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 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
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
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
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
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
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
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
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
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
An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyl...An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol （DES-CP） and mono-O-carboxymethyldiethylstilbestrol （DES-CM） with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carder proteins bovine serum albumin （BSA） by mixed-anhydride method for immunogen and conjugated with ovalbumin （OVA） by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection （LLD） of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.展开更多
Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in ...Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.展开更多
In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid （CMPA） derived from penicillin. Results from tests with immune rabbits indicate that： （i） A n...In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid （CMPA） derived from penicillin. Results from tests with immune rabbits indicate that： （i） A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; （ii） the immunogenic antigen （CMPA-BSA） can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and （iii） antibodies in antisera showed higher affinity to OVA-GSH-CuC1 than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples.展开更多
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 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.
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.
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.
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.
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.
文摘An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol （DES-CP） and mono-O-carboxymethyldiethylstilbestrol （DES-CM） with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carder proteins bovine serum albumin （BSA） by mixed-anhydride method for immunogen and conjugated with ovalbumin （OVA） by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection （LLD） of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.
文摘Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.
基金supported by the National Natural Science Foundation of China (Grant No. 30821005)National High Technology Research and Development Program of China (Grant No. 2007AA10Z401)
文摘In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid （CMPA） derived from penicillin. Results from tests with immune rabbits indicate that： （i） A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; （ii） the immunogenic antigen （CMPA-BSA） can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and （iii） antibodies in antisera showed higher affinity to OVA-GSH-CuC1 than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples.
