formulation methods,and the insufficient characterization of mAb-loaded nanocarriers.Here,we present a new nanocarrier consisting of hyaluronic acid-based nanoassemblies(HANAs)specifically designed to entrap mAbs with a high efficiency and an outstanding loading capacity(50%,w/w).HANAs composed by an mAb,modified HA and phosphatidylcholine(PC)resulted in sizes of~100 nm and neutral surface charge.Computational modeling identified the principal factors governing the high affinity of mAbs with the amphiphilic HA and PC.HANAs composition and structural configuration were analyzed using the orthogonal techniques cryogenic transmission electron microscopy(cryo-TEM),asymmetrical flow field-flow fractionation(AF4),and small-angle X-ray scattering(SAXS).These techniques provided evidence of the formation of core-shell nanostructures comprising an aqueous core surrounded by a bilayer consisting of phospholipids and amphiphilic HA.In vitro experiments in cancer cell lines and macrophages confirmed HANAs’low toxicity and ability to transport mAbs to the intracellular space.The reproducibility of this assembling process at industrial-scale batch sizes and the long-term stability was assessed.In conclusion,these results underscore the suitability of HANAs technology to load and deliver biologicals,which holds promise for future clinical translation.
基金SupportedbytheNationalNaturalScienceFoundationofChina (No .39870 473) .
文摘Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.
