Development of dipstick-based immuno-chemiluminescence techniques for the rapid detection of dichlorodiphenyltrichloroethane.

The occurrence of organochlorine pesticides in the environment has been a major concern, due to their high persistence and the possible impacts of their exposure to humans. Dichlorodiphenyltrichloroethane (DDT) is most hazardous and one of the most widely used organochlorine insecticides. DDT and its main metabolites are highly stable to physical, chemical and biological degradation and are therefore still being detected in many parts of the world. The present study describes dipstick-based immuno-chemiluminescence method for the detection of DDT with high sensitivity. Anti-DDT antibodies raised in chicken (IgY) were used as the biological sensing elements by immobilizing onto nitrocellulose membrane strips in a chemiluminescence (CL)-based dipstick technique. The photons generated during the biochemical interaction were directly proportional to the DDT concentration. A mean recovery of 81.2-95.6% was obtained for DDT-spiked fruit juice samples with 2.8-4.6% relative standard deviation (RSD). Using the proposed dipstick-based immuno-CL method, DDT was detected with linearity in the range 0.05-1 ng/mL, having a limit of detection (LOD) of 0.05 ng/mL. This method can be used for the rapid, reliable detection of DDT pesticide.

Targeted drug delivery to lymphocytes: a route to site-specific immunomodulation?

Lymphocytes are central to the progression of autoimmune disease, transplant rejection, leukemia, lymphoma and lymphocyte-resident viral diseases such as HIV/AIDs. Strategies to target drug treatments to lymphocytes, therefore, represent an opportunity to enhance therapeutic outcomes in disease states where many current treatment regimes are incompletely effective and promote significant toxicities. Here we demonstrate that highly lipophilic drug candidates that preferentially access the intestinal lymphatics after oral administration show significantly enhanced access to lymphocytes leading to improved immunomodulatory activity. When coadministered with such drugs, lipids enhance lymphocyte targeting via a three tiered action: promotion of drug absorption from the gastrointestinal tract, enhancement of lymphatic drug transport and stimulation of lymphocyte recruitment into the lymphatics. This strategy has been exemplified using a highly lipophilic immunosuppressant (JWH015) where coadministration with selected lipids led to significant increases in lymphatic transport, lymphocyte targeting and IL-4 and IL-10 expression in CD4+ and CD8+ lymphocytes after ex vivo mitogen stimulation. In contrast, administration of a 2.5-fold higher dose of JWH015 in a formulation that did not stimulate lymph transport had no effect on antiinflammatory cytokine levels, in spite of equivalent drug exposure in the blood. The current data suggest that complementary drug design and delivery strategies that combine highly lipophilic, lymphotropic drug candidates with lymph-directing formulations provide enhanced selectivity, potency and therapeutic potential for drug candidates with lymphocyte associated targets.

Analysis of DDT isomers with enzyme-linked immunosorbent assay and optical immunosensor based on rat monoclonal antibodies as biological recognition elements.

New rat monoclonal antibodies (mAbs) for DDT [1,1,1 -trichloro-2,2-bis (4-chlorophenyl) ethane], namely DDT 7C12, DDT 1C1, and DDT 1B2, were developed, characterized, and applied in ELISA both in coating antigen and in enzyme-tracer format. The latter used horseradish peroxidase (HRP) or glucose oxidase as enzymes. The lowest concentration of p,p'-DDT was determined with mAb DDT 7C12 and DDT-hapten HRP, with a test midpoint (IC50) of 0.5 +/- 0.2 microg/L (n=10) in 40 mM PBS (phosphate-buffered saline). The mouse anti-rat immunoglobulin lambda-light chain mAb LA1B12 was used as capture mAb. The best IC50 for o,p'-DDT in 40 mM PBS was 1.0 +/- 0.3 microg/L (n=12) and was obtained with mAb DDT 1C1 and DDT-hapten HRP, whereas mAb DDT 1B2 was very selective for p,p'-DDT with an IC50 of 4.2 + 1.6 microg/L (in 40 mM PBS, n=9). An optical immunosensor was optimized and applied for the analysis of DDT (or DDT equivalents). This immunosensor consists of a bench-top optical readout device and disposable sensor chips, which include the fluidic system. Evanescent field excitation and emission of the fluorophore Oyster-645 was used. An IC50 for p,p'-DDT [in 5% (v/v) isopropanol in 40 mM PBS] of 4 microg/L was obtained using DDT 7C12-Oyster-645. ELISA and immunosensor were used for the analysis of p,p'-DDT in unspiked and spiked surface water samples. Within the working ranges of these immunotechniques, recoveries ranged from 80 to 120%.

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: an organochlorine pesticide.

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL(-1) to 1000 ng mL(-1) to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL(-1) with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.

Immunization prevents DDT buildup in mouse tissues.

DDT is used for pest control, causing health and environmental hazards in some parts of the world. The goal of this study was to assess whether immunization against a toxic compound could reduce the toxicant uptake of an organism, specifically to develop a DDT immunization that promotes the production of specific antibodies and assess whether it reduces DDT levels in the bodies of mice that are exposed to DDT by intake. BALB/c mice were immunized with DDT-keyhole limpet hemocyanine (DDT-KLH) conjugate (n=10) or unconjugated KLH (n=10), which was used as a control. After the immunization specific DDT antibodies in the mouse serum were determined by ELISA and then the mice were fed chow containing 40 mg/kg of DDT for 45 days. Finally, the concentration of DDT and its metabolites, DDE and DDD, in various tissues was measured by gas chromatography. Specific DDT antibody levels were significantly higher in the DDT immunized group than in the control group. DDT, DDE and DDD levels in adipose tissue, blood, brain and spleen were significantly reduced in the DDT immunized animals relative to control animals. However, DDT and DDD levels were higher in the liver compared to the control group. The findings indicate that the DDT immunization reduces the total uptake of DDT in animal tissues, which is reflected by the lower levels in adipose tissue, blood, brain and spleen. The elevated levels in liver suggest that DDT-antibody complexes in mouse serum are delivered to the liver.

Development of nanomechanical biosensors for detection of the pesticide DDT.

We report the use of a novel technique for detection of the organochlorine insecticide compound dichlorodiphenyltrichloroethane (DDT) by measuring the nanometer-scale bending of a microcantilever produced by differential surface stress. A synthetic hapten of the pesticide conjugated with bovine serum albumin (BSA) was covalently immobilised on the gold-coated side of the cantilever by using thiol self assembled monolayers. The immobilisation process is characterised by monitoring the cantilever deflection in real-time. Then specific detection is achieved by exposing the cantilever to a solution of a specific monoclonal antibody to the DDT hapten derivative. The specific binding of the antibodies on the cantilever sensitised side is measured with nanomolar sensitivity. Direct detection is proved by performing competitive assays, in which the cantilever is exposed to a mixed solution of the monoclonal antibody and DDT. The future prospects and limitations to be overcome for the application of nanomechanical sensors for pesticide detection are discussed.

Associations of dichlorodiphenyltrichloroethane (DDT) 4.4 and dichlorodiphenyldichloroethylene (DDE) 4.4 blood levels with plasma IL-4.

DDT (dichlorodiphenyltrichloroethane) reportedly induces cancer in animals, mimics estrogen activity, induces antiandrogen effects, and impairs Natural Killer (NK) cells and T lymphocyte responses. In this study, the authors attempted to determine associations of DDT, dichlorodiphenyldichloroethylene (DDE), and dichlorodiphenyldichloroethane (DDD) blood levels with several immune parameters in patients occupationally exposed to insecticides. The study subjects were 49 patients who worked as farmers or farmhands in the former German Democratic Republic and who had been occupationally exposed to insecticides for at least 6 mo; 80% of them had been exposed for more than 20 yr. Blood levels of DDT, DDE, DDD, 2,3,4,5,6-pentachlorophenol (PCP), polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and gamma-hexachlorocyclohexanes (HCHs) were determined, and blood lymphocyte subpopulations, in vitro responses to mitogens or pooled allogeneic stimulator cells, plasma neopterin, and cytokine and soluble cytokine receptor levels were studied. The majority of patients were contaminated with more than 1 chemical--most commonly DDE, PCBs, and HCB. Linear-regression analysis showed that interleukin-4 (IL-4) plasma levels were associated with plasma levels of DDT 4.4 (p = .0001) and DDE 4.4 (p = .001). The data in this study suggest that PCBs, PCP, HCB, HCHs, DDE, and DDD suppress TH1 cytokines, such as IL-2 and interferon-gamma (IFN-gamma), and induce TH2 cytokines, such as IL-4. The authors hypothesized that clinical symptoms, such as the frequent infections reported by many patients, could be a consequence of these immunological abnormalities.

Effects of in vitro exposure of beluga whale leukocytes to selected organochlorines.

The effects of in vitro exposure to different organochlorines were evaluated on immune functions of beluga whale peripheral blood leukocytes and splenocytes. The effects of different concentrations of four different congeners of PCBs (138, 153, 180, and 169) as well as two DDT metabolites (p,p'-DDT and p,p'-DDE) were evaluated on phagocytosis and cell proliferation. The effects of dioxins and mixtures of organochlorines were also evaluated on cell proliferation. The different compounds tested had no marked effect on phagocytosis. PCB 138 and p,p'-DDT, but not PCB 153, PCB 180, PCB 169, and p,p'-DDE, reduced significantly the proliferative response of beluga splenocytes cultured either with or without phytohemagglutinin A (PHA). Proliferation of beluga splenocytes was not markedly affected by exposure to 5 ppm of PCB 138, 153, 180, and 169 separately. Exposure to a mixture of congeners 138, 153, and 180 (5 ppm each) significantly reduced splenocytes proliferation, but not the mixture of congeners 138, 153, 180, and 169 (5 ppm each). TCDD did not affect cell proliferation in our study. The reduced proliferation of beluga cells exposed in vitro to mixtures of organochlorines at concentrations in the range of those observed in tissues of St. Lawrence belugas might provide a basis to support the hypothesis that contaminants induce immunosuppression in these animals.

The immunotoxicity of pesticides in rodents.

Many pesticides that are widely used and have great potential for occupational and public exposure have received only a cursory examination with regards to their immunotoxic potential. Many of the studies that are available were done during the early period of immunotoxicology and many reports do not state whether or not any other toxic signs were observed and the dose-response relationships were not generally examined. Most studies were done in multiple species, through various routes of administration and using a variety of assays of immune function and do not allow a comparison. For the compounds extensively studied, the site of action has been determined at the cellular and perhaps the biochemical level, but the molecular site of action has not been determined. The approach to evaluate the mechanism of action will vary from compound to compound.

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT.

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.

A new, sensitive assay to determine immunological adjuvant activity based on the immunogenicity of neuraminidase-treated sheep erythrocytes.

A novel, sensitive system to determine immunological adjuvant activity is presented. It is based on the direct haemagglutinin response of mice to neuraminidase-treated sheep red blood cells (asialo-SRBC) seven days after i.p. immunization. For two model adjuvants it is shown that the response is more sensitive to stimulation than that to normal SRBC. Optimal stimulatory activity was measured at an antigen dose of 3 x 10(6) asialo-SRBC. Using this dose stimulation indices up to 100 were observed. The minimal effective dose of dextran sulphate, the so far most potent adjuvant in the model, was only 1 microgram. It is further shown that, in addition to substances with a rather general immunostimulatory activity, compounds with adjuvanticity which is commonly restricted to cellular responses are also effective in the system. The latter and reduced activity of the model adjuvants in nude mice strongly suggest that adjuvanticity in the asialo-SRBC model is T cell-dependent. Suppression of adjuvant activity in cobra venom factor-pretreated animals may indicate an involvement of complement in extrinsic immunostimulatory activity. Results show that the asialo-SRBC model is very suitable for evaluation and mechanistic study of immunological adjuvant activity.

Effects of immunological adjuvants on the mouse complement system--II. Anti-complementary effects of surface-active compounds.

The anti-complementary effects of the surface-active immunological adjuvants dimethyldioctadecylammonium bromide (DDA) and pluronic polyols L101 and L121 were investigated in the mouse system. All three adjuvants showed complement (C)-inactivating effects. DDA caused a time- and dose-dependent reduction of alternative pathway (AP) and overall C activity, which varied with the serum concentration. Polyols induced a preferential inactivation of the AP by a more direct mechanism. A rather general, causative relationship between anti-complementary and immunological adjuvant activities is suggested. This might involve interference with nonspecific elimination of antigen, counteraction of immunosuppression by terminal C components, and/or moderation of C3b-mediated reduction of Ia-expression, leading to a better antigen presentation.

Modulation of adjuvant-enhanced delayed-type hypersensitivity by the interferon inducers poly I:C and Newcastle disease virus.

The modulation by the interferon (IFN) inducers poly I:C and Newcastle disease virus (NDV) of the effector phase of adjuvant-enhanced delayed-type hypersensitivity (DH) was studied in mice. A strongly enhanced DH was induced in mice to ultraviolet (UV) light inactivated Semliki forest virus (SFV) by the use of the adjuvant dimethyldioctadecylammonium bromide. At day 6 after intracutaneous immunization, DH was elicited with SFV and measured 24 and 48 h later as increase in footpad thickness (footpad swelling test). Systemic, intravenous administration of either poly I:C, UV-inactivated NDV, or NDV-induced IFN prior to elicitation of DH with antigen resulted in a temporarily suppressed DH reaction. Both the poor swelling at 3 h and strong swelling at 24 h were suppressed, while the swelling at 48 h was enhanced. The model described provides a sensitive in vivo method to study modulating effects of drugs and microbial agents on the effector phase of DH.