A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.

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Abstract.A disintegrin and metalloprotease protein 15 (ADAM15), a membrane-anchored glycoprotein, is believed to function in cell-cell interactions via an integrin binding motif within its disintegrin domain. To screen its target proteins, the recombinant ADAM15 disintegrin domain (RADD) was expressed in Escherichia coli, biotinylated and used in a protein pull-down assay in vitro. A total of eight kinds of proteins were identified by 2DE/LC-MS-MS. One of them, p38 kinase, was selected for further investigation of its involvement in the anti-proliferative effect of RADD on melanoma cells. Phosphorylation of p38 kinase in melanoma cells was detected upon treatment with RADD. Furthermore, the suppression of p38 kinase activity resulted in a decrease in the RADD inhibitory effect on melanoma cell proliferation. These results provide evidence that RADD inhibits melanoma cell proliferation partly through p38 kinase activation. IntroductionADAMs (a disintegrin and metalloproteinases) are transmembrane glycoproteins that are implicated in diverse biological processes including proteolysis, cell adhesion, cell fusion, protein ecto-domain shedding and intracellular signaling (1). They contain multiple functional domains: a pro-domain, a metalloproteinase, a disintegrin domain and a cysteine-rich region containing an epidermal growth factor (EGF) repeat and a transmembrane domain (2). Among all of the known human ADAMs, ADAM15 (metargidin) is unique since it has an RGD (Arg-Gly-Asp) motif, an amino acid hairpin loop maintained by disulfide bridges, in its disintegrinlike domain. This RGD motif enables selective binding to integrins, a family of heterodimeric and transmembrane receptors that play key roles in the signaling networks (2,3).The interaction of ADAM15 with integrin α v ß 3 has been proven to be important in several biological processes, as α v ß 3 is implicated in diverse functions including angiogenesis, tumor cell metastasis and osteoporosis (2,4). Integrin α v ß 3 is expressed by various cell types and has several ligands, including extracellular matrix molecules such as vitronectin and cell surface proteins such as CD31 (5). ADAM15 was also found to adhere to melanoma cells via α v ß 3 and increased expression of α v ß 3 in these cells is positively correlated with increased malignancy (4). The human recombinant ADAM15 disintegrin domain (RADD) has been considered as a potential intrinsic inhibitor of angiogenesis, tumor growth and metastasis (6). One of the anti-cancer mechanisms involving the ADAM15 disintegrin domain was thought to be related to its integrin-binding activity. It can interact with integrin to loosen tumor cell adhesion to the underlying matrix and then prevent tumor progression (7). However, it is conceivable that the ADAM15 disintegrin domain might also interact with other cellular proteins to exert biological functions. The identification of novel binding proteins will facilitate the investigation of the anti-cancer mechanisms that engage the ADAM15 disintegrin domain. Although, few stu...

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This study was aimed at increasing the production of the recombinant human ADAM15 disintegrin domain (RADD) in Escherichia coli by releasing the rare codons and restricting amino acid residues. Three different strategies for increasing RADD production were examined: to select the suitable host strain, to optimize the rare codons, and to delete the amino acids residues. The total fusion protein glutathione-S-transferase (GST)-RADD concentration of 298 mg/l and 326 mg/l were achieved by selecting E. coli Rosetta (DE3) as the host strain and by changing GGA to GGC at the GST-RADD cassette, respectively. The RADD concentration was increased by 35.7% by eliminating the "Pro-Glu-Phe" residues at the GST-RADD junction. By combinational utilizing the preferred codon introduction and amino acid sequence optimization in E. coli Rosetta (DE3), the highest RADD concentration of 68 mg/l was achieved. The proposed strategy may provide an alternative approach for other enhanced recombinant protein production by E. coli.