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.
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.
To develop an accurate diagnosis of "new ulcer disease" in koi carp, we produced four monoclonal antibodies (mAbs) against a strain of atypical Aeromonas salmonicida isolated from koi carp. These mAbs did not cross-react with an isolate of atypical A. salmonicida from Japanese flounder and other pathogenic bacteria. Re-isolation from artificially infected koi carp was achieved by selecting the blue colonies on agar medium containing Coomassie brilliant blue, and some of the colonies were detected by immunofluorescent staining using the mAbs. These results suggested that the mAbs can distinguish atypical A. salmonicida from koi carp from resident aeromonads.
Two kinds of polyclonal antibodies against beef myoglobin were produced by immunizing rabbits with denatured myoglobin or with a peptide having the amino acid sequence unique to beef myoglobin. The latter peptide antibody reacted specifically with beef myoglobin without cross-reacting with other food proteins or with pork and chicken myoglobin. A sandwich ELISA, in which this peptide antibody was used as a capture antibody and the anti-denatured myoglobin antibody as an enzyme-labeled antibody, was also specific against beef myoglobin. When the beef content was determined in pork-or chickenbased model foods by this sandwich ELISA, the recoveries of beef protein agreed well with the actual mixing ratio of beef. Moreover, it could be confirmed by this method that the labeling of beef usage in several commercially processed foods was reasonable. Thus, a sandwich ELISA suitable for the evaluation of beef content in various foods was established.
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