Yukikatsu Itoh

Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai

Three murine monoclonal antibodies, OA-1, OA-2 and OA-3, against okadaic acid were prepared from hybridoma clones obtained by fusion of mouse 653 myeloma cells with mouse immune spleen cells sensitized to okadaic acid-ovalbumin conjugate. Each antibody reacted with dinophysistoxin-1 ( = 35-methylokadaic acid) as well as okadaic acid, but did not react with the other diarrhetic shellfish poisons or related compounds, such as 7-O-palmitoyl-okadaic acid (analogue of dinophysistoxin-3), pectenotoxin-1 and yessotoxin. A competitive inhibition enzymelinked immunosorbent assay which employed OA-3 antibody was performed and showed a sensitivity of about 10 ppb (10 ng/ml) for okadaic acid. This simple and time-saving ELISA assay system may be useful for the specific detection of diarrhetic shellfish poisons.

Determination of Ochratoxin a in coffee beans and coffee products by monoclonal antibody affinity chromatography

A clean‐up method using a monoclonal antibody affinity column was developed for the analysis of ochratoxin A (OTA) in coffee products. Monoclonal antibody specific for OTA was covalently bound to Sepharose‐4B and the resultant affinity column was used for the clean‐up of sample extracts. OTA was quantitatively recovered from the affinity column. The subsequent high performance liquid chromatography (HPLC) of the eluted sample revealed no marked interference peaks when compared with those extracts obtained by conventional approaches. The HPLC peak coinciding with OTA was confirmed by derivatization into ethyl and n‐propyl esters. Furthermore, the present affinity column could be regenerated at least 30 times without significant loss of the binding activity. The detection limit of OTA in the present affinity column‐HPLC procedure was 0.5 μg/kg for coffee beans and instant coffee powder, and 0.025 μg/kg for a canned coffee drink. The recoveries of OTA from coffee products were more than 98%, with coefficient...

Characterization of morphine-specific monoclonal antibodies showing minimal cross-reactivity with codeine

Six murine monoclonal antibodies against morphine were produced using N-(4-aminobutyl)normorphine as a hapten. Most of the antibodies obtained distinguished the substituents at the 3 and 6 positions of morphine. This property of the antibodies led to a reduction in cross-reactivity with codeine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) to negligible levels. However, one of the antibodies distinguished the substituent only at the 3 position of morphine, which cross-reacted with M-6-G, naloxone and naltrexone. In the competitive inhibition enzyme-linked immunosorbent assay, morphine was detected at concentrations as low as circa 100 pg/ml.