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.     

Rapid fluorescence polarization immunoassay for the mycotoxin deoxynivalenol in wheat

The fungus Fusarium graminearum, a pathogen of both wheat and maize, produces a toxin, deoxynivalenol (DON), that causes disease in livestock. A rapid test for DON in wheat was developed using the principle of fluorescence polarization (FP) immunoassay. The assay was based on the competition between DON and a novel DON-fluorescein tracer (DON-FL2) for a DON-specific monoclonal antibody in solution. The method, which is a substantial improvement over our previous DON FP immunoassay, combined a rapid (3 min) extraction step with a rapid (2 min) detection step. A series of naturally contaminated wheat and maize samples were analyzed by both FP immunoassay and liquid chromatography (HPLC-UV). For wheat the HPLC-UV and FP methods agreed well (linear regression r(2) = 0.936), but for maize the two methods did not (r (2) = 0.849). We conclude that the FP method is useful for screening wheat, but not maize, for DON.     

Extraction methods for quantitation of gentamicin residues from tissues using fluorescence polarization immunoassay

Sodium hydroxide digestion of unhomogenized kidney and skeletal muscle for 20 min at 70 degrees C was a superior method for extracting gentamicin from tissues, compared with simple homogenization, trichloroacetic acid precipitation of homogenized tissue, and sodium hydroxide digestion of homogenized tissue. Fluorescence polarization immunoassay was used to quantitate gentamicin. Sodium hydroxide digestion of unhomogenized tissue allowed for the recovery of 90.0 +/- 5.9% (means +/- SD) from renal cortex and 79.9 +/- 3.5% from skeletal muscle. The limit of sensitivity was 17.4 ng/g kidney tissue, 15.8 ng/g digested muscle, and 39.0 ng/g digested heart. The within-assay coefficient of variation (CV) at 100 ng/g kidney was 9.2%; at 500 ng/g kidney, the CV was 2.5%; and at 2000 ng/g kidney, the CV was 1.5%. The between-assay coefficient of variation was less than 7.5% for all concentrations from kidney, and the 99% confidence interval at 100 ng/g kidney was 71.7-112.4 ng gentamicin/g kidney. The within-assay coefficient of variation (CV) at 100 ng/g muscle was 15%; at 500 ng/g muscle, the CV was 2.6%; and at 2000 ng/g muscle, the CV was 2.3%. The between-assay coefficient of variation was less than 15% for all concentrations from muscle, and the 99% confidence interval at 100 ng/g muscle was 72.5-136.8 ng gentamicin/g muscle. Gentamicin-free milk could be distinguished from milk containing gentamicin concentrations of 10 ng/mL milk with 95% confidence, and from milk containing concentrations of 30 ng gentamicin/mL milk with 99% confidence. Quantitative results at or below the tolerance level can be obtained within 90 min of sample acquisition using these extraction and assay methods.     

Determination of ochratoxin A by monoclonal antibody-based enzyme immunoassay

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.     

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).     

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

Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 microg/kg) and lower than the detection limits of the usual chromatographic detection methods.     

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Development of a fluorescence polarization assay for the determination of aflatoxins in grains

Aflatoxins produced by Aspergillus flavus are commonly found in human and animal foods including grains, cereals, peanut products, sorghum, and soy seeds. Exposure to aflatoxins has been associated with carcinogenicity. This paper reports a simple, portable, and rapid fluorescence polarization (FP) assay for aflatoxin determination in grains. This immunoassay is field portable, homogeneous, and without any washing and cleaning steps. The assay is based upon the competition between free aflatoxin and an aflatoxin-fluorescein tracer for an aflatoxin-specific monoclonal antibody in solution. A series of naturally contaminated corn, sorghum, peanut butter, and peanut paste samples were analyzed by FP and compared with HPLC results. Similarly, spiked popcorn samples were analyzed by FP. FP results of naturally contaminated samples correlated well with HPLC (r (2) = 0.97). FP analysis of spiked popcorn samples (with a mixture of B(1)/B(2)/G(1)/G(2), 7/1/3/1, w/w) gave a good correlation with spiked values (r (2) = 0.99). However, FP consistently underestimated the aflatoxin contents. This was perhaps due to low cross-reactivity of the antibody used toward B(2), G(1), and G(2) aflatoxins. These results combined with the portability and simplicity of the assay suggest that the assay can be used for screening total aflatoxin in grains.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.     

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.     

A capillary electrophoresis laser-induced fluorescence method for analysis of potato glycoalkaloids based on a solution-phase immunoassay. 1. Separation and quantification of immunoassay products

Solution-phase immunoassays are typically faster and more precise than ELISAs. This research developed a solution-phase for the immunoassay of potato glycoalkaloids (GAs) based on quantification by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Solanidine coupled to 4'-(aminomethyl)fluorescein and a polyclonal antibody solution were used as the immunoreagents. Unbound fluorescent solanidine was detected by CE-LIF (excitation 488 nm, emission 520 nm). Optimum resolution of immunoassay products was achieved with a buffer consisting of 50 mM phosphate, 10% (v/v) methanol, and 1.5 mM SDS, pH 7.5. A plot of signal vs log [GA] produced a sigmoidal curve typical of immunoassays. Analysis of extracts of sprouted Yukon Gold potato tubers and nonsprouted Yukon Gold tubers resulted in total [GA] of 98 microg/g (RSD 9%) and 55 microg/g (RSD 9%), respectively. The findings indicated that CE-LIF coupled with a solution-phase immunoassay can be used to quantify total GA in potatoes.     

Development of a capillary electrophoresis-based immunoassay with laser-induced fluorescence for the detection of carbaryl in rice samples

A capillary electrophoresis-based competitive immunoassay (CEIA) with a laser-induced fluorescence (LIF) detector for the determination of carbaryl was developed. The method was based on the competitive reactions between fluorescently labeled carbaryl tracer (Ag*) and free carbaryl (Ag) with a limited amount of anticarbaryl antibody (Ab), and the relative amounts of each were separated and determined by capillary electrophoresis (CE) with an LIF detector. Using CEIA, equilibrium was reached in 30 min, and the analytical results were obtained within a further 8 min. The linear range and the detection limit for carbaryl were 0.16-50 ng/mL and 0.05 ng/mL, respectively. The sensitivity of this CEIA with an LIF detector was almost 14 times greater than that of ELISA, which used the same immuno-reagents. The method was also applied to the analysis of carbaryl in rice with rapid and simple sample pretreatment. The method is thus proposed as a fast and sensitive assay for the detection of carbaryl.     

Simultaneous determination of bisphenol A and its halogenated analogues in soil by fluorescence polarization assay

Purpose

Extensive use of bisphenol A (BPA) and its analogues has caused increasing concern over the potential adverse health impacts of these chemicals. The aim of this study was to develop a fluorescence polarization (FP) assay to detect bisphenol A, bisphenol AF (BPAF), tetrabromobisphenol A (TBBPA), and tetrachlorobisphenol A (TCBPA) simultaneously.

Materials and methods

A recombinant peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD*) was chosen as recognition element in developed FP procedure. The environmental soil samples were assayed for (1) analytical parameters, (2) matrix effect, (3) recovery experiment, and (4) HPLC validation. For statistical analysis, GraphPad Prism 5 software was used.

Results and discussion

Under the optimized conditions, this assay had an IC₅₀ of 2.80, 6.84, 4.67, and 4.66 μg g^?1 for BPA, BPAF, TBBPA, and TCBPA, respectively. The developed assay demonstrated the limits of detection (LODs) of 0.35, 0.21, 0.27, and 1.03 μg g^?1 for these four compounds, which could meet the requirement of environmental monitoring. For spiked soil samples, mean recoveries of the analytes ranged from 85.3 to 101.1%, yielding coefficient of variations (CVs) ranged from 9.8 to 17.5%.

Conclusions

In this study, a broad-specificity receptor protein for BPA, BPAF, TBBPA, and TCBPA was produced and was used to develop an FP assay in soil samples. The efficiency and simplicity of the proposed assay define its possible application as a screening tool for environmental monitoring.

     

A capillary electrophoresis laser-induced fluorescence method for analysis of potato glycoalkaloids based on a solution-phase immunoassay. 2. Performance evaluation

Glycoalkaloids (GAs) occur naturally in potatoes and are toxic to humans and animals. The objective of the present study was to evaluate the performance of a solution-phase immunoassay coupled to capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection for the determination of total glycoalkaloids in potatoes. The immunoassay was based on a competition between potato glycoalkaloids and fluorescently labeled solanidine. Reaction products were separated in the capillary zone electrophoresis mode. A calibration curve of signal vs log[GA] was linear from 50 to 400 nM. The CV for duplicate and day-to-day analyses averaged 5.7% and 12%, respectively. Spike recoveries ranged from 85 to 97% for spike levels ranging from 43 to 170 microg/g fresh potato. Potato samples with GA concentrations ranging from <40 to >200 microg/g were successfully analyzed, indicating that immuno-CE-LIF is a rapid alternative to traditional ELISA and HPLC methods.     

Hapten synthesis and development of polyclonal antibody-based multi-sulfonamide immunoassays

This paper reports the synthesis of five sulfonamide derivatives, the production of broad-specificity polyclonal antibodies for immunoassay of sulfonamides, and the analysis of milk samples by developed assay. The three-step synthesis procedure reported in most of the literature was adopted and modified in this study. In the procedure, the purification of the intermediate was avoided and the time of synthesis was shortened from >20 to 6-9 h with improved yields. This method is generally applicable to the synthesis of haptens containing the common structure of sulfonamides. Three haptens were coupled to keyhole limpet hemocyanin, and polyclonal antibodies were obtained from rabbits immunized with these conjugates. Using the antibodies obtained, from one of these was developed an enzyme-linked immunosorbent assay (ELISA) based on the competition between free sulfonamides and the hapten-horseradish peroxidase (HRP) conjugates. The hapten-HRP conjugate giving the best competitive results and 11 structurally different sulfonamides showed 50% inhibition at concentrations of <100 ng mL(-1). After removal of the protein with acetone, milk samples were analyzed by ELISA directly; a matrix effect could be avoided when a 1:20 dilution with phosphate-buffered saline was used, and 104-131% recoveries of spiked samples were obtained. The developed immunoassay is suitable to determine sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine, sulfapyridine, and sulfamethizole below the maximum residue limit in milk (100 ng mL(-1) of total sulfonamides) rapidly and reliably.     

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.     

Characterization of monoclonal antibodies for lead-chelate complexes: applications in antibody-based assays

Monoclonal antibodies against lead were generated by immunizing BALB/c mice with lead conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, S-2-(4-aminobenzyl)diethylenetriamine pentaacetic acid (DTPA). Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. One of the hybridomas generated from this fusion (4/7) synthesized and secreted an antibody that bound tightly to Pb2+-DTPA complexes but not to metal-free DTPA. The performance for a competitive inhibition enzyme-linked immunosorbent assay (ELISA) incorporating this antibody was assessed for its sensitivity to changes in pH, ionic strength, and blocking reagents. The cross-reactivities in this ELISA were less than 3% for Fe3+, Cd2+, Hg2+, and Cu2+ and less than 0.3% for Cr3+, Mn2+, Mg2+, In3+, Ag1+, Ni2+, Co2+, Zn2+, Ca2+, Cu1+, and Hg1+. The IC50 value achieved for lead was 2.72 +/- 0.034 microM, showing the detection range of 0.092-87.2 microM and the lowest detection limit of 0.056 +/- 0.005 microM. Recoveries from the analyte-fortified tap water and ultrapure water were in the range of 80-114% . These results indicate that the ELISA could be a convenient analytical tool for monitoring lead residues in drinking water.     

Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides

A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides.     

Enzyme immunoassay for mycophenolic acid in milk and cheese

Mycophenolic acid (MPA) was reacted with N-hydroxysuccinimide and conjugated to keyhole limpet hemocyanin (KLH), and to horseradish peroxidase (HRP), respectively. The MPA-KLH was used to produce anti-MPA antiserum in rabbits. A competitive direct enzyme immunoassay (EIA) for MPA was established with anti-MPA antiserum and MPA-HRP conjugate. The mean 50% inhibition and detection limit of MPA standard curves (n = 103) were 197 +/- 67 and 81 +/- 48 pg/mL, respectively. The EIA was specific for MPA and its synthetic 2-morpholinoethyl ester, mycophenolate mofetil (91% relative cross-reactivity). Raw bulk milk and pasteurized milk, with and without beta-glucuronidase pretreatment, were analyzed by EIA. No MPA was found in milk, at a detection limit of 100 pg/mL (recovery 58-66% at 0.125-2 ng/mL). Blue-veined cheese from the German market (n = 53) was analyzed by EIA, and the detection limit was at 0.5 ng/g (recovery 68-79% at 5-100 ng/g). All but two cheeses contained MPA, although mostly (66%) at levels of <10 ng/g. MPA at 400-1200 ng/g was found in Roquefort cheeses. Highest levels (4-11 microg/g) were found in a German soft cheese preparation. MPA levels in mycelium-rich parts of cheese were 3 times higher than in mycelium-free parts.