Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran

Nanocolloidal gold particles were prepared and labeled to an anti-carbofuran monoclonal antibody (Mab). This conjugate was dispensed on the conjugated pad of a porous glass fiber. Ovalbumin (OVA)-carbofuran and goat anti-mouse IgG were dispensed on the nitrocellulose (NC) membrane and served as the test line and control line, respectively. The carbofuran-containing sample migrated to the NC membrane and reacted with the anti-carbofuran Mab labeled with the colloidal gold. The mixture diffused along the membrane and passed through the OVA-carbofuran in the test line via capillary action. The more analyte present in the sample, the more effectively it will compete with the carbofuran immobilized on the test line for binding to the limited amount of antibody labeled with colloidal gold. An adequate amount of carbofuran could prevent attachment of the colored conjugate to the test line. The presence or absence of a colored band on the test line could indicate a negative or positive result, respectively. When measured to the water sample spiked with carbofuran, this was obtained at or above 0.25 mg/L of carbofuran. The major advantages of the one-step strip test are that the detection time needed was <10 min and all of the reagents are included in the test device.     

Development of an immunochromatographic lateral-flow test strip for rapid detection of sulfonamides in eggs and chicken muscles

A rapid immunochromatographic lateral-flow test strip was developed in the competitive reaction format for the detection of sulfonamides in eggs and chicken muscle. A monoclonal antibody against the common structure of sulfonamides was conjugated to colloidal gold particles as the detection reagent and an N-sulfanilyl-4-aminobenzoic acid (SUL)-bovine serum albumin (BSA) conjugate was immobilized to a nitrocellulose membrane as the capture reagent to prepare the test strip. With this method, it required only 15 min to accomplish the semiquantitative or quantitative detection of sulfonamides. The sensitivity to sulfonamides (sulfamonomethoxine, sulfamethoxydiazine, sulfadimethoxine, and sulfadiazine) was at least 10 ng/mL, as determined with an optical density scanner. By eye measurement, the sensitivity was 20 ng/mL for sulfamonomethoxine, sulfamethoxydiazine, and sulfadimethoxine and 40 ng/mL for sulfadiazine. On the basis of a sulfamonomethoxine standard curve, recoveries were from 89.5 to 95.6% for sulfamonomethoxine, from 89.5 to 95.1% for sulfamethoxydiazine, from 85.0 to 95.6% for sulfadimethoxine, and from 44.8 to 60.9% for sulfadiazine in egg and chicken muscle samples. A parallel analysis of 27 egg samples and 28 chicken muscle samples from the animal experiment showed that the differences between test strips and high-performance liquid chromatography (HPLC) were from 0.8 to 11.2% for egg samples and from 2.2 to 34% for chicken muscle samples for the quantitative detection, and the agreement rates between test strips and HPLC were 100%, based on the maximum allowed residue level of sulfadiazine (100 ng/g) established by the European Union and China. In conclusion, the method is rapid and accurate for the detection of sulfonamides in eggs and chicken muscles.     

Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection

A HPLC method using a modified sample preparation procedure was optimized and validated for the quantification of 10 quinolones (QNs), including marbofloxacin, ciprofloxacin, norfloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine, in swine, chicken, and shrimp tissues. In this method, only a small mass (相似文献   

Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA)

A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect a broad range of (fluoro)quinolones in various matrices. In the optimized generic test, anti-sarafloxacin antibodies in combination with norfloxacin conjugate showed 50% binding inhibition at 0.21 ng mL(-)(1) for sarafloxacin in buffer. Screening for this class of antibiotics is accomplished using a simple, rapid extraction carried out with a 1:1 mixture of methanol and phosphate-buffered saline adjusted to pH 7.4. This common extraction was able to detect 15 (fluoro)quinolone residues such as sarafloxacin, norfloxacin, difloxacin, ciprofloxacin, pefloxacin, ofloxacin, cinoxacin, danofloxacin, enrofloxacin, marbofloxacin, lomefloxacin, enoxacin, flumequine, oxolinic acid, and nalidixic acid in pig kidney, poultry muscle, egg, fish, and shrimp. The assay's detection capabilities (CCbeta) for most of these compounds were <10 microg kg(-)(1) except for the sarafloxacin-, oxolinic acid-, flumequine-, and cinoxacin-spiked matrices, the estimated CCbeta values of which were <4, <25, <100, and <200 microg kg(-)(1), respectively.     

Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect ciprofloxacin (CPFX) in food animal edible tissues. CPFX was converted by an active ester method into conjugates CPFX-bovine serum albumin (CPFX-BSA) and CPFX-human serum albumin (CPFX-HSA), which both allowed production of CPFX-specific rabbit antisera. In the ELISA, CPFX-HSA was coated onto the microtiter plate, followed by incubation with standard CPFX and anti-CPFX antibody. The indirect competitive ELISA revealed that the antisera have no cross-reactivity with penicillin, gentamicin, neomycin, sulfadiazine, and chlortetracycline. The antisera cross-reacted with enrofloxacin and norfloxacin about 69.8 and 44.6% as much as they did with CPFX. This ELISA was highly sensitive (0.32 ng/mL) to CPFX determination. Recovery of CPFX at 40 microg/kg was 75.58% in pork, 81.29% in chicken, and 84.30% in milk. The coefficients of variation varied from 3.7 to 9.2% over the range of CPFX concentrations studied. The linear detection range was between 1.6 and 1000 ng/mL. The results suggest that this ELISA is a specific, accurate, and convenient method for the detection of CPFX residues in food animal edible tissues.     

Application of quantum dot-antibody conjugates for detection of sulfamethazine residue in chicken muscle tissue

A new indirect competitive fluorescence-linked immunosorbent assay (cFLISA) method for the detection of sulfamethazine (SM2) in chicken muscle tissue was demonstrated using quantum dots (QDs) as the fluorescence label coupled with secondary antibody. The sensitivity of the cFLISA was compared with that of the HPLC method. The 50% inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 7.7 and 1.0 ng/mL, respectively. When SM2 was spiked at levels of 50, 100, and 200 ng/g, recoveries ranged from 80.6% to 117.4%, with a coefficient of variation of 6.9-9.6%. In the incurred sample analysis, the amounts of SM2 quantified by cFLISA were similar to the results obtained by the HPLC method. This study shows that cFLISA could be used as a screening method in practice.     

Portable surface plasmon resonance immunosensor for the detection of fluoroquinolone antibiotic residues in milk

An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 μg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 μg L(-1) and a LOD of 2.0 ± 0.2 μg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestle? Co.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl

Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase.     

Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the organophosphorus insecticide acephate, O,S-dimethyl acetylphosphoramidothioate, was developed using a polyclonal antibody. Five different haptens mimicking the analyte were synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) by the N-hydroxysuccinimide active ester and diazotization methods. Polyclonal antibodies raised against hapten-KLH conjugates in rabbits and hapten-BSA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems. The effects of various assay conditions such as detergent, organic solvents, pH, and preincubation of the mixture of the polyclonal antibody and the analyte on the sensitivity were evaluated. The IC(50) value for acephate was 25 ng/mL in an optimized heterologous system using hapten-4-BSA as a coating antigen and a polyclonal antibody no. 8377 against hapten-1-KLH, showing the detection range of 5-140 ng/mL and the lowest detection limit of 2 ng/mL. The cross-reactivities of the structurally related organophosphorus insecticides, including the major metabolite of the analyte, methamidophos, were less than 1%. Recoveries from the analyte-fortified tap water, mulberry leaves, and lettuce samples in the assay were in the range of 72-121% by simple extraction, concentration, and dilution. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring acephate residues in environmental and agricultural samples.     

Immunochromatography using colloidal gold-antibody probe for the detection of atrazine in water samples

An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.     

早期香蕉枯萎病Foc4双探针核酸纸基检测传感器研制

为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4, Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器。该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限。基于目标序列与双探针体系的碱基互补配对形成“金标探针-目标序列-T线探针”复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测。试验结果表明,双探针纸基传感器的检测限达到0.001 nmol/L,是传统纸基传感器的100倍,提高了检测灵敏度;在0.001～1000 nmol/L范围内,Foc4浓度与测试线光强度峰面积呈线性关系;使用高浓度非互补探针进行试验干扰,发现非互补序列对检测效果基本无影响,表明该传感器具有较好的特异性;香蕉叶片Foc4检测的平均回收率为77.6%～102.3%,相对标准偏差为7.4%～7.7%。与传统形态学观察等检测方法相比,该研究提出的双探针核酸纸基检测传感器可以及时、快速、准确地判断早期Foc4的存在,具有良好的推广性和实际应用价值。     

Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver

Pefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a labor-intensive instrumental method to detect the residue of pefloxacin in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen cationized bovine serum albumin-pefloxacin showed high sensitivity toward pefloxacin with an IC50 value of 6.7 ppb in buffer and was suitable for a screening assay to detect the residue of pefloxacin in food products. The antibody has been assessed using rapid enzyme immunoassays to exploit its specificity. The antibody prepared shows cross-reactivity with a few other (fluoro)quinolones including fleroxacin (116%), enrofloxacin (88%), and ofloxacin (10%). The assay measured drug residue in chicken liver spiked with pefloxacin with an interassay coefficient of variation of 13.6% or less and an intra-assay coefficient of variation of 10.9% or less. The average recovery rates at 0.5, 5, 10, 50, and 100 ppb were in the range of 86-106% for interassay and in the range of 87-103% for intra-assay, respectively.     

利用侧流核酸试纸条快速检测非洲猪瘟病毒

为满足基层普通实验室或养殖场等资源有限的实验室对非洲猪瘟病毒(African swine fever virus,ASFV)的检测需求,该研究开发了一种简单、快速、低成本的检测技术,可以用肉眼观察检测结果。研究将传统聚合酶链式反应(polymerase chain reaction,PCR)与胶体金试纸条技术结合,开发一种低成本的侧流核酸测定试纸条(lateral flow nucleic acid assay,LFNAA)。该体系设计了独特的尾引物,避免了传统核酸试纸条的半抗原标记及抗体的使用。经过PCR扩增后产生一端带着单链寡核苷酸尾巴的双链DNA产物,能够与胶体金标记的寡核苷酸捕获探针结合,从而在试纸条上形成可以用肉眼观察的目标产物。该LFNAA试纸条能够在猪瘟病毒(Classical swine fever virus,CFSV)、猪瘟病毒猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒1型(Porcine circovirus 1,PCV1)、猪圆环病毒2型(Porcine circovirus 2,PCV2)、猪伪狂犬病毒(Pseudorabies virus,PRV)、猪细小病毒(Porcine parvovirus virus,PPV)中特异的鉴定出ASFV的存在,其灵敏度与琼脂糖凝胶电泳的分析结果一致,均能达到103 copies/μL。因此,仅需要一台普通PCR仪,即可对ASFV进行快速灵敏的鉴定(<2 h),其低成本、操作简便的特点非常适合资源有限的实验室中非专业人员的操作。此外,该技术可进一步结合等温扩增技术,能够在食品安全和医学诊断中实现更快更简便的现场检测。     

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycin in Swine tissues

A monoclonal antibody (Mab) was produced by using the kanamycin-glutaraldehyde-bovine serum albumin (Kan-GDA-BSA) conjugate as the immunogen. The anti-Kan Mab exhibited high cross-reactivity with tobramycin (Tob) and slight or negligible cross-reactivity with other aminoglycosides. The specificity and cross-reactivity of this Mab are discussed regarding the three-dimensional, computer-generated molecular models of the aminoglycosides. Using this Mab, a rapid enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based strip test for Kan and Tob were developed. The rapid ELISA showed a 50% inhibition value (IC 50) of 0.83 ng/mL for Kan and 0.89 ng/mL for Tob with the analysis time less than 40 min, and the recoveries from spiked swine tissues at levels of 25-200 microg/kg ranged from 52% to 96% for Kan and 61% to 86% for Tob. In contrast, the strip test for Kan or Tob had a visual detection limit of 5 ng/mL in PBS, 50 microg/kg in meat or liver, and 100 microg/kg in kidney, and the results can be judged within 5-10 min. Observed positive samples judged by the strip test can be further quantitated by ELISA, hence the two assays can complement each other for rapid detection of residual Kan and Tob in swine tissues. Moreover, physical-chemical factors that affected the ELISA and strip test performance were also investigated. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was determined followed by a theoretical explanation of the effects.     

Molecular and immunochemical characteristics of monoclonal and recombinant antibodies selective for the triazine herbicide simetryn and application to environmental analysis

A monoclonal antibody (mab) selective for the thiomethyl-s-triazine herbicide simetryn was obtained and characterized in enzyme-linked immunosorbent assay (ELISA). An IC(50) value for simetryn was 8.5 ng/mL, and the detection range extended from 1.1 to 70 ng/mL in ELISA. The cDNAs encoding variable heavy chain (VH) and variable light chain (VL) of the mab were cloned to produce various recombinant antibodies. Single-chain variable fragment (scFv) antibodies derived from the mab were characterized in ELISA and showed similar reactivities and specificities to the parent mab. A urea denaturation test revealed that the scFv antibodies bound to simetryn were more stable than those in the absence of antigen. A sandwich ELISA based on VH and VL fragments of the mab was successfully developed and showed similar sensitivity to those based on the mab and scFv antibodies in ELISA. In the recovery experiments using spiked environmental samples, the results obtained in ELISA based on the mab were favorably correlated with those by HPLC.     

Development of ELISA and immunochromatographic assay for the detection of gentamicin

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.     

Development of monoclonal antibodies against pirimiphos-methyl and their application to IC-ELISA

To detect the organophosphorus (OP) pesticide pirimiphos-methyl in grain samples, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed and optimized. By the active esters method, pirimiphos-methyl hapten A was conjugated to keyhole limpet hemocyanin to be used as the immunogen for the production of monoclonal antibodies, and pirimiphos-methyl hapten B was conjugated to ovalbumin to be used as coating antigen. By using the monoclonal antibody and the coating antigen, an IC-ELISA has been developed. Under the established optimized conditions, the IC-ELISA showed an IC50 of 4.2 ng/mL with a detection limit of 0.07 ng/mL. The IC-ELISA showed negligible cross-reactivity with other OP pesticides except with pirimiphos-ethyl. Recoveries of pirimiphos-methyl from spiked grain samples ranged from 83 to 96%.     

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.     

Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay

A monoclonal antibody (Mab) against ochratoxin A (OTA) was produced from the hybridoma cell line C7G25, which was established by the fusion of Sp2/0-Ag14 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with the OTA-bovine serum albumin conjugate. This Mab belongs to the IgG(2a) heavy-chain subclass with a kappa-type light chain. The level of 50% inhibition concentration was 1.20 ng/mL in a competitive direct enzyme-linked immunosorbent assay (cdELISA), and the detection limit was 0.12 ng/mL. This antibody is specific for OTA but also shows cross-reactivity with ochratoxin B (31.7%) in a cdELISA. On the basis of the sandwich format using the produced Mab against OTA, a rapid immunochromatographic assay was developed to efficiently detect OTA. This method was able to detect up to 500 ng/mL of OTA in <10 min.     

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.