Rapid, on-site and quantitative paper-based immunoassay platform for concurrent determination of pesticide residues and mycotoxins.

Mycotoxins and pesticides are prevalent in cereal food. It is difficult to detect these two kinds of hazard factors simultaneously in rapid assay. In order to find a solution to the problem, carbamates and aflatoxins were selected in this study to establish a rapid, on-site, and quantitative paper sensor. Two novel monoclonal antibodies (mAbs) against carbaryl and carbofuran (1D2 and G11) were developed. The IC50 values (half maximal inhibitory concentration) were 0.8 ng/mL and 217.6 ng/mL for carbaryl and carbofuran, respectively. Based on the sensitive and specific mAbs, a multi-TRFICA (time-resolved fluorescence) paper sensor was developed, which simultaneously detected six types of hazardous chemicals, including AFB1, AFB2, AFG1, AFG2, carbaryl, and carbofuran. A universal sample pretreatment method for mycotoxins and pesticides was explored to apply on established competitive indirect enzyme-linked immunosorbent assay (icELISA) and multi-TRFICA-paper sensor. The established paper sensor can be easily observed with naked eyes, qualitatively under a UV lamp, and quantitated using a home-made device. It exhibited a calculated limit of quantity for AFTs, carbaryl, and carbofuran of 0.03, 0.02, and 60.2 ng/mL in corn samples, respectively. The spiking-recoveries and real sample studies proved that multi-TRFICA-paper sensor is an accurate, sensitive, and high throughput detection method for simple and low-cost analysis in corn samples.

Highly sensitive microcantilever-based immunosensor for the detection of carbofuran in soil and vegetable samples.

Microcantilever-based immunosensor is a next-generation electromechanical technique with broad application in biological detection. In this paper, we reported a microcantilever-based immunosensor that quantitatively detect the carbofuran, by using monoclonal antibodies to carbofuran as the receptor molecules. The surface of gold-coated microcantilever was chemically modified by the crosslinking of l-cysteine (l-cys)/glutaraldehyde (GA). The monoclonal antibodies to carbofuran were then immobilized on the side of the microcantilever to fabricate the immunosensor, the mechanical bending induced by antigen-antibody specific binding under an experimental environment. Under the optimized conditions, immunosensor detected carbofuran showed a good linear relationship over the range from 1.0×10-7 to 1.0×10-3g/L (R=0.998), with a detection limit of 0.1ng/mL. Moreover, the proposed immunosensor exhibited high sensitivity, specificity and good stability and can be successfully applied in the carbofuran determination in soil and vegetable samples with satisfactory results.

Cloning, expression, and identification of anti-carbofuran single chain Fv gene.

Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly(4)Ser)(3). The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC(50) value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF.

Gold immunochromatographic assay for simultaneous detection of carbofuran and triazophos in water samples.

Using a simple test for rapid identification and quantification of pesticide multiresidues in food and environmental samples is a long-cherished approach for practical monitoring purposes. Here two gold-based lateral-flow strips (strip A and strip B) were investigated for simultaneous detection of carbofuran and triazophos. For the strip A format, a bispecific monoclonal antibody (BsMcAb) against both carbofuran and triazophos was employed to prepare the immunogold probe. For the strip B format, anti-carbofuran monoclonal antibody (McAb) and anti-triazophos McAb separately labeled with colloidal gold were combined as detector reagents. By comparison of visual results from pesticide standard tests between the two formats, the strip B assay manifested higher sensitivities for both pesticides. Analysis of spiked water samples by the preferable strip indicated that the detection limits for carbofuran and triazophos were 32 and 4 microg/L, respectively. The strength of the portable one-step strip assay was in the simultaneous screening for two pesticides within a short time (8-10 min) without any equipment.

Development of a bispecific monoclonal antibody to pesticide carbofuran and triazophos using hybrid hybridomas.

A mouse hybrid hybridoma (tetradoma) was derived from fusing hybridomas producing monoclonal antibody to N-methylcarbamate pesticide carbofuran with hybridomas producing monoclonal antibody to organophosphorus pesticide Triazophos. The prepared tetradoma line (12C1 to 2H12) secreted hybrid immunoglobulin exhibiting parental and bispecific binding characteristics. The effect of relevant physicochemical factors on the immunoassay based on the 12C1 to 2H12 bispecific monoclonal antibody had been studied to optimize the ELISA performance. The developed immunoassay showed that the detection limit (I(20)) were 0.36 and 1.89 ng/mL for triazophos and carbofuran, respectively, without obvious cross-reactivity to other related compounds. Water samples spiked with triazophos at 0.5, 1, and 5 ng/mL or carbofuran at 5, 10, and 20 ng/mL were directly analyzed by the developed ELISA format. The mean recovery of triazophos and carbofuran were 108.1% and 107.5%, with variation coefficient of 15.9% and 17.7%, respectively.

[Studies of immunoaffinity chromatography for carbofuran].

The purified anti-carbofuran antibody was conjugated to carbonyl diimidazole (CDI)-activated Sepharose CL-4B to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to carbofuran. The conditions of IAC were optimized as follows: pH 7.2 phosphate buffer (PB) was used as equilibrium and adsorbent medium, and methanol-water (60:40, v/v) as eluent. The results showed that the dynamic column capacity was up to 1.58 mg/L bed volume. The efficiency of enrichment of IAC was more than 167 times when the initial concentration of carbofuran in a standard sample solution was lower than 2 microg/L. The spiked river water was cleaned up and enriched by IAC, and carbofuran in eluate was determined by enzyme linked immunosorbent assay (ELISA). The average recovery of carbofuran from river water was 89.8% with the relative standard deviation of 4.8% at the spiked level of 0.1 mg/L. Meanwhile the eluate was determined by high performance liquid chromatography (HPLC), the results from HPLC correlated well with those from ELISA. The IAC method of carbofuran was successfully established.

Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables.

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine 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 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran 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 carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, 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 = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.

Determination of carbofuran by on-line immunoaffinity chromatography with coupled-column liquid chromatography/mass spectrometry.

Development of a new technology for combined on-line sample preparation and analyte confirmation is presented. This technology involves the use of antibodies for trace analyte extraction and enrichment directly from a complex matrix. The antibodies are used in columns designed for use with ordinary high-pressure liquid chromatographic equipment. The immunoaffinity columns are combined with conventional reversed-phase LC columns by use of column-switching techniques and coupled directly to an atmospheric pressure ionization quadrupole ion trap mass spectrometer equipped with a pneumatically assisted electrospray (ion spray) interface. A column of aldehyde-activated silica was used to prepare a column specific to carbofuran. This column demonstrated excellent specificity toward carbofuran and showed no binding of another, unrelated compound, fluometuron. Direct extraction and detection of carbofuran was demonstrated at low levels (40 pg/mL) in spiked water, but the real utility of immunoaffinity chromatography (IAC) is demonstrated by the on-line extraction and detection of carbofuran from a chemically complex, crude potato extract. Samples extracted using an IAC column indicate that superior purification is obtained with IAC in comparison with samples pumped directly onto a reversed-phase trapping column. A detection limit for carbofuran of approximately 2.5 ng/g of potato was obtained using an atmospheric pressure ionization quadrupole ion trap mass spectrometer.