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
Neu5Gc is a common sialic acid in mammals,but humans cannot synthesize it independently.When Neu5Gc in the diet enters the body,the body produces antibodies against Neu5Gc,which in turn promotes the immune response an...Neu5Gc is a common sialic acid in mammals,but humans cannot synthesize it independently.When Neu5Gc in the diet enters the body,the body produces antibodies against Neu5Gc,which in turn promotes the immune response and chronic inflammation and may be closely related to embryo transfer rejection.Therefore,Neu5Gc has gained widespread interest from gynecologists,nutritionists,and immunologists.The same problem occurs during embryo transfer,where the embryo carries paternal gene products and is a hapten relative to the maternal endometrium.Successful implantation requires a receptive endometrial environment.Moreover,with the widespread application of in vitro fertilization and embryo transfer technology,nonhuman amino acids may be introduced into the embryo culture medium,mainly the genetic engineering-derived essential serum albumin in embryo culture medium,which may introduce the nonhuman sialic acid Neu5Gc and cause embryo transfer rejection.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
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
High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Tr...High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.展开更多
The relative molecular mass of Arbutin is small. Both fluorolabeling and radiolabeling may affect its properties and functions. Therefore, the immunoassay of Arbutin was studied. Arbutin was coupled to bovine serum al...The relative molecular mass of Arbutin is small. Both fluorolabeling and radiolabeling may affect its properties and functions. Therefore, the immunoassay of Arbutin was studied. Arbutin was coupled to bovine serum albumin to get the Arbutin\|BSA conjugate with high molar ratio of Arbutin to BSA. Two rabbits were injected with the conjugate to develop the anti\|Arbutin serum. Ammonium sulfate precipitation and affinity chromatography were used to purify the antibody. Double agar diffusion test and enzyme\|linked immunosorbent assay (ELISA) were adopted to identify the antibody titer. The results demonstrated that the purity and activity of the antibody are high. The method proposed is satisfactory for the immunological detection of Arbutin.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supported by the National Natural Science Foundation of Fujian(nos.2023J011312,2023Y9052,and 2021J02036)Wu Jieping Medical Foundation Research Special Project(320.6750.2021-19-4).
文摘Neu5Gc is a common sialic acid in mammals,but humans cannot synthesize it independently.When Neu5Gc in the diet enters the body,the body produces antibodies against Neu5Gc,which in turn promotes the immune response and chronic inflammation and may be closely related to embryo transfer rejection.Therefore,Neu5Gc has gained widespread interest from gynecologists,nutritionists,and immunologists.The same problem occurs during embryo transfer,where the embryo carries paternal gene products and is a hapten relative to the maternal endometrium.Successful implantation requires a receptive endometrial environment.Moreover,with the widespread application of in vitro fertilization and embryo transfer technology,nonhuman amino acids may be introduced into the embryo culture medium,mainly the genetic engineering-derived essential serum albumin in embryo culture medium,which may introduce the nonhuman sialic acid Neu5Gc and cause embryo transfer rejection.
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.
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.
基金This work was supported by the National High Technology Research and Development Program of China(No.2006AA06Z411)China Postdoctoral Science Foundation(No.20100471297)Jiangsu Planned Projects for Postdoctoral Research Funds(No.0902065C).
文摘High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.
文摘The relative molecular mass of Arbutin is small. Both fluorolabeling and radiolabeling may affect its properties and functions. Therefore, the immunoassay of Arbutin was studied. Arbutin was coupled to bovine serum albumin to get the Arbutin\|BSA conjugate with high molar ratio of Arbutin to BSA. Two rabbits were injected with the conjugate to develop the anti\|Arbutin serum. Ammonium sulfate precipitation and affinity chromatography were used to purify the antibody. Double agar diffusion test and enzyme\|linked immunosorbent assay (ELISA) were adopted to identify the antibody titer. The results demonstrated that the purity and activity of the antibody are high. The method proposed is satisfactory for the immunological detection of Arbutin.
