Metallothionein Antibody Research Articles

Detection of tilapia metallothionein using antibody-immobilized quartz crystal microbalance sensor

Metallothionein (MT) was induced in tilapia by three subcutaneous injections of CdCl2 and the recovered MT protein was purified to homogeneity through DEAE-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. For use as biological component, a polyclonal anti-tilapia MT antibody was prepared using the purified tilapia MT. During the preparation of the tilapia MT sensor, topological changes pertinent to immobilization of the thiolated antibody, subsequent blocking of the surface with BSA and final immune reaction were observed by AFM imaging. Specificity of the sensor was confirmed through comparison of the responses of defective and intact sensor chips, and the responses of the sensor chips that were immobilized with the antibody and BSA. Concentration-dependent responses of the sensor showed a linear relationship of Y (log10 sensor response)=0.3438X (log10 tilapia MT)+0.7368 (r=0.9606) in double-logarithmic plot with a limit of detection of 20ng/mL. When the serum from a tilapia induced with CdCl2 was compared with the control serum, induction of MT was detectable even at 20,000-fold dilution of tilapia serum.

Responses of label-free metallothionein sensor immobilized with thiolated antibody

Metallothionein (MT) is a low molecular weight metalloprotein that is induced by extraneously introduced heavy metals to the blood and organs of living organisms and is generally regarded as a useful biomarker of possible heavy metal contamination. In this study, a polyclonal anti-rabbit MT antibody was manufactured using a high-purity MT antigen and exploited as the biological component to develop a label-free quartz crystal microbalance immunosensor via the thiolation with sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate. When measured at each step of antibody immobilization by the batchwise operation using a dip cell, the biggest sensor response was obtained in the case of the whole course of immobilization, which indicated that the immobilization procedure of this study was effective and the obtained sensor signal was the output of the corresponding immune reaction. When determined for MT at two antibody dilutions for immobilization, concentration-dependent sensor responses were more conspicuous at the antibody dilution of 1:5 and the limit of detection of 20 ng/mL was observed.

Quantitative immunochemical evaluation of fish metallothionein upon exposure to cadmium

Efficient implementation of an environmental biomarker requires multi-annual comparability over a wide geographical range. The present study improved the comparability of a quantitative competitive metallothionein (MT) enzyme-linked-immuno-sorbent-assay (ELISA) in the sentinel fish Lithognathus mormyrus by introducing to the assay recombinant MT and beta-actin standards. Commercial antibodies for cod MT and mammalian actin were implemented. In addition, a sensitive anti L. mormyrus MT antibody was produced, adequate only for solid phase immunochemical assays. Cadmium was applied to the fish through injection and feeding to serve as a testing platform of the ELISA. The results demonstrated high potential protective capacity of the liver against toxic levels of transition metals through increasing MT levels. MT transcript levels were evaluated also from fish sampled at polluted and relatively clean natural sites, indicating applicability of MT as biomarker of exposure to a multi-factorial pollution, in comparison to its low revealed sensitivity to controlled cadmium exposure.

Plasma Metallothionein Antibody, Urinary Cadmium, and Renal Dysfunction in a Chinese Type 2 Diabetic Population

It has been reported that diabetes may increase the risk of cadmium-induced kidney damage. The presence of metallothionein antibody (MT-Ab) increased the susceptibility for tubular damage among cadmium workers. This study focused on the relationships between levels of MT-Ab, urinary cadmium, and kidney function in a Chinese type 2 diabetic population. A cross-sectional study was performed on 229 type 2 diabetic patients (92 men and 137 women) who were recruited from two community centers in one district of Shanghai City in China. Information was obtained from interviews, health records, and blood and urine samples. Levels of the tubular biomarker beta2-microglobulin increased significantly when the levels of MT-Ab and urinary cadmium were elevated in male and female subjects; in contrast, the levels of urinary albumin, a glomerular biomarker, did not display such a pattern. After adjusting for potential confounding covariates, logistic regression showed that the odds ratios (ORs) of tubular dysfunction increased upon 1) increasing the MT-Ab concentration from a low to high level (OR 5.56 [95% CI 2.25-13.73]) and 2) increasing the level of urinary cadmium from <1 to >or=1 microg/g creatinine (3.34 [1.17-9.53]); the OR of patients currently smoking was 3.51 (1.14-10.80) relative to that of those who had never smoked. This study proves that the presence of MT-Ab can potentiate tubular dysfunction among diabetic subjects and that patients with high MT-Ab levels are more prone to development of tubular damage.

Plasma Metallothionein Antibody and Cadmium-Induced Renal Dysfunction in an Occupational Population in China

It has been reported that anti-metallothionein (a metallothionein antibody) is present in the circulation of healthy subjects and in patients suffering from atopic dermatitis. The aim of this study was to investigate whether cadmium-induced renal dysfunction is related to the presence of the plasma metallothionein antibody (MT-Ab) in workers exposed to cadmium (Cd) occupationally. Plasma metallothionein antibody was determined by enzyme linked immunosorbent assay (ELISA) techniques, and both exposure assessment and risk assessment were conducted in cadmium-exposed workers in China. We demonstrate that there is a significantly increased prevalence of renal dysfunction with respect to the level of urinary cadmium in a dose-dependent manner. We found no significant correlations between the levels of MT-Ab and the external or internal exposure doses of cadmium (p > 0.05), but the levels of MT-Ab did correlate positively with two biomarkers of renal dysfunction-urinary beta2-microglobulin (UB2M; r = 0.218, p < 0.05) and N-acetyl-beta-D-glucosaminidase (UNAG; r = 0.302, p < 0.001)-in the cadmium-exposed workers. Workers who have high levels of MT-Ab display cadmium-induced tubular nephrotoxicity more frequently than those possessing low levels of MT-Ab; odds ratio (OR) 4.2; 95% confidence intervals 1.2-14.5 (p < 0.05). This study suggests that subjects that have higher MT-Ab levels more readily develop cadmium-induced renal dysfunction. Thus, the levels of plasma MT-Ab can be used as a biomarker of susceptibility to renal dysfunction in occupational cadmium exposure.

Early mouse preimplantation development is unaffected by microinjection of metallothionein antibodies.

Polyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.

The significance of the nuclear and cytoplasmic localization of metallothionein in human liver and tumor cells.

Metallothioneins are a group of low-molecular-weight intracellular proteins present in high levels in fetal mammalian livers, bound to zinc and copper. They are also present in two major isoforms in low basal levels in various organs of adults in several species. Although a number of functions have been proposed for metallothioneins, their major biological roles may be in the storage of zinc and copper during rapid growth and development, and also in the detoxification of certain toxic metals. In adult liver, metallothionein is mainly localized in the cytoplasm, it is localized also in the hepatocyte nuclei in human fetal liver and fetal and neonatal rat liver, as determined by immunohistochemical staining with a specific metallothionein antibody. Because of its high expression in fetal development, the potential role of metallothioneins in human tumors was investigated. The cellular localization of metallothionein was demonstrated in various human tumors such as thyroid tumors, testicular germ cell carcinoma, bladder transitional cell carcinomas, and salivary gland tumors. In most of these tumor tissues, metallothioneins were found in high levels in nucleus and cytoplasm in both benign and malignant tumors, although the proliferating edge of the malignant tumors showed most intense metallothionein staining. The expression of metallothionein is not universal to all tumor growth; its presence may depend on various factors, such as the type of tumor, cellular origin, morphological heterogeneity, or stage of growth. Human testicular seminomas, which are well differentiated, showed little expression of metallothionein irrespective of the staging, as compared to less well-differentiated embryonal carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

The incorporation of 35S-cysteine and 3H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of 35S-cysteine over 3H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.

A radioimmunoassay for perch ( Perca fluviatilis) metallothionein

A sensitive radioimmunoassay (RIA) for the measurement of metallothionein (MT) from perch ( Perca fluviatilis) has been developed. The method is a double-antibody RIA with rabbit anti-perch MT serum as first antibody, goat anti-rabbit immunoglobulin G as second antibody, and perch MT conjugated to 125I-labeled Bolton-Hunter reagent as tracer. The rabbit antiserum raised against perch MT recognizes rainbow trout ( Oncorhynchus mykiss) MT, but shows little cross-reactivity with horse MT. At a dilution of 1:2000, the MT antibodies bind 36% of the tracer when no cold ligand is present. The sensitivity of the assay is 15 pg perch MT per tube and the practical working range is 0.15–250 ng perch MT per tube. The RIA allows determination of MT in plasma or lysed blood cells at concentrations as low as 3.0 ng/ml and in tissues at levels above 9.0 ng/g (wet weight). Intra- and interassay coefficients of variation were 6 and 10%, respectively.

Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein

The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophpresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, p I values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-activity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6–12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.

A radioimmunoassay for metallothionein in fish

A sensitive radioimmunoassay (RIA) for metallothionein (MT) from perch ( Perca fluviatilis) has been developed. The method described is a double antibody RIA with rabbit anti-perch-MT as first antibody, goat anti-rabbit-immunoglobulin G as second antibody and perch MT conjugated to [ 125I] Bolton-Hunter reagent as tracer. The antiserum cross-reacts with MT from four fish species tested in addition to perch, but has low avidity for horse kidney MT. At a dilution of 1:2000, the MT antibodies were able to bind 36% of the tracer when no cold ligand was present. The sensitivity of the assay was 15 pg perch MT/tube and the practical working range was found to be 0·15–250 ng perch MT/tube. The RIA allows determination of MT in plasma or lysed blood cells at concentrations as low as 3·0 ng/ml and in tissues at levels over 9·0 ng/g (wet weight). Intra- and interassay coefficients of variation were 6 and 10% respectively.

Formation and characterization of aurothioneins: Au,Zn,Cd-thionein, Au,Cd-thionein, and (thiomalato-Au)chi-thionein.

Three gold-containing thioneins (Au,Zn,Cd-Th, Au,Cd-Th, and (TmSAu)chi Th, where Th = thionein and TmS = thiomalate) have been prepared by the reactions of horse kidney Zn,Cd-thionein with gold thiomalate (AuSTm). When thionein was present in excess, the thiomalate ligand was displaced and the protein chelated the gold in a bidentate fashion. Primarily zinc but also some cadmium was displaced to form Au,Zn,Cd-Th or Au,Cd-Th. Excess AuSTm reacted to form (TmSAu)chi-thionein with monodentate coordination of the protein to each bound gold, retention of the thiomalate, loss of zinc and cadmium, and an increase in the Stokes radius of the product. EXAFS/XANES studies of Au,Zn,Cd-Th and (TmSAu)chi Th established that the oxidation states and coordination environments of gold were Au(I)S2 and that the gold-sulfur bond distances were 229 and 230 pm, respectively. Radioimmunoassay established that the aurothioneins retained their antigenicity to native metallothionein antibodies. Metal exchange reactions with gold were complete within 5-10 min when Zincon or 4-(2-pyridylazo) resorcinol was used to monitor Cd2+ and Zn2+ displacement.

Cross-reactivity of metallothioneins from different origins with rabbit anti-rat hepatic metallothionein antibody

Rat hepatic Cd-metallothionein was purified and isolated into its two components, metallothionein 1 and 2, by disc electrophoresis. Antibodies to metallothionein 2 were generated in rabbits. The antiserum reacted with the protein and formed a single precipitin band on a double diffusion plate. By ammonium sulfate precipitations, it was found that the antiserum cross-reacted with rat hepatic metallothionein 1. Cross-reactivity of the antiserum was also observed for components of rat renal Cd-metallothionein, rabbit hepatic Cd-metallothionein and human renal metallothionein.