Design and synthesis of an isopenicillin N synthase mimic

Towards the aim of creating a functional mimic of isopenicillin N synthase, a small molecule designed to coordinate around iron(II) and model the enzyme active site has been prepared in nine synthetic steps from 2,6-bis(hydroxymethyl)pyridine, (S)-(+)-mandelic acid and pivaldehyde. One aspartate, two histidines and a water ligand in the natural enzyme are replaced by an α-hydroxy acid, pyridine and aniline in the model compound. Additionally, a free thiol designed to simulate the enzyme substrate, δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine, is linked to the ligand by a three carbon chain. We postulate that in the presence of molecular oxygen, the complex formed between this synthetic ligand and iron(II) will display oxidative chemistry similar to that observed in the active site of isopenicillin N synthase.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide diazinon

Four haptens of the organophosphorus (OP) insecticide diazinon were synthesized to develop enzyme-linked immunosorbent assays (ELISAs) for this pesticide. One of them was conjugated to KLH to be used as the immunogen for production of monoclonal antibodies. By using the antibodies and a coating antigen, an indirect competitive ELISA was developed, which showed an IC₅₀ of 4.0 ng/mL with a detection limit of 0.7 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 6.0 ng/mL with a detection limit of 0.9 ng/mL. The antibodies in both assays showed negligible cross-reactivity with metabolites of diazinon and other OP pesticides. Recovery of diazinon from fortified lettuce and rice samples was satisfactory except at the fortified concentration of 100 ppb.     

Synthesis of haptens of organophosphorus pesticides and development of enzyme-linked immunosorbent assays for parathion-methyl

A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed. While the previous synthetic approach for this type of haptens requires seven steps, the present method involves only two steps. Using this method, four haptens of the OP insecticide parathion-methyl were synthesized. Rabbits were immunized with either one of the two haptens coupled to bovine serum albumin for production of polyclonal antibodies. Using the serum with the highest specificity, an antigen-coated ELISA was developed, which showed an IC₅₀ of 6.4 ng/ml with a detection limit of 0.2 ng/ml. An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 3.5 ng/ml with a detection limit of 0.3 ng/ml. The antibodies showed negligible cross-reactivity with other OP pesticides tested except with the insecticides parathion and paraoxon only in the antigen-coated ELISA.     

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I₅₀ of the assay ranged from 0.64 to 20.98 μg mL⁻¹ for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I₅₀ and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL⁻¹, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.     

A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N′N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC₅₀ values of 16 ng mL⁻¹ and 19 ng mL⁻¹, respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC₅₀ value for eight standard curves was in the range 12–18 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 ± 0.11 ng mL⁻¹. The ELISA tolerated 5% methanol without significant influence on IC₅₀ value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3–119.0% and the intra-assay relative standard deviation (RSD) was within 4.7–33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense. Figure Polyclonal antibody based ELISA for detection of olaquindox     

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.     

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.     

酶联免疫吸附分析法测定水产品及水中孔雀石绿和无色孔雀石绿

本文报道一种同时测定水产品及水样中孔雀石绿(MG)和无色孔雀石绿(LMG)的间接竞争酶联免疫吸附分析法。对无色孔雀石绿分子进行修饰,使其与载体蛋白交联,得到免疫原和包被抗原,经过多次免疫动物制得抗无色孔雀石绿的多克隆抗体。在优化的实验条件下,IC50值(标准曲线中吸光度抑制至最大吸光度值的50%时所对应的待测物浓度)为0.9~2.6μg/L,检出限为0.02~0.10μg/L,无色孔雀石绿在水样及水产品中的回收率为76.2~95.0%,与孔雀石绿的交叉反应率为95.25%。真实样品测定中,两种食用鱼养殖水样及一个鱼样中未检出孔雀石绿和无色孔雀石绿,但在观赏鱼养殖水样及另一鱼样中检出孔雀石绿和无色孔雀石,浓度分别为1.84μg/L和1.38μg/L。     

Determination of beta-conglycinin in soybean and soybean products using a sandwich enzyme-linked immunosorbent assay

Soybean protein has long been recognized as a source of dietary allergens for humans and animals with β-conglycinin being the major allergen. This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows for the detection of trace amount of β-conglycinin in soybean and soybean products. In the sandwich ELISA, mouse anti-β-conglycinin monoclonal antibody (Mab 5C5) was used as coating antibody, and rabbit anti-β-conglycinin polyclonal antibody (Pab) was used as secondary antibody. The assay showed high specificity for β-conglycinin with minimum cross-reactions with other soy proteins. The practical working range for the determination of β-conglycinin using the developed assay was 3–100 ng mL⁻¹ and the limit of determination (LOD) was 1.63 ng mL⁻¹. The recoveries of β-conglycinin in spiked soybean samples were between 88.1% and 106.6% with relative standard deviation less than 8.9% (intra-day) and 13.1% (inter-day). The developed method was used to analyze 469 soybean seed samples from different sources as well as five soybean products treated with different processing techniques. The data showed that the concentration of β-conglycinin decreased significantly after processing, especially for soybean protein isolation, where the concentration of β-conglycinin dropped to nearly zero. The assay provides a specific and sensitive method for the screening of β-conglycinin and allows for further investigation into hypersensitive mechanisms of soybean proteins and development of soybean processing techniques to reduce their negative effects.     

Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin

Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 μg mL⁻¹. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.     

Application of a modified enzyme-linked immunosorbent assay for 3-amino-2-oxazolidinone residue in aquatic animals

Furazolidone has been banned from use in food animals because of its carcinogenicity and mutagenicity, but its continued misuse is widespread in aquacultures. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in aquatic products. In this regard, we modified a simplified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to address this need. A good linearity was achieved over a concentration range of 0.05-12.15 μg L⁻¹, and the IC₅₀ value was 0.96 μg L⁻¹. The sample preparation was simple and effective included water bath treatments, acid hydrolysis combined with overnight derivatization of AOZ by benzaldehyde. The limit of detection and the limit of quantification were 0.15 and 0.3 μg kg⁻¹. The recoveries of AOZ in all tissues were between 78.0-95.3% at the levels of 0.3, 1.0, and 2.0 μg kg⁻¹. The inter-assay variability was less than 19.1%. The modified ic-ELISA was applied in quantification of AOZ elimination in carp. The results showed that AOZ was quite difficult to eliminate. Good correlations of the results obtained by ELISA and LC-MS/MS were observed in incurred carp muscle (r = 0.9923) and carp plasma (r = 0.9915) at the levels of 2.5-571.8 μg kg⁻¹ (μg L⁻¹). Better results were obtained by modified ic-ELISA when compared with commercial ELISA kit. Therefore, the present assay is considered a rapid, accurate, reliable, and inexpensive method for the detection of furazolidone-residues in the edible tissues of aquatic animals.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay

In this work, we report Escherichia coli O157:H7 detection using antibody-immobilized capillary reactors, enzyme-linked immunosorbent assay (ELISA), and a biochip system. ELISA selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody-immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system. The detection limits of E. coli O157:H7 using the ELISA and Cy5 label-based immunoassays were determined to be 3 and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.     

High performance enzyme-linked immunosorbent assay for determination of miroestrol,a potent phytoestrogen from Pueraria candollei

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73–3000 ng mL⁻¹, which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80–104.37% and 98.31–106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R² = 0.9996) (Yusakul et al. [18]) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695 ± 0.037–12.108 ± 0.285 μg g⁻¹ dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.     

Development and evaluation of a loop-mediated isothermal amplification method in conjunction with an enzyme-linked immunosorbent assay for specific detection of Salmonella serogroup D

Loop-mediated isothermal amplification in conjunction with enzyme-linked immunosorbent assay (LAMP-ELISA) provides a sensitive, specific and cost-effective method for detection of etiological causes of infections. The present study developed a reliable LAMP-ELISA diagnostic kit for identification of Salmonella serogroup D strains and evaluated its potential use in the detection of Salmonella serovars Enteritidis and Typhi. The LAMP-ELISA assay used a serogroup D/A-specific primer set to amplify a region of the prt gene, followed by hybridization of the digoxigenin-labeled products to a highly specific oligonucleotide probe for exact identification of serogroup D serovars. Among the bacteria tested, a positive reaction was only observed for strains belong to Salmonella serogroup D. The detection limit of the LAMP-ELISA assay was 4 CFU per tube, which was lower than PCR-ELISA assay with the same target gene (50 CFU per tube). Finally, the technique was successfully applied to an artificially contaminated meat sample with a detection limit 10³ CFU mL⁻¹, which was 10 times more sensitive than PCR-ELISA. Overall, the LAMP-ELISA assay could be used as a sensitive alternative method to PCR-ELISA for the specific detection of Salmonella serogroup D serovars in routine food microbiology or clinical laboratories worldwide.     

Detection of domoic acid in rat serum and brain by direct competitive enzyme-linked immunosorbent assay (cELISA)

In 1987 a large-scale incident of human poisoning in Canada was traced to commercial mussels contaminated with domoic acid (DOM). Since then, routine screening of shellfish domoic acid content has been carried out using a variety of assays, with liquid chromatography using ultraviolet absorbance detection (LC–UV) or mass spectrometric detection (LC–MS) being the currently accepted standard methodologies. Recently, a highly specific competitive enzyme-linked immunosorbent assay (cELISA) has been developed for the detection and analysis of DOM in commercial shellfish, but its accuracy relative to LC methods has not been independently verified in mammalian tissues. In this study we demonstrate that measurement of rat serum DOM concentration by cELISA gives a good correlation (r ²=0.993) across a broad range of concentrations when compared to LC–MS analysis, with only a small (15%) overestimation of sample DOM content. In addition, we have developed an extraction method for analysis of DOM in rat brain by cELISA which yields complete recovery across a range of sample dilutions.     

Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)–ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)–EDTA–BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)–EDTA–BSA–horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator–protein complexes such as EDTA–BSA and EDTA–BSA–HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.     

Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

A highly sensitive and selective immunoassay for the detection of tetrabromobisphenol A in soil and sediment

Tetrabromobisphenol A is the most widely used brominated flame retardant. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) for the detection of tetrabromobisphenol A was developed. The limit of detection and the inhibition half-maximum concentration of tetrabromobisphenol A in phosphate buffered saline with 10% methanol were 0.05 and 0.87 ng mL⁻¹, respectively. Cross-reactivity values of the ELISA with a set of important brominated flame retardants including tetrabromobisphenol A-bis(2,3-dibromopropylether), 2,2′,6,6′-tetrabromobisphenol A diallyl ether, hexabromocyclododecane, 1,2-bis(pentabromodiphenyl) ethane, 1,2-bis(2,4,6 tribromophenoxy) ethane, bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate, and polybrominated diphenyl ethers were <0.05%. Concentrations of tetrabromobisphenol A determined by ELISA in the soils from farmlands, the soils from an e-waste recycling site, and the sediments of a canal were in the range of non-detectable–5.6 ng g⁻¹, 26–104 ng g⁻¹ and 0.3–22 ng g⁻¹ dw, respectively, indicating the ubiquitous pollution of tetrabromobisphenol A. The results of this assay for 16 real world samples agreed well with those of the liquid chromatography–tandem mass spectrometry method, indicating this ELISA is suitable for screening of tetrabromobisphenol A in environmental matrices.