Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion

Five haptens with different spacer-arm attachment sites on the structure of the organophosphorus insecticide fenthion were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and three haptens containing all or most of the structure of fenthion were conjugated with bovine serum albumin (BSA) for the immunogen. Six polyclonal antisera were raised against the three BSA conjugates, and 30 antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for fenthion. The study revealed the best combination with high sensitivity (I₅₀ of 0.08 ng mL⁻¹) and high assay specificity, which indicated that when structural difference between the analyte and an immunizing hapten is less than that between a coating hapten and the immunizing hapten, a high sensitive enzyme-linked immunosorbent assay (ELISA) in the heterologous system may stand a good chance to be developed. The immunity results showed that heterology in the hapten spacer-arm attachment site of the immunogen could achieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody, and that the moiety of an analyte, which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.     

Hapten synthesis and antibody production for the development of a melamine immunoassay

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by ¹H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC₅₀ of 70.6 ng mL⁻¹, a LOD of 2.6 ng mL⁻¹ and a LOQ of 7.6 ng mL⁻¹. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.     

Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity

A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion and fenthion. Three haptens with different spacer-arms were synthesized. The haptens were conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens. Rabbits were immunized with the immunogens and six polyclonal antisera were produced and screened against each of the coating antigens using competitive indirect enzyme-linked immunosorbent assay for selecting the proper antiserum. The effect of hapten heterology on immunoassay sensitivity was also studied. The antibody-antigen combination with the most selectivity for malathion was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC₅₀ values, under optimum conditions, were estimated to be 30.1 μg L⁻¹for malathion, 28.9 μg L⁻¹ for dimethoate, 88.3 μg L⁻¹ for phenthoate, 159.7 μg L⁻¹ for phosmet, 191.7 μg L⁻¹ for methidathion, 324.0 μg L⁻¹ for fenitrothion, 483.9 μg L⁻¹ for methyl parathion, and 788.9 μg L⁻¹ for fenthion. Recoveries of malathion, dimethoate, phenthoate, phosmet and methidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%. This assay can be used in monitoring studies for the multi-residue determination of O,O-dimethyl organophosphorus pesticides.     

Hapten design and indirect competitive immunoassay for parathion determination: correlation with molecular modeling and principal component analysis

A novel procedure for parathion hapten design is described. The optimal antigen for parathion was selected after molecular modeling studies of six types of potentially immunizing haptens with the aim to identify the best mimicking target analyte. Heterologous competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed after screening a battery of competitors as coating antigens. The relationship between the heterology degree of the competitor and the resulting immunoassay detectability was investigated according to the electronic similarities of the competitor haptens and the target analyte. Molecular modeling and principal component analysis were performed to understand the electronic distribution and steric parameters of the haptens at their minimum energetic levels. The results suggested that the competitors should have a high heterology to produce assays with good detectability values. An indirect competitive ELISA was finally selected for further investigation. The immunoassay had an IC₅₀ value of 4.79 ng mL⁻¹ and a limit of detection of 0.31 ng mL⁻¹. There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%.     

Preparation of a multi-hapten antigen and broad specificity polyclonal antibodies for a multiple pesticide immunoassay

A multi-determinant artificial antigen was prepared by haptens of four pesticides (chlorpyrifos, triazophos, carbofuran and parathion methyl) conjugating to the carrier protein BSA in turn. Male New Zealand white rabbits were immunized with this multi-determinant immunogen to produce the polyclonal antibodies (PAbs), which can recognize the four pesticides. The PAbs displayed high level for each relative hapten-OVA conjugate, with the favorable titers of 4.49 × 10⁴, 8.98 × 10⁴, 2.24 × 10⁴ and 1.86 × 10⁴, for CHBu-OVA, THHe-OVA, BFNB-OVA and MP5-OVA, respectively. Characterization studies of the PcAbs showed that it has high affinity and specificity to the four relative pesticides. An indirect competitive ELISA was developed for multi-residue determination. The I₅₀ value for the four pesticides was 0.290, 0.065, 0.582 and 2.824 μg mL⁻¹, with the detection limit (I₁₀) of 0.022, 0.005, 0.015 and 0.115 μg mL⁻¹ for carbofuran, triazophos, chlorpyrifos and parathion methyl, respectively. The linear rang was 0.016-2.000, 0.005-0.500, 0.010-2.000 and 0.063-5.000 μg mL⁻¹, respectively, for carbofuran, triazophos, chlorpyrifos and parathion methyl. Results indicated that, this study provided a new strategy to develop immunoassays through artificial antigen design for pesticides multi-residue determination.     

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO₂ groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC₅₀ values for atrazine and 2,4-D equal to 0.8 ng mL⁻¹ and 7 ng mL⁻¹, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL⁻¹ respectively in the optimum working range between 0.01 and 1000 ng mL⁻¹ with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.     

Immunoassay based on a polyclonal antibody for sex steroid hormones produced by a heterogeneous hapten-conjugated immunogen: Estimation of its potentiality and antibody characteristics

A method in which antibodies are produced by using an immunogen heterogeneously conjugated with two or more kinds of haptens having unlike chemical structures against a same carrier protein was offered as an efficient approach for development of antibody to low molecular compounds. To appreciate the potentiality of the approach, 17β-estradiol (E2) and testosterone were selected as model compounds. The I₅₀ values of antiserum developed were 6 and 8 μg L⁻¹ with the detection limits of 0.02 and 0.15 μg L⁻¹ for E2 and testosterone, respectively. Antiserum owned an interesting characteristic that it was possible to independently analyze E2 and testosterone without mutual interference by making proper use of coating antigens. When using β-estradiol 17-hemisuccuinate (EH) conjugated with bovine serum albumin (BSA) as a coating antigen, the enzyme-linked immunosorbent assay (ELISA) was very selective to E2 and some estrogen analogues. Therefore, if testosterone coexisted in the ELISA for E2 detection, it showed no interference with it. From these findings, it was suggested that the verified method was an efficient and rational approach in development of polyclonal antibody to low molecular compounds.     

Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC₅₀) was 348 ng mL⁻¹ for parathion, 13 ng mL⁻¹ for coumaphos, 22 ng mL⁻¹ for quinalphos, 35 ng mL⁻¹ for triazophos, 751 ng mL⁻¹ for phorate, 850 ng mL⁻¹ for dichlofenthion, and 1301 ng mL⁻¹ for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure-activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, E_HOMO, and energy of the lowest unoccupied molecular orbital, E_LUMO) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC₅₀) = −63.274E_HOMO + 15.985E_LUMO + 0.556 log P − 25.015, with a determination coefficient (r²) of 0.908.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples

The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ng mL⁻¹. The values of IC₅₀ for nine standard curves were in the range of 1.1-2.0 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.07-0.14 ng mL⁻¹. The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀ value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n = 9). The mAb-based ELISA proven to be a feasible quantitative/screening method for Sudan I analysis in food samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput and low expense.     

Enzyme-linked immunosorbent assays for the insecticide fenitrothion. Influence of hapten conformation and sample matrix on assay performance

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC₅₀ of 14 ng mL⁻¹ with a detection limit of 3.0 ng mL⁻¹. A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 17 ng mL⁻¹ with a detection limit of 1.6 ng mL⁻¹. The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Development of a Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Analysis of the Organophosphorous Pesticide Fenthion in Real Samples Based on Monoclonal Antibody

A new specific monoclonal antibody against the organophosphorous pesticide fenthion was produced based on our previous study. A sensitive and specific Enzyme-Linked Immunosorbent Assay (ELISA) for fenthion based on the new immunizing/coating hapten combination was developed. In this study, the H₁ which attempts to expose the aromatic ring group was conjugated with bovine serum albumin (BSA) for the immunogen. All of the haptens that have been synthesized were conjugated with ovalbumin (OVA) for the coating antigen. The efficient antibody/coating conjugated combinations were selected, and the optimized ELISA was developed. In the optimized system, the IC₅₀ value was 5.8 ng · mL^?1 with the detection limit (IC₂₀) of 0.028 ng · mL^?1. The cross reactivity with all of the tested pesticides was lower than the 0.5%. Also, the system can detect the fenthion addition to the real samples such as soil, water, rice, and Chinese cabbages.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

Facile, sensitive and selective fluorescence turn-on detection of HSA/BSA in aqueous solution utilizing 2,4-dihydroxyl-3-iodo salicylaldehyde azine

A novel fluorescence turn-on detection method of human serum albumin (HSA) and bovine serum albumin (BSA) in aqueous solution is investigated using 2,4-dihydroxyl-3-iodo salicylaldehyde azine (DISA). Upon the addition of DISA to HSA/BSA solution, a fluorescence turn-on effect at 529 nm can be observed with a large stokes shift of ∼129 nm based on hydrophobic binding-mode between protein and dye. Under the optimal condition, the linear ranges of fluorescence intensity for HSA and BSA are 0.1-30 μg mL⁻¹ with the relative correlation coefficient of R² = 0.991 (n = 10) and 0.3-50 μg mL⁻¹ with R² = 0.997 (n = 10); and the detection limits for HSA and BSA based on IUPAC (C_DL = 3S_b/m) are 20 ng mL⁻¹ and 50 ng mL⁻¹, respectively.     

Indirect competitive immunoassay for trichlorophenol determination: Rational evaluation of the competitor heterology effect

An improved competitive indirect immunoassay for the detection of 2,4,6-trichlorophenol (2,4,6-TCP) has been developed and optimized by preparing heterologous haptens that have been evaluated as coating antigens. The relation between the degree of heterology and immunoassay detectability has been investigated according to the geometric and electronic distribution similarities between the haptens and the analyte using molecular modeling tools. The assay has been characterized according to different physicochemical parameters such as the incubation time, the ionic strength, the effect of detergents and the pH. The resulting assay has an IC₅₀ of 1.44 μg l⁻¹ and a limit of detection (LOD) of 0.2 μg l⁻¹ and it shows a good accuracy and suitability to analyze trichlorophenol in drinking water.     

Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays

Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4′-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG₁ secondary antibody with ABTS as a chromogenic substrate. For X the IC₅₀ value derived from the standard curve was 62.91 ng mL⁻¹, and for both IX and 8-PN 37.15 ng mL⁻¹. The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.     

Biotin-avidin amplified enzyme-linked immunosorbent assay for determination of isoflavone daidzein

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed and optimized for the determination of a weakly estrogenic isoflavone daidzein in serum, urine and Puerariae radix. Specific polyclonal antibody was produced against daidzein by immunization of rabbits with a conjugate of 7-O-(carboxymethyl)-daidzein and bovine serum albumin (BSA). The polyclonal antibody showed specific recognition of daidzein, while cross-reactivities to coumarin, 4-hydroxycoumarin, phenol, and other isoflavones such as puerarin and rutin were all lower than 1%. The linear range of daidzein calibration curve was 0.1-1000 ng mL⁻¹. The detection limit was found to be 0.04 ng mL⁻¹, and the intra-assay and inter-assay coefficients of variation were 7 and 16%, respectively. Human serum and urine samples were spiked with known amounts of daidzein and measured by the established BA-ELISA. Recoveries were between 91 and 107%. Daidzein in P. radix was determined by the BA-ELISA method and HPLC method, and the content of daidzein was determined to be 0.0219 and 0.0194%, respectively. The results indicated that there was a good agreement between the two methods. The established method is very useful for monitoring daidzein in biological samples and traditional Chinese medicine.     

Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of carbamate pesticides in water samples by high performance liquid chromatography

A novel ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) coupled with high performance liquid chromatography-diode array detection has been developed for the extraction and determination of six carbamate pesticides (metolcarb, carbofuran, carbaryl, pirimicarb, isoprocarb and diethofencarb) in water samples. In the UASEME technique, Tween 20 was used as emulsifier, and chlorobenzene and chloroform were used as dual extraction solvent without using any organic dispersive solvent that is normally required in the previously described common dispersive liquid–liquid microextraction method. Parameters that affect the extraction efficiency, such as the kind and volume of the extraction solvent, the type and concentration of the surfactant, ultrasound emulsification time and salt addition, were investigated and optimized for the method. Under the optimum conditions, the enrichment factors were in the range between 170 and 246. The limits of detection of the method were 0.1–0.3 ng mL⁻¹ and the limits of quantification were between 0.3 and 0.9 ng mL⁻¹, depending on the compounds. The linearity of the method was obtained in the range of 0.3–200 ng mL⁻¹ for metolcarb, carbaryl, pirimicarb, and diethofencarb, 0.6–200 ng mL⁻¹ for carbofuran, and 0.9–200 ng mL⁻¹ for isoprocarb, with the correlation coefficients (r) ranging from 0.9982 to 0.9998. The relative standard deviations varied from 3.2 to 4.8% (n = 5). The recoveries of the method for the six carbamates from water samples at spiking levels of 1.0, 10.0, 50.0 and 100.0 ng mL⁻¹ were ranged from 81.0 to 97.5%. The proposed UASEME technique has demonstrated to be simple, practical and environmentally friendly for the determination of carbamates residues in river, reservoir and well water samples.     

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA–AZb6, which displayed an IC₅₀ value of 0.102 μg L⁻¹ and a LOD of 0.017 μg L⁻¹, was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L⁻¹.     

An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates

Based on direct hapten coated format a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for bisphenol A (BPA) was developed. Polystyrene surface was modified by 3-Aminopropyltriethoxysilane (APTES) to produce amino groups after H₂SO₄/HNO₃-pre-treatment. 4,4-bis (4-hydroxyphenyl) valeric acid (BVA) which is analogue of BPA, was successfully immobilized on the surface of microtiter plates by N,N′-dicyclohexylcarbodiimide (DCC) method. The essential steps of the assay were optimized, especially blocking procedure which is key step to prevent unspecific binding of antibody. The results indicated that compared with hapten-protein coated format (IC₅₀ = 176.67 ng ml⁻¹, LOD = 15.90 ng ml⁻¹), the direct hapten coated format (IC₅₀ = 23.50 ng ml⁻¹, LOD = 0.27 ng ml⁻¹) could improve assay sensitivity and the detection ranges were 2.30 ng∼157.60 ng ml⁻¹ with good signal reproducibility (P value > 0.05) after careful optimization of assay conditions. Tap water samples and seawater samples were spiked with a known amount of BPA and measured by ciELISA. The average recoveries were between 70 and 142%. As far as we are aware this is the most sensitive ELISA for BPA yet reported.