Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.     

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.     

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.     

Development of monoclonal antibody-based immunoassays to the N-methylcarbamate pesticide carbofuran

To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently characterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterologous assay haptens. Depending on the immunoreagent combination and assay format, competitive assays with I(50) values in the 1.2-10.2 nM (0.27-2.27 ng/mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFNH, coupled either to horseradish peroxidase or to ovalbumin, was used to develop a direct and an indirect enzyme-linked immunosorbent assay, respectively. Optimized immunoassays displayed very similar analytical characteristics, with an I(50) value around 0.7 ng/mL and a limit of detection around 0.08 ng/mL. Both immunoassays were able to tolerate the presence of methanol up to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pesticides were not recognized by the MAb. These immunoassays should reasonably allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practical importance.     

Hapten syntheses and antibody generation for a new herbicide, metamifop

To develop a competitive indirect enzyme-linked immunosorbent assay for metamifop, a new aryloxyphenoxypropionic acid herbicide, three structurally related haptens were synthesized. Hapten conjugates to keyhole limpet hemocyanin and bovine serum albumin were used as immunogens and plate-coating antigens, respectively. Various sets of polyclonal antibodies from rabbits and the coating antigens were screened for the assay in simple homologous and heterologous ELISA formats. A selected heterologous ELISA was optimized to show an average IC50 value as low as 20.1 ng/mL, detection ranges of 1.0-350 ng/mL, and a lowest detection limit of 0.1 ng/mL. The cross-reactivities of other aryloxyphenoxypropionic acid herbicides to the antibodies were less than 0.5% in the assays except fenoxaprop-P and fenoxaprop-P ethyl, having a diaryl ether group identical to that of metamifop. Molecular modeling studies revealed that the physicochemical properties of the diaryl ether group are the most important determinants of sensitivity and selectivity. The results strongly indicate that the selected set of ELISA is a highly sensitive and convenient tool for detecting metamifop.     

Production and characterization of monoclonal antibodies against the antibiotic tilmicosin

Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocalization of tilmicosin.     

Hapten synthesis and monoclonal antibody-based immunoassay development for detection of the fungicide trifloxystrobin

High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.     

Immunoreagent generation and competitive assay development for cyprodinil analysis

Cyprodinil is an anilinopyrimidine fungicide applied worldwide for the prevention and treatment of highly destructive plant diseases in a large variety of crops, including cereals, fruits, and vegetables. This paper describes the development of the first reported immunoassays for cyprodinil. Two original haptens have been synthesized and conjugated to different carrier proteins, and polyclonal antibodies have been produced. Moreover, competitive enzyme-linked immunosorbent assays have been developed and characterized for the analysis of this widely used pesticide. The influence of organic solvents and buffer conditions over the assay analytical parameters was studied. The IC(50) values of the optimized immunoassays were 1.6 and 2.8 ng/mL for the direct and indirect formats, respectively. Quantitative recoveries were found using spiked apple and grape juice samples after a simple direct dilution, and a limit of quantification of 20 ng/mL for both fruit matrices was achieved. These immunoreagents could be very valuable for the sensitive, straightforward, and rapid monitoring of cyprodinil residues in foodstuffs.     

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.     

Development of an ELISA for the detection of the residues of the fungicide iprovalicarb

A competitive indirect enzyme-linked immunosorbent assay was developed for the fungicide iprovalicarb, using a polyclonal antibody produced against a hapten conjugated through the carboxyl group on the benzene ring to keyhole limpet hemocyanin. Under an optimized condition using a heterologous format, an IC(50) of 3.51 ng/mL and the lowest detection limit of 0.065 ng/mL were obtained. When the isopropoxy group was removed from the iprovalicarb structure for the synthesis of a hapten, the resulting hapten was not successful as an immunogen, indicating that the isopropyl moiety was an important epitope, as evidenced by the cross-reactivities of some structurally related compounds. When applied to the real crop and water samples, the recoveries were in the range of 80.52-144.70% (n = 4) and 72.11-100.43% (n = 4), respectively. Accordingly, this ELISA can be used as a useful method for monitoring iprovalicarb residues in crop and water samples.     

Development of an immunoassay for the residues of the herbicide bensulfuron-methyl

To develop a competitive indirect enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of the sulfonylurea herbicide bensulfuron-methyl, seven structurally related haptens were synthesized. Four of them mimicking the target analyte were conjugated to keyhole limpet hemocyanin by the N-hydroxysuccinimide activated ester method to use as immunogens, and all of them were conjugated to bovine serum albumin to use as plate-coating antigens. Polyclonal antibodies raised in rabbits and the coating antigens were screened and selected for the assay in simple homologous and heterologous ELISA formats. Three sensitive heterologous ELISAs were selected and optimized, showing the average IC(50) values of bensulfuron-methyl as low as 0.17, 0.09, and 0.09 ng/mL, the detection ranges of 0.04-0.60, 0.01-0.60, and 0.04-0.25 ng/mL, and the lowest detection limits of 0.03, 0.002, and 0.03 ng/mL, respectively. The cross-reactivities of other sulfonylurea herbicides and metabolites of bensulfuron-methyl to the antibodies were less than 15% in the two assays. Recoveries from the analyte-fortified water samples in assay I were in the range of 81-125% by simple dilution. The correlation between the ELISA and HPLC was 0.999 (n = 15) with a slope of 1.37 in the analysis of groundwater samples fortified with bensulfuron-methyl. The results obtained strongly indicate that the ELISA can be a highly sensitive and convenient tool for detecting bensulfuron-methyl residues in agricultural and environmental samples.     

检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立

本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗,并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng／mL,待检血清最佳稀释度为1：2,酶标单抗工作浓度为1：3200,血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测,竞争ELISA的检出率为40.2％,细胞毒性中和试验检出率为36.6％,两者符合率达91.5％。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。     

Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate

This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.     

Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals

To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.     

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.     

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.     

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.     

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.     

Enzyme immunoassay for hen egg white lysozyme used as a food additive

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.