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.     

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     

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.     

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.     

酶联免疫吸附分析法测定水中双氯芬酸钠

本研究报道一种高灵敏度的测定自来水和表面水中药物残留双氯芬酸钠含量的间接酶联吸附分析法(ELISA)。在包被抗原为20μg/L,抗体为1∶20000倍稀释,辣根过氧化物酶-羊抗兔IgG为1∶20000倍稀释的优化条件下,双氯芬酸钠的检出限(LOD)为0.004μg/L~0.008μg/L,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0.03μg/L~0.12μg/L。双氯芬酸钠在自来水中和表面水中的回收率分别为96%和113%。表面水存在一定的基体效应,但仅需要简单过滤稀释就可以消除。     

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。     

Biological monitoring of 3-phenoxybenzoic acid in urine by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6000 for the enzyme conjugate. Urine samples were hydrolyzed with concentrated hydrochloric acid; extracted with dichloromethane and solvent-exchanged into a methanol/buffer solution, prior to analysis in a 96-microwell plate immunoassay. Quantitative recoveries of 3-PBA were obtained for fortified urine samples by ELISA (92 ± 18%) as well as by gas chromatography/mass spectrometry (GC/MS) (90 ± 13%). The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. Analytical results from over one hundred urine samples showed that the ELISA and GC/MS data were highly correlated, with a correlation coefficient of 0.95. At the 10 ng/mL comparative concentration level, the false positive rate was 0% and the false negative rate was 0.8% for ELISA when using GC/MS as the reference method. The ELISA method has a suitable low detection limit for 3-PBA to assess pyrethroid exposures in non-occupational settings.     

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.     

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.     

蓖麻毒素的间接酶联免疫快速检测

建立了间接酶联免疫法快速检测蓖麻毒素的方法。方法的线性范围在0.08~1.25 mg/L之间,相关系数r=0.992 3,检出限为0.02 mg/L。将该法用于检测实际水样、土壤、奶粉和血液蓖麻毒素加标样品,回收率在60%~98%范围。该方法简单、快速、假阳性率低,非常适合蓖麻毒素的快速筛选、定性和半定量分析。     

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 of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K_aff) of 7.3 × 10⁷ L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2 μg/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000 ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06 ng/mL, respectively.     

酶联免疫吸附分析法测定环境水样中痕量药物吲哚美辛

将吲哚美辛与蛋白质载体结合,制成完全免疫抗原,经过多次动物免疫得到了性能优良的兔抗吲哚美辛抗体,在优化实验条件的基础上,建立了灵敏度高、特异性强、简便、稳定的测定水样中吲哚美辛的酶联免疫吸附分析方法(ELISA).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0.09μg/L～0.17 μg/L,最低检出限为 0.005μg/L～0.01 μg/L.真实水样中,均发现含有吲哚美辛,浓度在0.016 μg/L~0.083 μg/L之间,水样的加标回收率在84.4%～127.0%之间.     

Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay optimization and investigation of matrix effects from different food samples

In this study, a panel of haptens was synthesized for immunoconjugate preparation, and several haptens for heterologous tracer conjugates were also prepared. A highly sensitive polyclonal antibody against the organophosphorus insecticide phosmet was obtained and competitive direct enzyme-linked immunosorbent assays (cd-ELISA) for this pesticide were developed. In the cd-ELISA, the limit of detection (IC₁₅) was 0.6 μg kg⁻¹ and the sensitivity (IC₅₀) was 20 μg kg⁻¹. The suitability of the ELISA for pesticide quantification in peach, apple, orange juice, and apple juice was also studied. Good accuracy and precision were obtained with mean recoveries between 78% and 102.3% and mean coefficients of variation below 13.63%. Validation of the ELISA was conducted by high-performance liquid chromatography. The correlation between the data obtained using the microwell assay and the high-performance liquid chromatography was good (R ² = 0.9849). The developed immunoassay methods were suitable for the rapid quantitative or qualitative determination of phosmet in food samples.     

Determination of the insecticide fenoxycarb in apple leaf samples by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was used for the determination of fenoxycarb in apple leaf samples. Single step extraction procedures with phosphate-citrate buffered solution containing different amounts of methanol were tested showing that a solvent percentage of 20% (v/v) was the best condition, with recoveries between 85 and 100% in the working range of 25-500 μg kg⁻¹ and a negligible matrix effect. The low detection limit reached, 1 μg kg⁻¹ against 50 μg kg⁻¹ for the recommended liquid chromatographic method, makes the ELISA more suitable for determinations of the fenoxycarb residues in apple leaf samples. The reliability of the ELISA was evaluated by assaying the insecticide in spiked and contaminated samples by three different approaches: direct determination, standard addition method with a calibration graph, and the dilution test. The corresponding coefficients of variation were, respectively, 11, 22 and 27%. The direct determination on the (1+1) diluted apple leaf extract was used to measure the insecticide residues in samples collected in the north-eastern Italian regions of Veneto and Trentino-Alto Adige.     

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.     

Development and validation of a highly sensitive ELISA for the determination of pharmaceutical indomethacin in water samples

The development and validation of a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of pharmaceutical indomethacin in water samples was presented. The immunogen and coating antigen were prepared by covalently linking indomethacin to bovine serum albumin and ovalbumin by anhydride ester method. Two rabbits were immunized by standard immunization processes and the superior antibody was characterized in terms of sensitivity, specificity, precision, accuracy and stability. Under optimal experimental conditions, the standard curve was constructed in the concentration range of 0.01-10 ng/mL. For 10 consecutive standard curves run in 2 weeks, IC₅₀ value (the concentration of analyte producing 50% of inhibition) were found within 0.10-0.25 ng/mL, and the detection limit (DL) at a signal-to-noise ratio of 3 (S/N = 3) was about 0.01 ng/mL. The antiserum recognized acemetacin, a precursor of indomethacin with 92.3% cross-reactivity, while the cross-reactivity values of antiserum with other tested compounds were very low. From the spiking experiments, the recoveries were found within 98-123%. The ELISA was applied for the determination of indomethacin in different water samples and the results were confirmed by conventional HPLC. The correlation coefficient of 0.988 was obtained, demonstrating a good correlation of ELISA with HPLC.     

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.     

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.     

玉米赤霉醇人工抗原合成及其多克隆抗体的制备

对玉米赤霉醇(ZER)16-OH进行改造,设计合成了模拟玉米赤霉醇特征结构的半抗原———ZER-16-羧丙基丁醚。用混合酸酐法将其与载体蛋白BSA连接,用于免疫新西兰大白兔,制备的抗血清效价达1∶102 400。基于活性酯法合成的包被抗原建立了CI-ELISA检测方法,该法对玉米赤霉醇检测的线性范围为18.39~1744.70 ng/mL,检出限为8.61 ng/mL。