The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins,due to a robust immune response.Elucidating how hapten repeated immunization shapes...The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins,due to a robust immune response.Elucidating how hapten repeated immunization shapes the rabbit B cell receptor(BCR)repertoire is crucial to understanding rabbit immune response to small molecules and assisting rare antibody discovery/engineering.In this study,we enriched and sequenced chloramphenicol(CAP)-specific rabbit B cells following repeated immunization,and analyzed both CAP-specific repertoires combined with the structure and affinity features of V1S69/V1S37 germline-based BCRs.The length of rabbit complementarity-determining region 3 of heavy chain(CDRH3)increased after hapten immunization.Repeated immunization significantly reduced the diversity of CAP-specific rabbit BCR clonotypes,and changed the frequency of VDJ usage and the type of V(D)J recombination.The average number of mutations among VL is notably higher than that of VH genes in rabbits,however,they are both not changed along with repeated immunization.Moreover,repeated immunization resulted in an increase surface charge and a decrease in solvent accessible surface area,leading to improvement in the stability of the most abundant V1S69/V1S37 germline-based BCR,along with an affinity increase from an IC50 of 898.2 ng mL^(−1)at the 1st immunization to 4.16 ng mL^(−1)at the 6th immunization.The study provides a benchmark for rabbit repertoire-scale analyses and offers a method for antibody discovery of small molecules.展开更多
Staphylococcal food poisoning is a significant foodborne illness caused by staphylococcal enterotoxins(SEs).Immu-noassays have become the primary method for rapidly detecting harmful bacteria and toxins because of the...Staphylococcal food poisoning is a significant foodborne illness caused by staphylococcal enterotoxins(SEs).Immu-noassays have become the primary method for rapidly detecting harmful bacteria and toxins because of their excel-lent sensitivity and specificity.However,these assays have limitations in that they cannot differentiate between types of SEs and do not provide rapid,on-site,quantitative testing.In this study,a time-resolved fluorescence immuno-chromatography assay(TRFICA)was developed specifically for detecting staphylococcal enterotoxin E(SEE),which is commonly found in dairy products.Compared with a double antibody sandwich enzyme-linked immunosorbent assay,which had a detection limit of 0.028 ng/mL,TRFICA demonstrated comparable sensitivity,enabling SEE quan-tification with a detection limit as low as 0.081 ng/mL in infant formula.Validation by spiking infant formula samples confirmed no cross-reactivity with analogs(recoveries ranged from 93.17%to 128.77%).Furthermore,with an 8-min reaction time and interpretation delivered by a portable TRFICA strip reader,our method demonstrates potential for use in mobile and on-site detection.This study describes a rapid,easy,and reliable method for detecting trace lev-els of SEE in infant formula,which could serve as an early screening tool toward preventing food poisoning in infants and children.展开更多
Fluoroacetamide(FAM)has been employed as a rodenticide for an extended duration,leading to a multitude of inci-dents involving human ingestion poisoning.Currently,FAMs have been prohibited by nations globally;however,...Fluoroacetamide(FAM)has been employed as a rodenticide for an extended duration,leading to a multitude of inci-dents involving human ingestion poisoning.Currently,FAMs have been prohibited by nations globally;however,there are still instances of their illegal usage.Conventional instrument methods are characterized by their time-consuming nature and complex operational procedures,rendering them inadequate for meeting urgent diagnostic needs in patients with acute FAM poisoning.Therefore,there is an immediate need to develop a prompt,user-friendly,and precise immunoassay method for the diagnosis of acute poisoning induced by FAM.A lateral flow immunochro-matography assay(LFIA)was developed in this study for the visual detection of FAMs in blood samples,representing the first report of such an approach.The method exhibited a cut-off value of 0.5 mg/mL under the optimized condi-tions,enabling the entire FAM detection process in blood samples to be completed within a mere 8 min without any pretreatment requirements.Notably,the results were easily discernible by visual inspection alone.These results indi-cate that the developed LFIA holds great promise as a convenient and rapid diagnostic tool for FAM poisoning diag-nosis,thereby offering valuable support for subsequent treatment strategies.展开更多
基金approved by the Animal Ethics Committee of China Agricultural University and strictly conducted by Chinese laws and guidelines(AW32602202-2-2).
文摘The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins,due to a robust immune response.Elucidating how hapten repeated immunization shapes the rabbit B cell receptor(BCR)repertoire is crucial to understanding rabbit immune response to small molecules and assisting rare antibody discovery/engineering.In this study,we enriched and sequenced chloramphenicol(CAP)-specific rabbit B cells following repeated immunization,and analyzed both CAP-specific repertoires combined with the structure and affinity features of V1S69/V1S37 germline-based BCRs.The length of rabbit complementarity-determining region 3 of heavy chain(CDRH3)increased after hapten immunization.Repeated immunization significantly reduced the diversity of CAP-specific rabbit BCR clonotypes,and changed the frequency of VDJ usage and the type of V(D)J recombination.The average number of mutations among VL is notably higher than that of VH genes in rabbits,however,they are both not changed along with repeated immunization.Moreover,repeated immunization resulted in an increase surface charge and a decrease in solvent accessible surface area,leading to improvement in the stability of the most abundant V1S69/V1S37 germline-based BCR,along with an affinity increase from an IC50 of 898.2 ng mL^(−1)at the 1st immunization to 4.16 ng mL^(−1)at the 6th immunization.The study provides a benchmark for rabbit repertoire-scale analyses and offers a method for antibody discovery of small molecules.
基金supported by Beijing Municipal Science and Technology Commission(Z211100007021007)the Key R&D Program of Ningxia Hui Autonomous Region(2021BBF02036).
文摘Staphylococcal food poisoning is a significant foodborne illness caused by staphylococcal enterotoxins(SEs).Immu-noassays have become the primary method for rapidly detecting harmful bacteria and toxins because of their excel-lent sensitivity and specificity.However,these assays have limitations in that they cannot differentiate between types of SEs and do not provide rapid,on-site,quantitative testing.In this study,a time-resolved fluorescence immuno-chromatography assay(TRFICA)was developed specifically for detecting staphylococcal enterotoxin E(SEE),which is commonly found in dairy products.Compared with a double antibody sandwich enzyme-linked immunosorbent assay,which had a detection limit of 0.028 ng/mL,TRFICA demonstrated comparable sensitivity,enabling SEE quan-tification with a detection limit as low as 0.081 ng/mL in infant formula.Validation by spiking infant formula samples confirmed no cross-reactivity with analogs(recoveries ranged from 93.17%to 128.77%).Furthermore,with an 8-min reaction time and interpretation delivered by a portable TRFICA strip reader,our method demonstrates potential for use in mobile and on-site detection.This study describes a rapid,easy,and reliable method for detecting trace lev-els of SEE in infant formula,which could serve as an early screening tool toward preventing food poisoning in infants and children.
基金supported by Beijing Municipal Science and Technology Commission(Z211100007021007)the earmarked fund for CARS36,the Key R&D Program of Ningxia Hui Autonomous Region(2021BBF02036)the program of Beijing Municipal Education Commission(KM202212448002).
文摘Fluoroacetamide(FAM)has been employed as a rodenticide for an extended duration,leading to a multitude of inci-dents involving human ingestion poisoning.Currently,FAMs have been prohibited by nations globally;however,there are still instances of their illegal usage.Conventional instrument methods are characterized by their time-consuming nature and complex operational procedures,rendering them inadequate for meeting urgent diagnostic needs in patients with acute FAM poisoning.Therefore,there is an immediate need to develop a prompt,user-friendly,and precise immunoassay method for the diagnosis of acute poisoning induced by FAM.A lateral flow immunochro-matography assay(LFIA)was developed in this study for the visual detection of FAMs in blood samples,representing the first report of such an approach.The method exhibited a cut-off value of 0.5 mg/mL under the optimized condi-tions,enabling the entire FAM detection process in blood samples to be completed within a mere 8 min without any pretreatment requirements.Notably,the results were easily discernible by visual inspection alone.These results indi-cate that the developed LFIA holds great promise as a convenient and rapid diagnostic tool for FAM poisoning diag-nosis,thereby offering valuable support for subsequent treatment strategies.
