Five PAHs haptens 3 a～3 e were synthesized from naphthalene, anthracene, chrysene, pyrene and acenaphthene via two steps including Friedel-Crafts acylation and WolffKishner-Huang reduction. The sixth hapten 4-(acenap...Five PAHs haptens 3 a～3 e were synthesized from naphthalene, anthracene, chrysene, pyrene and acenaphthene via two steps including Friedel-Crafts acylation and WolffKishner-Huang reduction. The sixth hapten 4-(acenaphthylen-3-yl)butanoic acid(3 f) was prepared from hapten 3 e via three steps including esterification, bromination and elimination. Their structures were confirmed by melting point, ~1H NMR and ^(13)C NMR. Their optical properties and crystal structures were also investigated. The results of density functional theory(DFT) calculation provided the supports that the size, shape(geometry) and electronic properties at the corresponding parts of 3 a～3 f did not change significantly, compared to those of PAHs. The haptens 3 a～3 f were coupled with bovine serum albumin(BSA) to make antigens. The coupling ratios were 1:20～1:38. These results show that the haptens could be used to induce specific antibodies for PAHs.展开更多
A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone （AOZ）. Haptens designed newly were achieved facilely in good yield by using LiCI-N（Et）3 as new ...A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone （AOZ）. Haptens designed newly were achieved facilely in good yield by using LiCI-N（Et）3 as new catalysis system, the structure of which was elucidated by spectroscopy analysis, such as NMR and MS. Novel antigens for AOZ were prepared successfully by convenient active ester method. The ratios of haptens 3 and 4 to carrier proteins were proven respectively as 41 ： 1 （5a）, 39：1 （6a）, 11：1 （5b） and 9：1 （6b） by trinitrobenzene sulfonic acid （TNBS） method. The results of indirect competitive ELISA （ic-ELISA） of antiserums indicated that the haptens with a short unsaturated side chain can evoke specific immune response effectively.展开更多
The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody. In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of...The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody. In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of cortisol, different tether lengths of 6-corticosteroid haptens and their BSA conjugates were designed and synthesized.展开更多
Glutathione was modified selectively by 2,4 dinitrochlorobenzene,giving S substituted dinitrophenyl glutathione(GSH S DNP).GSH S DNP was further esterified by iso butanol,hexanol,cyclohexanol and benzyl alcohol.Four h...Glutathione was modified selectively by 2,4 dinitrochlorobenzene,giving S substituted dinitrophenyl glutathione(GSH S DNP).GSH S DNP was further esterified by iso butanol,hexanol,cyclohexanol and benzyl alcohol.Four haptens used to generate abzyme with glutathione peroxidase(GPX)activity were synthesized.They are GSH DNP biesters:GSH DNP di iso butyl ester(GSH DNP IBU),GSH DNP bihexyl ester(GSH DNP HE),GSH DNP bicyclohexyl ester(GSH DNP CH),GSH DNP bibenzylmethyl ester(GSH DNP BE).The structures of the haptens were characterized by means of elemental analysis,IR and 1H NMR.展开更多
A facile procedure was described for the hapten design of the N-methylcarbamate insecticide propoxur.Two new haptens of propoxur(hapten 1a and hapten 1b) were synthesized by introducing appropriate spacers in the pest...A facile procedure was described for the hapten design of the N-methylcarbamate insecticide propoxur.Two new haptens of propoxur(hapten 1a and hapten 1b) were synthesized by introducing appropriate spacers in the pesticide aromatic moiety of the analyte molecular structure.First,the propoxur reacted with nitric acid to yield the intermediate product.Then hapten 1a was prepared via the reduction of the intermediate product,and hapten 1b was formed by the acylation of hapten 1a with succinic anhydride.In addition,we have also developed a simple method to prepare hapten 2 for the pyrethroid insecticide cyhalothrin,which only requires two steps including the reactions of nitration and reduction.Three haptens were coupled to carrier proteins to prepare the corresponding artificial antigens.The molecular structures of these three haptens were identified by 1H NMR and MS,and those of the artificial antigens were confirmed by UV-vis.展开更多
T helper 2 （Th2） polarization is a major pathological feature in allergic diseases; its etiology is not fully understood. This study aims to elucidate the adjuvant effect of the microbial product-derived small pepti...T helper 2 （Th2） polarization is a major pathological feature in allergic diseases; its etiology is not fully understood. This study aims to elucidate the adjuvant effect of the microbial product-derived small peptides in the initiation of antigen-specific Th2 polarization. In this study, a clinical survey of patients with chronic rhinosinusitis （CRS） and food allergy （FA） was carried out. The Staphylococcal enterotoxin B （SEB）-derived small peptides （Ssps） were examined in the human stool extracts. The formation of Ssp/antigen adducts was tested in a protein-protein combination assay. The bone marrow-derived dendritic cells （BMDCs） were employed to test the role of Ssp/ovalbumin （OVA） adducts in the dendritic cell （DC） maturation. A mouse model was developed to test the role of Ssp/OVA adducts in the initiation of ＂l＇h2 polarization in the intestine. The results showed that 54 （18.2%} patients with FA were diagnosed among 296 patients with SEB＋ CRS; only eight （2.9%） FA patients were identified among 272 patients with SEB- CRS. Ssps were detected in the stool protein extracts from FA patients with SEB＋ CRS, but not in those with SEB- CRS. Ssp/OVA adducts induced DC maturation, speeded up DC migration, activated CD4＋ T cells in the regional lymph nodes and induced skewed Th2 polarization in the local tissue. We conclude that patients with SEB ＋ CRS are prone to suffering from FA. SEB can be degraded to Ssps in the gastrointestinal tract. The Ssps can bind macromolecular antigens to form adducts to promote the antigenicity of the antigens and induction of the antigen-specific Th2 polarization and inflammation in the local tissue.展开更多
Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were lo...Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay(ic ELISA). Under optimized conditions, the half maximal inhibitory concentration(IC50) value of the best polyclonal antibody was 2.1 mg·L^(-1), based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible crossreactivity with bisphenol B and bisphenol E. A sensitive ic ELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples(5, 10 and 20 mg·L^(-1)), the recovery ranged from 80% to 102% with a coefficient of variation(CV) value below 15.8%. The limit of detection of ic ELISA was1.95 mg·L^(-1). These results indicate that the ic ELISA method is suitable for the detection of BPA in milk.展开更多
Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three result...Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens. Three of the resultant polyclonal antibodies （Pabs） were obtained. The antiserum exhibited relatively high antibody titres （1：32-64） in double agar diffusion.展开更多
Baicalein, baicalin, scutellarin and Chrysin-7-O-β-D-glucuronide are the major flavonoids of the Shuang-huang-lian powder injection. These flavonoids are thought to be haptens that can induce allergic reactions. The ...Baicalein, baicalin, scutellarin and Chrysin-7-O-β-D-glucuronide are the major flavonoids of the Shuang-huang-lian powder injection. These flavonoids are thought to be haptens that can induce allergic reactions. The interaction mechanism of these haptens with bovine serum albumin （BSA） was investigated by surface plasmon resonance （SPR） and molecular modeling method. The SPR study indicated that these compounds could specifically bind to the BSA with one binding site and equilibrium dissociation constant （KD） values were determined. Molecular modeling explored the mechanism of interaction under simulated physiological condition. The result of molecular modeling indicated that flavonoids could bind with BSA in the hydrophobic pocket of sub-domain II with hydrogen bonding as the main acting force.展开更多
A new hapten, aldicarb oxime succinic ester （AOSE）, was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA （bovine serum a...A new hapten, aldicarb oxime succinic ester （AOSE）, was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA （bovine serum albumin） conjugate. Enzyme-linked immunosorbent assays （ELISAs） showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.展开更多
Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine seru...Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method. Infrared spectra（IR） showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra（UV） and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）. The calculated average binding ratios of PBA/BSA and PBA/OVA were 18：1 and 10：1 by UV, and 31：1 and 22：1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons（PAHs）.展开更多
A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Tw...A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.展开更多
Antigen 12, containing a phosphonyl peptide hapten with free C-terminal carboxylic group, was synthesized by 11 reaction steps. The design of the hapten was based on the transition state of peptide hydrolysis catalyze...Antigen 12, containing a phosphonyl peptide hapten with free C-terminal carboxylic group, was synthesized by 11 reaction steps. The design of the hapten was based on the transition state of peptide hydrolysis catalyzed by carboxypeptidase A.展开更多
The thiol group of glutathione (GSH) reacts specifically with 2.4-di-m-trochlorobenzene to give S-substituted dinitrophenyl glutathione (GSH-S-DNP);two carboxyl groups of GSH-S-DNP were further esterified by n-butanol...The thiol group of glutathione (GSH) reacts specifically with 2.4-di-m-trochlorobenzene to give S-substituted dinitrophenyl glutathione (GSH-S-DNP);two carboxyl groups of GSH-S-DNP were further esterified by n-butanol to produce the hapten,multisubstrate analog GSH-S-DNP Butyl Ester (GSH-S-DNP BE).The primary structure of the hapten was characterized by the free ammo group analysis,1H NMR,IR determinations and the elemental analysis.The hapten was then conjugated to bovine serum albumin (BSA) in the presence of glutaraldehyde.The reaction mixture was purified by Ultrogel AcA54 coluni chromatography to give the antigen.On an average,25 haptens were bound to each BSA molecule.Electrophoresis analysis showed that the average molecular weight of the antigen was 87 KD.CD spectrum showed that the α-helix content of the antigen increased.展开更多
This study reported that the hapten of the organophosphorus insecticide chlorpyrifos, O,O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl)thio-2-pyridyl]phosphorothioate(named AR) wassynthesized by using technical grade chl...This study reported that the hapten of the organophosphorus insecticide chlorpyrifos, O,O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl)thio-2-pyridyl]phosphorothioate(named AR) wassynthesized by using technical grade chlorpyrifos reacted with 3-marcapropanoic acid in hotalkaline solution. The hapten was conjugated to bovine serum albumin (BSA) with the modifiedactive ester method to form artificial immune antigen. The ratio of AR:BSA was 39:1. Theartificial coating antigen for chlorpyrifos was synthesized by conjugating AR to ovalbumin(OVA) with the mixed-anhydride method, and the ratio was 13:1. The anti-chlorpyrifos polyclonalantibodies were obtained by using the artificial immune antigen (AR-BSA) to immune in therabbits.展开更多
Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural ana...Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues,the preparation of highly specific antibodies is crucial.In this study,a novel hapten DEX-GA was designed and synthesized,and the monoclonal antibody(mAb)3D1 was successfully prepared based on the hapten.The mAb 3D1 has the 50%inhibitory concentration of 0.38 ng/mL for dexamethasone,with cross-reactivity below 7.45%against twelve glucocorticoids including betamethasone,prednisone,triamcinolone and beclomethasone.Based on this antibody,indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)and colloidal gold immunochromatography assay(GICA)were developed for accurate detection of dexamethasone in milk and animal tissues.The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215μg/kg,which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method.The GICA method established in this study had a visual limit of detection of 0.3μg/L in milk.Notably,this constitutes the first application of GICA in animal tissues,demonstrating a visual detection limit of 0.2μg/kg.The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.展开更多
Cyclosulfamuron(CS)is a widely used sulfonylurea herbicide in plant cultivation and food production,and poses significant threats to ecological balance and food safety.To address this concern,we designed a novel CS ha...Cyclosulfamuron(CS)is a widely used sulfonylurea herbicide in plant cultivation and food production,and poses significant threats to ecological balance and food safety.To address this concern,we designed a novel CS hapten using computer-simulated semi-antigens.A monoclonal antibody(mAb)exhibiting high specificity and affinity was generated,with an affinity constant of 1.049×10^(9) L/mol and a semi-inhibitory concentration(IC50)of 4.94 ng/mL.Leveraging this anti-CS mAb,we constructed a rapid,visually detectable,quantitative lateral flow immunoassay(LFIA)to detect CS in cereals.Under the optimal conditions,the cut-off values were determined to be 100μg/kg for brown rice and corn samples and 50μg/kg for rice samples.The corresponding limits of detection values(LODs)were found to be 1.93μg/kg in brown rice,2.18μg/kg in corn and 1.93μg/kg in rice,respectively.The recovery rates ranged from 96.2%to 104.8%with coefficients of variation ranging from 3.5%to 6.4%,showing good agreement with the results obtained by liquid chromatography–tandem mass spectrometry(LC–MS/MS).The proposed assay demonstrated robust analytical performance,with significant application potential for rapid,on-site detection.展开更多
Formaldehyde is a well-known carcinogen that can occur naturally or be illicitly added as an adulterant to food,calling for rapid and reliable detection methods.In this work,leveraging the facile and selective transfo...Formaldehyde is a well-known carcinogen that can occur naturally or be illicitly added as an adulterant to food,calling for rapid and reliable detection methods.In this work,leveraging the facile and selective transformation of formaldehyde into 5-nitro-benzimidazole(5-nitro-BZM)using 4-nitro-o-phenylenediamine,two haptens were designed to target the BZM structure,leading to the production of a monoclonal antibody specifically recognizing BZM.Utilizing this antibody,a lateral flow immunoassay(LFIA)was developed for the detection of formaldehyde in grass carp,bighead carp,and basal shrimp,following a rapid pre-analysis derivatization.The detection limits were 0.15 mg/kg in grass carp,0.11 mg/kg in bighead carp,and 0.14 mg/kg in kiwi shrimp.The recoveries were in the range from 75.3%to 89.2%,and the LFIA results showed good correlation with those obtained from the HPLC.This marks the first report concerning an immunoassay for formaldehyde,offering a more rapid and cost-effective alternative for routine formaldehyde monitoring.展开更多
Carmoisine(CMS)is a synthetic azo colorant that can be used as a food colorant in products such as candy.However,due to potential health risks associated with carcinogenic,child development and allergic reactions,its ...Carmoisine(CMS)is a synthetic azo colorant that can be used as a food colorant in products such as candy.However,due to potential health risks associated with carcinogenic,child development and allergic reactions,its use is strictly regulated.We have developed a colloidal gold immunochromatographic assay(CG-ICA)based on a highly sensitive monoclonal antibody(mAb)for the rapid and sensitive detection of CMS in food products.This study proposes a novel hapten and evaluates its structure through computer-aided analysis based on molecular structural similarity.Then,a mAb was successfully prepared,with a half-maximal inhibitory concentration of 0.20 ng/mL and a limit of detection(LOD)of 0.03 ng/mL.The CG-ICA strip demonstrated a LOD of 0.48–0.58μg/L for three soft drink types,achieving recovery rates between 90.78%and 109.61%and a coefficient of variation(CV)between 2.39%and 5.24%.The accuracy of the method was further validated by liquid chromatography–tandem mass spectrometry(LC–MS).Without requiring complicated purification steps,the simple operation and rapid result-yielding ability of CG-ICA within 20 min demonstrate its suitability for field rapid screening of CMS in soft drinks.The method developed in this study represents a new exploration for CMS detection and provides a direction for the rapid screening of similar colorants.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province(BK20161542)the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province(16KJB550005 and 17KJB150006)+1 种基金Jiangsu Overseas Visiting Scholar Program for University Prominent Young&Middle-aged Teachers and Presidents(2017)the Youth Foundation of Southeast University Chengxian College(y0004)
文摘Five PAHs haptens 3 a～3 e were synthesized from naphthalene, anthracene, chrysene, pyrene and acenaphthene via two steps including Friedel-Crafts acylation and WolffKishner-Huang reduction. The sixth hapten 4-(acenaphthylen-3-yl)butanoic acid(3 f) was prepared from hapten 3 e via three steps including esterification, bromination and elimination. Their structures were confirmed by melting point, ~1H NMR and ^(13)C NMR. Their optical properties and crystal structures were also investigated. The results of density functional theory(DFT) calculation provided the supports that the size, shape(geometry) and electronic properties at the corresponding parts of 3 a～3 f did not change significantly, compared to those of PAHs. The haptens 3 a～3 f were coupled with bovine serum albumin(BSA) to make antigens. The coupling ratios were 1:20～1:38. These results show that the haptens could be used to induce specific antibodies for PAHs.
文摘A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone （AOZ）. Haptens designed newly were achieved facilely in good yield by using LiCI-N（Et）3 as new catalysis system, the structure of which was elucidated by spectroscopy analysis, such as NMR and MS. Novel antigens for AOZ were prepared successfully by convenient active ester method. The ratios of haptens 3 and 4 to carrier proteins were proven respectively as 41 ： 1 （5a）, 39：1 （6a）, 11：1 （5b） and 9：1 （6b） by trinitrobenzene sulfonic acid （TNBS） method. The results of indirect competitive ELISA （ic-ELISA） of antiserums indicated that the haptens with a short unsaturated side chain can evoke specific immune response effectively.
基金Fanicial support from the Grand-in-aid from Ministry of Education Sports Science and Culture of Japan is greatly acknowledged.
文摘The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody. In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of cortisol, different tether lengths of 6-corticosteroid haptens and their BSA conjugates were designed and synthesized.
文摘Glutathione was modified selectively by 2,4 dinitrochlorobenzene,giving S substituted dinitrophenyl glutathione(GSH S DNP).GSH S DNP was further esterified by iso butanol,hexanol,cyclohexanol and benzyl alcohol.Four haptens used to generate abzyme with glutathione peroxidase(GPX)activity were synthesized.They are GSH DNP biesters:GSH DNP di iso butyl ester(GSH DNP IBU),GSH DNP bihexyl ester(GSH DNP HE),GSH DNP bicyclohexyl ester(GSH DNP CH),GSH DNP bibenzylmethyl ester(GSH DNP BE).The structures of the haptens were characterized by means of elemental analysis,IR and 1H NMR.
基金supported by the National High Technology Research and Development Program of China(2006AA10Z449)the National Natural Science Foundation of China(20801024)Wu Jieping Medical Foundation(32067500615)
文摘A facile procedure was described for the hapten design of the N-methylcarbamate insecticide propoxur.Two new haptens of propoxur(hapten 1a and hapten 1b) were synthesized by introducing appropriate spacers in the pesticide aromatic moiety of the analyte molecular structure.First,the propoxur reacted with nitric acid to yield the intermediate product.Then hapten 1a was prepared via the reduction of the intermediate product,and hapten 1b was formed by the acylation of hapten 1a with succinic anhydride.In addition,we have also developed a simple method to prepare hapten 2 for the pyrethroid insecticide cyhalothrin,which only requires two steps including the reactions of nitration and reduction.Three haptens were coupled to carrier proteins to prepare the corresponding artificial antigens.The molecular structures of these three haptens were identified by 1H NMR and MS,and those of the artificial antigens were confirmed by UV-vis.
文摘T helper 2 （Th2） polarization is a major pathological feature in allergic diseases; its etiology is not fully understood. This study aims to elucidate the adjuvant effect of the microbial product-derived small peptides in the initiation of antigen-specific Th2 polarization. In this study, a clinical survey of patients with chronic rhinosinusitis （CRS） and food allergy （FA） was carried out. The Staphylococcal enterotoxin B （SEB）-derived small peptides （Ssps） were examined in the human stool extracts. The formation of Ssp/antigen adducts was tested in a protein-protein combination assay. The bone marrow-derived dendritic cells （BMDCs） were employed to test the role of Ssp/ovalbumin （OVA） adducts in the dendritic cell （DC） maturation. A mouse model was developed to test the role of Ssp/OVA adducts in the initiation of ＂l＇h2 polarization in the intestine. The results showed that 54 （18.2%} patients with FA were diagnosed among 296 patients with SEB＋ CRS; only eight （2.9%） FA patients were identified among 272 patients with SEB- CRS. Ssps were detected in the stool protein extracts from FA patients with SEB＋ CRS, but not in those with SEB- CRS. Ssp/OVA adducts induced DC maturation, speeded up DC migration, activated CD4＋ T cells in the regional lymph nodes and induced skewed Th2 polarization in the local tissue. We conclude that patients with SEB ＋ CRS are prone to suffering from FA. SEB can be degraded to Ssps in the gastrointestinal tract. The Ssps can bind macromolecular antigens to form adducts to promote the antigenicity of the antigens and induction of the antigen-specific Th2 polarization and inflammation in the local tissue.
基金supported by the National Natural Science Foundation of China (31622057)
文摘Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay(ic ELISA). Under optimized conditions, the half maximal inhibitory concentration(IC50) value of the best polyclonal antibody was 2.1 mg·L^(-1), based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible crossreactivity with bisphenol B and bisphenol E. A sensitive ic ELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples(5, 10 and 20 mg·L^(-1)), the recovery ranged from 80% to 102% with a coefficient of variation(CV) value below 15.8%. The limit of detection of ic ELISA was1.95 mg·L^(-1). These results indicate that the ic ELISA method is suitable for the detection of BPA in milk.
基金supported by the National Natural Science Foundation of China(No.20677008)The Research Fund for the Doctoral Program of Higher Education(20060255004)Shanghai Leading Academic Discipline Project(No.B604).
文摘Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens. Three of the resultant polyclonal antibodies （Pabs） were obtained. The antiserum exhibited relatively high antibody titres （1：32-64） in double agar diffusion.
基金Nature Science Foundation of Beijing,China(Grant No.7142088)
文摘Baicalein, baicalin, scutellarin and Chrysin-7-O-β-D-glucuronide are the major flavonoids of the Shuang-huang-lian powder injection. These flavonoids are thought to be haptens that can induce allergic reactions. The interaction mechanism of these haptens with bovine serum albumin （BSA） was investigated by surface plasmon resonance （SPR） and molecular modeling method. The SPR study indicated that these compounds could specifically bind to the BSA with one binding site and equilibrium dissociation constant （KD） values were determined. Molecular modeling explored the mechanism of interaction under simulated physiological condition. The result of molecular modeling indicated that flavonoids could bind with BSA in the hydrophobic pocket of sub-domain II with hydrogen bonding as the main acting force.
文摘A new hapten, aldicarb oxime succinic ester （AOSE）, was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA （bovine serum albumin） conjugate. Enzyme-linked immunosorbent assays （ELISAs） showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.
基金Supported by National High-Tech Research and Development Program of China(No.2002AA649180)PhD Programs Foundation of Ministry of Education of China(No.20010055005)
文摘Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method. Infrared spectra（IR） showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra（UV） and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）. The calculated average binding ratios of PBA/BSA and PBA/OVA were 18：1 and 10：1 by UV, and 31：1 and 22：1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons（PAHs）.
基金This work was financially supported by National Natural Science Foundation of China （20447003）.
文摘A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
文摘Antigen 12, containing a phosphonyl peptide hapten with free C-terminal carboxylic group, was synthesized by 11 reaction steps. The design of the hapten was based on the transition state of peptide hydrolysis catalyzed by carboxypeptidase A.
文摘The thiol group of glutathione (GSH) reacts specifically with 2.4-di-m-trochlorobenzene to give S-substituted dinitrophenyl glutathione (GSH-S-DNP);two carboxyl groups of GSH-S-DNP were further esterified by n-butanol to produce the hapten,multisubstrate analog GSH-S-DNP Butyl Ester (GSH-S-DNP BE).The primary structure of the hapten was characterized by the free ammo group analysis,1H NMR,IR determinations and the elemental analysis.The hapten was then conjugated to bovine serum albumin (BSA) in the presence of glutaraldehyde.The reaction mixture was purified by Ultrogel AcA54 coluni chromatography to give the antigen.On an average,25 haptens were bound to each BSA molecule.Electrophoresis analysis showed that the average molecular weight of the antigen was 87 KD.CD spectrum showed that the α-helix content of the antigen increased.
文摘This study reported that the hapten of the organophosphorus insecticide chlorpyrifos, O,O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl)thio-2-pyridyl]phosphorothioate(named AR) wassynthesized by using technical grade chlorpyrifos reacted with 3-marcapropanoic acid in hotalkaline solution. The hapten was conjugated to bovine serum albumin (BSA) with the modifiedactive ester method to form artificial immune antigen. The ratio of AR:BSA was 39:1. Theartificial coating antigen for chlorpyrifos was synthesized by conjugating AR to ovalbumin(OVA) with the mixed-anhydride method, and the ratio was 13:1. The anti-chlorpyrifos polyclonalantibodies were obtained by using the artificial immune antigen (AR-BSA) to immune in therabbits.
基金funded by the National Natural Science Founda-tion of China(32573424)the National Key Research and Development Programs of China(2023YFD1301001,2024YFF1105705)the Hubei Provincial Natural Science Foundation(2024AFB146).
文摘Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues,the preparation of highly specific antibodies is crucial.In this study,a novel hapten DEX-GA was designed and synthesized,and the monoclonal antibody(mAb)3D1 was successfully prepared based on the hapten.The mAb 3D1 has the 50%inhibitory concentration of 0.38 ng/mL for dexamethasone,with cross-reactivity below 7.45%against twelve glucocorticoids including betamethasone,prednisone,triamcinolone and beclomethasone.Based on this antibody,indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)and colloidal gold immunochromatography assay(GICA)were developed for accurate detection of dexamethasone in milk and animal tissues.The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215μg/kg,which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method.The GICA method established in this study had a visual limit of detection of 0.3μg/L in milk.Notably,this constitutes the first application of GICA in animal tissues,demonstrating a visual detection limit of 0.2μg/kg.The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.
基金support received from the National Natural Science Foundation of China(No.32460627)the Guizhou Provincial Basic Research Program(Natural Science)(No.QianKeHeJiChu-ZK[2024]YiBan103)the Special Research Fund of Natural Science(Special Post)of Guizhou University(2022)54.
文摘Cyclosulfamuron(CS)is a widely used sulfonylurea herbicide in plant cultivation and food production,and poses significant threats to ecological balance and food safety.To address this concern,we designed a novel CS hapten using computer-simulated semi-antigens.A monoclonal antibody(mAb)exhibiting high specificity and affinity was generated,with an affinity constant of 1.049×10^(9) L/mol and a semi-inhibitory concentration(IC50)of 4.94 ng/mL.Leveraging this anti-CS mAb,we constructed a rapid,visually detectable,quantitative lateral flow immunoassay(LFIA)to detect CS in cereals.Under the optimal conditions,the cut-off values were determined to be 100μg/kg for brown rice and corn samples and 50μg/kg for rice samples.The corresponding limits of detection values(LODs)were found to be 1.93μg/kg in brown rice,2.18μg/kg in corn and 1.93μg/kg in rice,respectively.The recovery rates ranged from 96.2%to 104.8%with coefficients of variation ranging from 3.5%to 6.4%,showing good agreement with the results obtained by liquid chromatography–tandem mass spectrometry(LC–MS/MS).The proposed assay demonstrated robust analytical performance,with significant application potential for rapid,on-site detection.
基金supported by the National Nature Science Foundation of China(32472423)the Guangdong Basic and Applied Basic Research Foundation(2023A1515030011)+1 种基金the“Climbing Plan”Project of Guangdong College Students Science and Technology Innovation Cultivation Special Fund(pdih2023b0386)College Student Innovation and Entrepreneurship Training Program Project of South China Agricultural University(202310564023).
文摘Formaldehyde is a well-known carcinogen that can occur naturally or be illicitly added as an adulterant to food,calling for rapid and reliable detection methods.In this work,leveraging the facile and selective transformation of formaldehyde into 5-nitro-benzimidazole(5-nitro-BZM)using 4-nitro-o-phenylenediamine,two haptens were designed to target the BZM structure,leading to the production of a monoclonal antibody specifically recognizing BZM.Utilizing this antibody,a lateral flow immunoassay(LFIA)was developed for the detection of formaldehyde in grass carp,bighead carp,and basal shrimp,following a rapid pre-analysis derivatization.The detection limits were 0.15 mg/kg in grass carp,0.11 mg/kg in bighead carp,and 0.14 mg/kg in kiwi shrimp.The recoveries were in the range from 75.3%to 89.2%,and the LFIA results showed good correlation with those obtained from the HPLC.This marks the first report concerning an immunoassay for formaldehyde,offering a more rapid and cost-effective alternative for routine formaldehyde monitoring.
基金funded by the National Key R&D Program(2019YFC1604602)grants from Jiangsu Agriculture Science and Technology Innovation Fund(CX(21)2036)supported by the Fundamental Research Funds for the Central Universities(JUSRP622012).
文摘Carmoisine(CMS)is a synthetic azo colorant that can be used as a food colorant in products such as candy.However,due to potential health risks associated with carcinogenic,child development and allergic reactions,its use is strictly regulated.We have developed a colloidal gold immunochromatographic assay(CG-ICA)based on a highly sensitive monoclonal antibody(mAb)for the rapid and sensitive detection of CMS in food products.This study proposes a novel hapten and evaluates its structure through computer-aided analysis based on molecular structural similarity.Then,a mAb was successfully prepared,with a half-maximal inhibitory concentration of 0.20 ng/mL and a limit of detection(LOD)of 0.03 ng/mL.The CG-ICA strip demonstrated a LOD of 0.48–0.58μg/L for three soft drink types,achieving recovery rates between 90.78%and 109.61%and a coefficient of variation(CV)between 2.39%and 5.24%.The accuracy of the method was further validated by liquid chromatography–tandem mass spectrometry(LC–MS).Without requiring complicated purification steps,the simple operation and rapid result-yielding ability of CG-ICA within 20 min demonstrate its suitability for field rapid screening of CMS in soft drinks.The method developed in this study represents a new exploration for CMS detection and provides a direction for the rapid screening of similar colorants.
