Synthesis and Density Functional Theory(DFT) Calculation of Haptens for PAHs

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.

Design and efficient synthesis of novel haptens and complete antigens for the AOZ,a toxic metabolite of furazolidone

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.

Design and Synthesis of 6α-Corticosteroid Haptens and Their Bovine Serum Albumin (BSA) Conjugates

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.

Synthesis and Characterization of Specific Haptens Used to Generate Abzyme with Glutathione Peroxidase Activity

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.

Syntheses of haptens and hapten-protein conjugates for insecticide propoxur and cyhalothrin

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.

Staphylococcal enterotoxin B-derived haptens promote sensitization

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.

Synthesis of haptens and production of antibodies to bisphenol A

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.

Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

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.

A New Hapten for Immunoassay of Aldicarb

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.

Characterization of Hapten-Protein Conjugates for Immunoassay of Polycyclic Aromatic Hydrocarbons(PAHs)

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）.

Development of Anti-Isoproturon Polyclonal Antibody

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.

The Synthesis of a Novel Phosphorus Containing Antigen

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.

Studies on Increasing the Activity of Selenium-Containing Abzyme (Ⅰ)——Synthesis, Characterization and Properties of Hapten and Antigen

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.

Development of the Artificial Antigens for the Organophosphorus Insecticide Chlorpyrifos

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.

Hydrophobic Pocket Modification and Humanized Catalytic Antibodies with Glutathione Peroxidase Activity(Ⅰ)——Preparation and Characterization of Haptenes and Conjugates

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Rational hapten design and establishment of broad-spectrum indirect competitive enzyme-linked immunosorbent assay for benzimidazoles monitoring in milk

Objectives Benzimidazoles(BZs)are commonly used for the treatment of soil-transmitted helminth infections in veterinary clinics;however,misuse and overdosing of BZs will cause residual problems and have the potential to damage human health through the food chain.Thus,the existence of BZs in foods needs more attention.This study aims to establish a broad-spectrum immunoassay for rapid detection and to simultaneously monitor BZs in milk.Materials and Methods Based on structure analysis,a‘zero epitope loss’strategy,which introduced a spacer arm into the imino group of the imidazole ring of albendazole,was first adopted for hapten modification to obtain an ultra-sensitive and broad-spectrum antibody.An indirect competitive enzyme-linked immunosorbent assay(icELISA)was established for the detection of 18 BZs in milk sample with a single-step pretreatment.A quantitative structure–activity relationship model was constructed to interpret and predict the recognition.Results The antibody could recognize 20 BZs and the half-inhibitory concentrations ranged from 0.054 to 417.58 ng/mL,the limits of detection of icELISA ranged from 0.4 to 89.4 ng/mL,and the mean recovery rates ranged from 76.49%to 120.40%,with a coefficient of variation<20%.Substituent R1 of BZs was considered to be the main influencing factor for recognition,and the comparative molecular field analysis model(q2=0.724,r2=0.998)was finally chosen for further prediction.Conclusions The results indicated that the established icELISA could simultaneously identify 18 BZs,with good accuracy and precision,which was suitable for rapid detection of BZs in milk.

Hapten design to prepare monoclonal antibodies and establishment of immunoassay for direct screening of oxamichydrazide in chicken,the metabolite of nifuraldezone

In this study,two novel haptens of oxamichydrazide(OXZ),the metabolite of nifuraldezone,were designed and synthesized by derivatization with ethyl-4-bromobutanoate and ethyl-2-bromoacetate for the first time.The rationality of the haptens was enlightened by conformational alignment and electronic evaluations based on computional chemistry.The synthesized haptens were efficient in generating antibody response to OXZ.A monoclonal antibody(mAb)named 2D9 with high affinity was obtained and used to establish an indirect competitive enzyme-linked immunosorbent assay(icELISA)for the direct determination of OXZ in chicken samples without the need for derivatization step.Under optimum conditions,the developed icELISA showed an IC50 value of 0.66 ng mL^(−1),and limit of detection of 0.11μg kg^(−1)in chicken.The average recoveries were found to be 80.4-91.3%with a coefficient of variation less than 9.0%.In conclusion,we have successfully desigened haptens,prepared mAbs and developed an immunoassay for the direct determination of OXZ in chicken samples without the need for a derivatization step for the first time.This provides a potentially rapid,accurate,and sensitive screening tool for nifuraldezone residue in animal-derived food.

Preparation of a highly specific and sensitive monoclonal antibody against tilmicosin and its application in lateral flow immunoassay

To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues,it is crucial to prepare a high specificity and sensitivity antibody against target for developing an accurate immunoassay.In this study,tilmicosin(TM)was selected as a model molecule.Firstly,two-dimensional similarity,electrostatic potential energy,mulliken atomic charges and overlapping of different haptens with TM were calculated using Gaussian 09W and Discovery studio,and the newly designed TM-HS was selected as the optimal hapten.Furthermore,a monoclonal antibody(mAb 12C8)was produced with the half maximal inhibitory concentration(IC50)of 0.36 ng/mL,and negligible cross-reactivity(CR)with other antibiotics.Finally,a lateral flow immunoassay(LFA)for the detection of TM based on amorphous carbon nanoparticles(ACNPs)labeled mAb 12C8 was developed by the reflectance value under natural light.The recoveries of TM ranged from 83.18%to 103.25%with a coefficient of variation(CV)<12.47%.The results showed that the cut-off value of TM in milk samples was 1 ng/mL,and the limits of detection(LODs)for chicken muscle,bovine muscle,porcine muscle and porcine liver samples were 5.23,5.98,6.85 and 7.31μg/kg,respectively.In addition,40 real samples were tested by the LFA,and the detection results were consisted with that of high-performance liquid chromatography-UV detector(HPLC-UV).Those results indicated that the developed LFA is an accurate and useful tool for on-site screening of TM in milk and animal tissues.

Analysis on molecular interaction mechanism of four hapten flavonoids in Shuang-huang-lian powder injection with bovine serum albumin

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.

Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

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%.