Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

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.

Development of a one-step strip for the detection of triazophos residues in environmental samples.

Environmental and food safety issues now are recognized internationally, and pesticide residues play key roles as environment and food pollutants. It is crucial to develop methods for rapid determination of pesticide residues in environments and foods. A one-step strip based on nanocolloidal-gold-labeled monoclonal antibodies for detection of triazophos residue was developed. The nanocolloidal gold, with an average particle diameter of 25 nm (G25), was labeled to an antitriazophos monoclonal antibody. This conjugate was dispensed on the conjugate pad of a porous glass fiber. Ovalbumin hapten and goat anti-mouse IgG were dispensed on the nitrocellulose membrane and served as the test line (T-line) and control line (C-line), respectively. After conditions optimization, the one-step strip was finally developed for the residue determination of triazophos. The limit of detection (LOD) of the strip was 4 ng/mL for standard. The detection was not affected by the pH of the liquid sample but low total ion concentration will induce illegible C-line and T-line. The LOD for spiked samples of soil and water was 5ng/mL, with run time of no more than 10min.

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.

Effects of O,S,S-trimethyl phosphorodithioate on immune function.

The effect of acute administration of 20-80 mg/kg O,S,S-trimethyl phosphorodithioate (OSS-TMP) to C57BL/6 female mice on the murine immune system was determined. The parameters examined to evaluate overt toxicity of the compound included body weight, plasma cholinesterase levels, splenic nucleated cell number and thymic weight and nucleated cell number. Acute administration of 60 or 80 mg/kg OSS-TMP led to a 75 or 63% decrease, respectively, in plasma cholinesterase levels and a decrease in thymic size. At a dose of 80 mg/kg OSS-TMP, the animals also exhibited some lethargy and body weight loss. Below 60 mg/kg OSS-TMP, no overt toxic manifestations were observed. These studies were carried further to determine the effect of OSS-TMP on the generation of in vivo primary and in vitro secondary cellular and humoral immune responses. At nontoxic doses of the compound, i.e. 20 and 40 mg/kg OSS-TMP, the in vivo generation of a primary cytotoxic T lymphocyte (CTL) response to alloantigen was significantly elevated, but this response was unaffected following restimulation of the splenocytes by alloantigen in vitro. The generation of an in vivo primary and in vitro secondary humoral responses to sheep red blood cells (SRBC) was elevated following a single dose of 40 mg/kg OSS-TMP. Administration of toxic doses of OSS-TMP, i.e. 60 and 80 mg/kg, did not alter the ability of splenocytes to generate a primary or secondary CTL response, but suppressed the generation of humoral immune responses. These results differ significantly from those observed in a similar system following acute administration of a structural analog, O,O,S-trimethyl phosphorothioate which was previously shown to have potent immunosuppressive activity at nontoxic doses.

Modulation of cellular and humoral immune responses by O,S,S-trimethyl phosphorodithioate, an impurity of commercial malathion.

We studied the effect of O,S,S-trimethyl phosphorodithioate (OSS-Me) on macrophage function and the immune responses of both T- and B-cells using an in vitro system. The involvement of glutathione (GSH) in the bioactivation of OSS-Me to suppress such responses was investigated after either direct culture with spleen or B-cells and/or co-culture of B-cells with either B- or T-cells that were or were not preincubated with OSS-Me and glutathione enriched cytosol. Our results show that OSS-Me preincubated with GSH suppresses immune responses through an inhibitory effect on the cell function. Both cytotoxic T-lymphocyte (CTL) generation and antibody production were impaired. Antigen presentation by macrophages was also inhibited. In mixing experiments of pretreated and normal cells, the inhibitory effect was also observed on macrophages and helper T-cells responding to a T-dependent antigen. We concluded that the immunosuppressive effect of OSS-Me is due to impairment of cell function and not mediated through generation of regulatory suppressor T-cells. This inhibitory effect was only detectable in the presence of glutathione in the preincubation mixture, suggesting that an OSS-Me glutathione adduct might be responsible for the toxic effect.

Effects of subchronic treatment with O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems.

The effect of a 14-day treatment with low doses of O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity in technical malathion, on the generation of cell-mediated and humoral immune responses was examined in female C57B1/6 mice. At a dose of 0.5 mg/kg/day OOS-TMP, the generation of antibody-secreting cells to sheep red blood cells (SRBC), the production of interleukin 2 (IL-2), and proliferative responses to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) were elevated. In contrast, the cytotoxic T-lymphocyte (CTL) response to alloantigen was unchanged. At 5.0 mg/kg/day OOS-TMP, both the CTL and specific antibody response were unchanged, but all other immune parameters examined were elevated. Data from cell separation and reconstitution experiments indicated that both macrophages and B cells were affected by this treatment regime. These data suggest that long-term exposure to low amounts of OOS-TMP may enhance the ability of an animal to generate an immune response.