Preparation of hapten-specific monoclonal antibody for cadmium and its ELISA application to aqueous samples

High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.

Expression, Purification and Characterization of C-FADD

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecuiar structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain a FADD variant that containing C-terminal region. In this study, mouse FADD （80-205） containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni^2＋ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical a-helix spectrum in CD （circular dichroism） assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ia-binding activity which was important for its non-apoptotic function.

Surface property and in vitro toxicity effect of insoluble particles given by protein corona:Implication for PM cytotoxicity assessment

In vitro toxicological assessment helps explore key fractions of particulate matter(PM)in association with the toxic mechanism.Previous studies mainly discussed the toxicity effects of the water-soluble and organic-soluble fractions of PM.However,the toxicity of insoluble fractions is relatively poorly understood,and the adsorption of proteins is rarely considered.In this work,the formation of protein corona on the surface of insoluble particles during incubation in a culture medium was investigated.It was found that highly abundant proteins in fetal bovine serum were the main components of the protein corona.The adsorbed proteins increased the dispersion stability of insoluble particles.Meanwhile,the leaching concentrations of some metal elements(e.g.,Cu,Zn,and Pb)from PM increased in the presence of proteins.The toxicity effects and potential mechanisms of the PM insoluble particle–protein corona complex on macrophage cells RAW264.7 were discussed.The results revealed that the PM insoluble particle–protein corona complex could influence the phagosome pathway in RAW264.7 cells.Thus,it promoted the intracellular reactive oxygen species generation and induced a greater degree of cell differentiation,significantly altering cell morphology.Consequently,this work sheds new light on the combination of insoluble particles and protein corona in terms of PM cytotoxicity assessment.