Inhibiting NF-κB-Mediated Inflammation by Catechol-Type Diphenylbutadiene via an Intracellular Copper- and Iron-Dependent Pro-Oxidative Role.

Chronic inflammation mediated by nuclear factor-κB (NF-κB) plays a crucial role in the development of cancer. As part of our continuous efforts placed on investigating anticancer mechanisms of dietary catechols, we further applied catechol-type diphenylbutadiene (3,4-DHB) as a model molecule to probe whether it inhibits inflammation by its pro-oxidative role. Employing lipopolysaccharide-stimulated RAW264.7 cells as a model of inflammation, we validated that benefiting from its catechol moiety, 3,4-DHB inhibited significantly the LPS-induced formation of NO (11.48 ± 0.39 µM) compared with the only LPS-stimulated group (31.8 ± 1.78 µM) with an inhibitory rate of 64% at 5 µM, expression of iNOS and COX-2 proteins, phosphorylation of IkB kinase and IkBα, and nuclear translocation of NF-κB. Noticeably, its inhibitory activity against the NF-κB-mediated inflammation can be obviously revised by pretreatment of the cells with dithiothreitol (a quencher of both electrophilic o-quinone and ROS), neocuproine (a specific chelating agent for copper ions), and deferoxamine (a specific chelating agent for iron ions). The above results support that depending on intracellular copper and iron ions, 3,4-DHB, a pro-electrophile, can be converted into its corresponding o-quinone electrophile together with the generation of ROS, a pro-oxidative event that mediates its inhibitory activity against NF-κB signaling and inflammation. The copper- and iron-dependent inhibition against inflammation supports that dietary catechols are probably pro-oxidative anti-inflammatory agents.

Light-triggered OVA release based on CuS@poly(lactide-co-glycolide acid) nanoparticles for synergistic photothermal-immunotherapy of tumor.

The immunotherapy played a vital role in the treatment of metastatic tumor. To further enhance the effect of the immunotherapy, the combination of photothermal effect can not only eradicate the tumor cells by hyperthermia, but also improved the antigen release in vivo to achieve enhanced immune responses. In this study, a core-shell structured nanocomplex was developed by loading of ovalbumin (OVA) and copper sulfide nanoparticles (CuS-NPs) into the poly(lactide-co-glycolide acid) nanoparticles (PLGA-NPs). The CuS-NPs exhibited favorable photothermal effect, which significantly kill the 4T1 tumor cells in vitro. The photothermal effect of the CuS-NPs accelerated the OVA release, which led to higher levels of IL-6, IL-12 and TNF-α, and activation of CD8+ T cells. Both of the OVA-PLGA-NPs and CuS-NPs with NIR light irradiation contributed inhibited primary tumor while the growth of the distant tumors was not hindered. The irradiated CuS@OVA-PLGA-NPs exhibited a minimal primary tumor because of the combined effect of photothermal therapy and immunotherapy. Moreover, the irradiated CuS@OVA-PLGA-NPs showed the most extensive distribution of CD8+ T cells in the primary and distant tumor, which blocked the rise of the distant tumor. In conclusion, the CuS@OVA-PLGA-NPs presented as a promising strategy for metastatic tumor therapy.

Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.

Immunostimulatory effect of chitosan conjugated green copper oxide nanoparticles in tumor immunotherapy.

Current study demonstrates the immunogenic role of biopolymer coated green synthesized copper oxide nanoparticles by the induction of cellular immunity through the activation immune cells. Alongside humoral immunity response was triggered by the surface coated NPs through IgG response which indicate the adjuvanic role of the nano conjugate. Th1 (Type 1 and Type 2 helper T cells) and Th2 cells were activated after the treatment with nano conjugate and act as an immunostimulant which would inhibit the proliferation of breast cancer (MCF-7) and cervical cancer (HeLa) cells in in vitro. Solid tumor induced by 4 T1 cells were also inhibited in in vivo Balb/C mice model. Secretion of pro-inflammatory cytokines and the increase in CD + 4 populations indicate the activation of immune cells in the current study. Immunotherapy by the help of metal nano conjugate can be an effective tool to eradicate the cancer cells from the system.

Chronic exposure of Oreochromis niloticus to sub-lethal copper concentrations: Effects on growth, antioxidant, non-enzymatic antioxidant, oxidative stress and non-specific immune responses.

BACKGROUND: Trace elements of copper (Cu) are one of the main forms of ecological noxious waste in freshwater systems that affect the survival and development of organisms. The objective of the current study was to investigate the effects of chronic exposure to Cu on the growth, oxidative stress, immune and biochemical response in the Nile tilapia, Oreochromis niloticus. METHODS: Three groups of O. niloticus were tested as follows; the first group was used as the control (not treated with Cu in water), while the 2nd and 3rd groups were exposed to (low) 40 µg L-1 and (high) 400 µg L-1 concentrations of Cu added to water, respectively. The duration of the experiment, which was conducted in triplicate, was 60 d. End points were evaluated on days 30 and 60. Following 30 d and 60 d of exposure to Cu, the fish were removed from experimental tanks to determine growth. Consequently, blood samples were collected from caudal veins at the end of the trial period (30 d and 60 d) and serum was separated to evaluate different immunological parameters, such as lysozymes (LYZ), respiratory burst activity (RBA) and myeloperoxidase (MPO). Gill and liver tissues were collected for evaluation of Cu and certain biochemical parameters as follows: antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST); non-enzymatic antioxidants such as glutathione (GSH) and metallothionein (MT), and oxidative stress indicators such as malondialdehyde (MDA) and protein carbonyl (PCO). The results pertaining to treatments and the control were compared using two-way ANOVA and Tukey's HSD test. The level of significance was set at P ≤ 0.05. Data were expressed as mean ±â€¯SD. RESULTS: Chronic exposure to Cu did not induce any mortality in fish during the test period. However, following exposure to Cu, growth of fish in the exposed groups was affected more than that in the control group (unexposed to Cu). In addition, accumulation of Cu in the liver tissue was higher than that in the gill tissues of fish exposed to Cu, compared to that in the control. Gill and liver tissues of Cu-exposed fish showed a significant (P ≤ 0.05) reduction in the activities of the antioxidant enzymes, SOD, CAT, GPx, and GST, compared to those of unexposed fish. Non-enzymatic antioxidants, GSH and MT, in gill and liver tissues were significantly increased (P ≤ 0.05) in fish exposed to both concentrations of Cu, compared to those in unexposed fish. Oxidative stress indicators, MDA and PCO in gills and liver of Cu-exposed fish was significantly (P ≤ 0.05) at both tested concentrations, when compared to control group. Non-specific immune response of LYZ, RBA, and MPO activity in serum decreased significantly (P ≤ 0.05) in Cu-exposed fish, compared with that of unexposed fish. CONCLUSION: Overall, the present results highlighted that chronic exposure to Cu ions may exert a strong effect on the antioxidant and immune responses of O. niloticus. Changes in antioxidant enzymes, oxidative stress effects and immune parameters during post-chronic metal exposure may indicate the potential of these parameters as biomarkers of metal toxicity in aquatic ecosystems.

Reduced Neutrophil Extracellular Trap (NET) Formation During Systemic Inflammation in Mice With Menkes Disease and Wilson Disease: Copper Requirement for NET Release.

Neutrophil extracellular traps (NETs) contribute to pathological disorders, and their release was directly linked to numerous diseases. With intravital microscopy (IVM), we showed previously that NETs also contribute to the pathology of systemic inflammation and are strongly deposited in liver sinusoids. Over a decade since NET discovery, still not much is known about the metabolic or microenvironmental aspects of their formation. Copper is a vital trace element essential for many biological processes, albeit its excess is potentially cytotoxic; thus, copper levels are tightly controlled by factors such as copper transporting ATPases, ATP7A, and ATP7B. By employing IVM, we studied the impact of copper on NET formation during endotoxemia in liver vasculature on two mice models of copper excess or deficiency, Wilson (ATP7B mutants) and Menkes (ATP7A mutants) diseases, respectively. Here, we show that respective ATP7 mutations lead to diminished NET release during systemic inflammation despite unaltered intrinsic capacity of neutrophils to cast NETs as tested ex vivo. In Menkes disease mice, the in vivo effect is mostly due to diminished neutrophil infiltration of the liver as unmutated mice with a subchronic copper deficiency release even more NETs than their controls during endotoxemia, whereas in Wilson disease mice, excess copper directly diminishes the capacity to release NETs, and this was further confirmed by ex vivo studies on isolated neutrophils co-cultured with exogenous copper and a copper-chelating agent. Taken together, the study extends our understanding on how microenvironmental factors affect NET release by showing that copper is not a prerequisite for NET release but its excess affects the trap casting by neutrophils.

Roles of Prion Protein in Virus Infections.

The normal cellular prion protein, designated PrPC, is a membrane glycoprotein expressed most abundantly in brains, particularly by neurons, and to a lesser extent in non-neuronal tissues including lungs. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. We recently found that PrPC has a protective role against infection with influenza A viruses (IAVs) in mice by reducing reactive oxygen species in the lungs after infection with IAVs. The antioxidative activity of PrPC is probably attributable to its function to activate antioxidative enzyme Cu/Zn-superoxide dismutase, or SOD1, through regulating Cu content in lungs infected with IAVs. Oxidative stress could play a pivotal role in the pathogenesis of a wide range of viral infections. Here, we introduce our and others' studies on the role of PrPC in viral infections, and raise the attractive possibility that PrPC might be a novel target molecule for development of antioxidative therapeutics against not only IAV infection but also other viral infections.

A sensitive, colorimetric immunosensor based on Cu-MOFs and HRP for detection of dibutyl phthalate in environmental and food samples.

A sensitive and artful colorimetric immunosensor based on horseradish peroxidase (HRP) was designed by labelling metal-organic frameworks (Cu-MOFs) on the second antibody (Cu-MOFs@Ab2) as signal amplification for the detection of trace dibutyl phthalate (DBP). In this system, when Cu-MOFs@Ab2 was captured by antigen- primary antibody (Ab1) complex, tremendous Cu(II) will be released from Cu-MOFs in the presence of nitric acid (HNO3), and Cu(II) will be further reduced to Cu(I) after the addition of sodium ascorbate (SA), consequently, inhibiting the HRP to catalyse the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (ox TMB). Under the optimized conditions, the limit of detection (LOD) was 1 µg L-1, which was almost 60 times lower than that using a conventional ELISA with the same antibody. In addition, our method showed good accuracy and reproducibility (recoveries of 87.73-103.4%; CV values of 1.46-5.95%) through a spike-recovery analysis. The proposed immunosensor indicated great potential for trace DBP determination from environmental and food samples.

Comparison of the effect of dietary copper nanoparticles and one copper (II) salt on the metabolic and immune status in a rat model.

The aim of the study was to evaluate the effects of a diet containing different levels of Cu in two different chemical forms (carbonate and nanoparticles) on metabolic, immune and antioxidant status in a rat model. Five experimental treatments (8 rats in each) were used to test different dosages of Cu added to the diet (standard -6.5 mg/kg, half the standard dosage -3.25 mg/kg, and no added Cu as a negative control) and two sources of added copper (standard -CuCO3 and copper nanoparticles -CuNPs). Blood and urine samples were collected from all the animals after four weeks of treatment. Metabolic and immune parameters were determined in blood and urine samples. The study has shown that a dietary Cu deficiency (negative control) decreases rat's plasma levels of Cu, Fe, CREAT, BIL and IL-6, whereas reducing the level of Cu from the recommended 6.5 mg/kg to 3.25 mg/kg decreases only the plasma concentration of TG, IgE and IL-6. Replacing CuCO3 with CuNPs in rat diets affects their metabolism, as indicated by decreased Ca, CREAT, BIL, ALB and IL-6 plasma levels. To sum up, CuNP added to a diet of rats have a more beneficial effect on metabolic indices (indicative of kidney and liver function) and inhibit inflammatory processes more effectively than CuCO3.

Role of essential trace elements in tuberculosis infection: A review article.

Malnutrition is one of the risk factors in tuberculosis (TB) infection. Mineral levels perturbation is seen in patients with TB. Moreover there are some strategies to starve pathogens of essential metals. Here we decided to conclude association between some essential elements and TB. Copper, calcium and iron are essential for hosts' immune system although calcium and iron are necessary for Mycobacterium tuberculosis vitality. Changing these elements alongside with anti-TB therapy is suggested for better treatment outcomes.

Heavy metal and tattoo: an allergy and legislative problem.

We presented an interesting clinical case of a 23 years old man presented with a 2-week history of pruritus, erythema and papules on legs, arms and trunk. These lesions developed 2 months after tattooing. It showed positive patch test reaction to Copper and Disperse Blu.

Dietary magnesium and copper affect survival time and neuroinflammation in chronic wasting disease.

Chronic wasting disease (CWD), the only known wildlife prion disease, affects deer, elk and moose. The disease is an ongoing and expanding problem in both wild and captive North American cervid populations and is difficult to control in part due to the extreme environmental persistence of prions, which can transmit disease years after initial contamination. The role of exogenous factors in CWD transmission and progression is largely unexplored. In an effort to understand the influence of environmental and dietary constituents on CWD, we collected and analyzed water and soil samples from CWD-negative and positive captive cervid facilities, as well as from wild CWD-endozootic areas. Our analysis revealed that, when compared with CWD-positive sites, CWD-negative sites had a significantly higher concentration of magnesium, and a higher magnesium/copper (Mg/Cu) ratio in the water than that from CWD-positive sites. When cevidized transgenic mice were fed a custom diet devoid of Mg and Cu and drinking water with varied Mg/Cu ratios, we found that higher Mg/Cu ratio resulted in significantly longer survival times after intracerebral CWD inoculation. We also detected reduced levels of inflammatory cytokine gene expression in mice fed a modified diet with a higher Mg/Cu ratio compared to those on a standard rodent diet. These findings indicate a role for dietary Mg and Cu in CWD pathogenesis through modulating inflammation in the brain.

Copper oxide nanoparticles recruit macrophages and modulate nitric oxide, proinflammatory cytokines and PGE2 production through arginase activation.

AIM: In the present study, we examine the effects of copper oxide nanoparticles (CuNP) on macrophage immune response and the signaling pathways involved. MATERIALS & METHODS: A peritonitis model was used to determine in vivo immune cells recruitment, while primary macrophages were used as an in vitro model for the cellular and molecular analysis. RESULTS: In vivo, CuNP induce significant macrophages recruitment to the site of injection. In vitro, in LPS-stimulated primary macrophages, the co-treatment with CuNP inhibited the production of NO in a dose-dependent manner. The mechanism underlying NO and proinflammatory cytokines inhibition was associated with an increased arginase activity. Macrophage stimulation with CuNP did not provoke any cytokine secretion; however, arginase inhibition promoted TNFα and MIP-1ß production. In addition, CuNP induced the expression of COX-2 and the production of PGE2 through arginase activation. CONCLUSION: Our results demonstrate that CuNP activate arginase and suppress macrophage innate immune response.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis.

RATIONALE: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

Copper hypersensitivity.

The world production of copper is steadily increasing. Although humans are widely exposed to copper-containing items on the skin and mucosa, allergic reactions to copper are only infrequently reported. To review the chemistry, biology and accessible data to clarify the implications of copper hypersensitivity, a database search of PubMed was performed with the following terms: copper, dermatitis, allergic contact dermatitis, contact hypersensitivity, contact sensitization, contact allergy, patch test, dental, IUD, epidemiology, clinical, and experimental. Human exposure to copper is relatively common. As a metal, it possesses many of the same qualities as nickel, which is a known strong sensitizer. Cumulative data on subjects with presumed related symptoms and/or suspected exposure showed that a weighted average of 3.8% had a positive patch test reaction to copper. We conclude that copper is a very weak sensitizer as compared with other metal compounds. However, in a few and selected cases, copper can result in clinically relevant allergic reactions.

A copper chelate selectively triggers apoptosis in myeloid-derived suppressor cells in a drug-resistant tumor model and enhances antitumor immune response.

Myeloid-derived suppressor cells (MDSCs), one of the major orchestrators of immunosuppressive network are present in the tumor microenvironment suppress antitumor immunity by subverting Th1 response in tumor site and considered as a great obstacle for advancement of different cancer immunotherapeutic protocols. Till date, various pharmacological approaches have been explored to modulate the suppressive functions of MDSCs in vivo. The present study describes our endeavor to explore a possibility of eradicating MDSCs by the application of a copper chelate, namely copper N-(2-hydroxy acetophenone) glycinate (CuNG), previously found to be a potential immunomodulator that can elicit antitumorogenic Th1 response in doxorubicin-resistant EAC (EAC/Dox) bearing mice. Herein, we demonstrated that CuNG treatment could reduce Gr-1+CD11b+ MDSC accumulation in ascitic fluid and spleen of EAC/Dox tumor model. Furthermore, we found that CuNG mediated reduction in MDSCs is associated with induction of Th1 response and reduction in Treg cells. Moreover, we observed that CuNG could deplete MDSCs by inducing Fas-FasL mediated apoptotic cell death where death receptor Fas expression is enhanced in MDSCs and FasL is provided by activated T cells. However, MDSC expansion from bone marrow cells and their differentiation was not affected by CuNG. Altogether, these findings suggest that the immunomodulatory property of CuNG is attributed to, at least in part, by its selective cytotoxic action on MDSCs. So, this preclinical study unveils a new mechanism of regulating MDSC levels in drug-resistant cancer model and holds promise of translating the findings into clinical settings.

Immunotoxicity of aluminum.

Aluminum (Al) is present in the daily life of all humans. With the incidence of Al contamination increased in recent years, the toxicity of Al on the immune function has attracted more attention. Even with this increased attention, the mechanism of Al immunotoxicity still remains unclear. The mechanism of Al immunotoxicity reviewed herein focused on the effects of Al on the splenic trace elements, the status of α-naphthyl acetate esterase (ANAE) cells, cytokines, complement and immunoglobulins, as well as macrophages. The studies in the literature showed that Al decreased splenic iron (Fe) and zinc (Zn) levels, but the effects of Al on splenic copper (Cu) level was ambiguous and controversial. Al exposure inhibited levels of ANAE(+) cells, the production of interleukin (IL)-2 and the functions of macrophages. With respect to other key cytokines, studies showed that Al suppressed the production of tumor necrosis factor (TNF)-α in vitro; effects of Al on TNF-α formation in vivo were less overt. Al exposure reduced complement 3 (C3) level, but effects of Al exposure on complement 4 (C4) level were not as clear-cut. Lastly, the effects of Al exposure on the IgG, IgM and IgA levels were conflicting. Taken in totality, the results of several studies in the literature demonstrated that Al could impart adverse effects on immune function.

Copper induced immunotoxicity promote differential apoptotic pathways in spleen and thymus.

Inorganic copper, such as that in drinking water and copper supplements, largely bypasses the liver and enters the free copper pool of the blood directly and that promote immunosuppression. Nevertheless, the signaling pathways underlying copper-induced immune cell death remains largely unclear. According to our previous in vivo report, to evaluate the further details of the apoptotic mechanism, we have investigated how copper regulates apoptotic pathways in spleen and thymus. We have analyzed different protein expression by western blotting and immunohistochemistry and mRNA expression by RT-PCR and gel electrophoresis. We also have measured mitochondrial trans-membrane potential, ROS and CD4(+) and CD8(+) population by flow cytometry. Sub lethal doses of copper in spleen and thymus of in vivo Swiss albino mice promote different apoptotic pathways. In case of spleen, ROS generation and mitochondrial trans-membrane potential changes promotes intrinsic pathway of apoptosis that was p53 independent, ultimately leads to decrease in CD4(+) T cell population and increase in CD8(+) T cell population. However in case of thymus, ROS generation and mitochondrial trans-membrane potential changes lead to death receptor that regulate extrinsic and intrinsic pathways of apoptosis and the apoptotic mechanism which was p53 dependent. Due to copper treatment, thymic CD4(+) T cell population decreased and CD8(+) T cell population was increased or proliferated. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules like p27, p73, p62, poly (ADP-ribose) polymerase (PARP) that differentially changed due to copper treatment in spleen and thymus of Swiss albino mice. Present study firstly demonstrates how apoptotic pathways differentially regulate copper induced immunosuppression.

Preparation and characterization of monoclonal antibody specific for copper-chelate complex.

Monoclonal antibody (MAb) against chelated Cu(2+) was developed. The conjugate employed as immunogens in BALB/c mice to raise antibodies was synthesized using Cu(2+) coupling to keyhole limpet hemocyanin (KLH) via a bifunctional chelator (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, p-SCN-Bn-DOTA). One of the hybridomas secreting antibody that bound tightly to Cu(2+)-ethylenediamine tetraacetic acid (EDTA) complex but not to metal-free EDTA was isolated from the fusion between murine splenocytes and SP2/0 myeloma cells. The performance of a competitive enzyme-linked immunosorbent assay (ELISA) was assessed for its sensitivity to changes in pH and ionic strength. The cross-reactivities (CR) with other metals were below 1%, except for Hg(2+) with a CR of 7.19 %. The IC(50) value achieved for copper was 0.89 µg/mL, showing a detection range of 0.25-8.13 µg/mL and the lowest detection limit of 0.032 µg/mL. The concentration of Cu(2+) in environmental water and serum samples obtained by ELISA correlated well with Atomic Absorption Spectrometry (AAS), and the mean recovery was 93.7%. These results indicated that ELISA could be a convenient analytical tool for monitoring copper in drinking water and human serum.