ABSTRACT
An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.
Subject(s)
Antibodies, Monoclonal , Food Contamination/analysis , Fungicides, Industrial/analysis , Nitriles/analysis , Surface Plasmon Resonance , Vegetables/chemistry , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Maximum Allowable Concentration , Nitriles/immunologyABSTRACT
Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.
Subject(s)
Gene Expression Regulation , Glycoproteins/genetics , Histone Demethylases/metabolism , Carcinogenesis , DNA Methylation/drug effects , Endodeoxyribonucleases , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , HEK293 Cells , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/deficiency , Histone Demethylases/genetics , Histones/metabolism , Humans , Promoter Regions, Genetic/geneticsABSTRACT
We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490-2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and beta-mercaptoethanol for extraction was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed were discussed in connection with allergen labeling.
