Akihiro Hasegawa

Survey of microcystins in environmental water by a highly sensitive immunoassay based on monoclonal antibody

By using a highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody, microcystin (MC) concentration was analyzed in environmental water samples (total, 134), collected in 1993-1995 from ponds, lakes, reservoirs, and rivers in Japan, Thailand, Germany, and Portugal. MCs detected in the water samples filtered over a glass filter were designated as free MCs, and those samples that were freeze-thawed twice before the filtration were designated as total MCs. MCs (> 50 pg/ml) were detected in 14 of 24 samples collected from the lakes that were used as recreation and water supply in Japan in different regions. In the MC-positive samples, the concentration of free MCs was only a few percentages of the total MCs, indicating that the most part of MCs found in the water samples was present in algal cells. An additional trial on 33 samples collected continuously from Lake Inbanuma, Japan, during June-September 1994-1995 revealed that the total MCs were in a range of 52-52,000 pg/ml. In Chiang Mai, Thailand, 6 of 10 samples were positive, with the mean and highest of 161 and 354 pg/ml, respectively. In the Frankfurt area. Germany, 4 of 10 and 7 of 8 samples collected in the same lakes for recreation in July 1993 and November-December 1994 showed the presence of MCs, with their mean and highest values of 257 and 407 pg/ml, respectively. Another survey of MCs in dense bloomed samples collected with plankton net revealed a contamination of MCs up to 36,000 pg/ml. In Portugal, 28 of 29 samples from 4 lakes, 20 rivers, and 5 reservoirs were positive for MCs, with the respective means of 13,664, 11,048, and 2,278 pg/ml. These data indicated that MCs contaminate environmental water in ponds, rivers, lakes, and reservoirs worldwide. The present ELISA is considered to be a reliable tool for the mass monitoring and risk assessment of MCs in water supplies.

Immunoaffinity column as clean-up tool for determination of trace amounts of microcystins in tap water

Trace amounts of microcystins (MCs) in drinking water should be monitored because of their potential hazard for human health as an environmental tumor promoter. We describe here a new clean-up tool with immunoaffinity column (IAC) for determination of trace amounts of MCs (from pg to microg/litre) in tap water. The water samples were concentrated with IAC clean-up and MCs levels were determined by HPLC with UV detection or enzyme-linked immunosorbent assay (ELISA). In the combination with HPLC analysis, mean recovery of microcystin-LR (MCLR),-RR and-YR spiked to tap water were 91.8%, 77.3% and 86.4%, respectively, in the range 2.5-100 microg/litre. The chromatogram of MCs-spiked tap water sample cleaned up with IAC showed effective elimination of the impurities compared to that with octadecyl silanized cartridge, which had been cleaned up with a conventional method. Also, in the combination with highly sensitive ELISA, mean recovery of MCLR spiked to tap water was 80% in the range 0.1-1000 ng/litre. The combined methods developed here can detect pg to microg/litre of MCs in tap water. The overall results indicated that IAC will be suitable as a clean-up tool for trace amounts of MCs in tap water.

Further Survey of Aflatoxin M1 in Milk Powders by ELISA

A survey of AFM1 residues in 58 commercial milk powder samples was carried out using an enzyme‐linked immunosorbent assay (ELISA) based on a monoclonal antibody against aflatoxin M1 (AFM1). The samples were collected from the USA (10), China (28), Italy (14), New Zealand (3) and Poland (3). The ELISA was performed without the need for clean‐up procedures. The data revealed that 4 (US), 21 (Chinese) and 1 (Polish) samples were positive for AFM1, with an average of 95.5, 102.8 and 85.0 pg g‐1 of the AFM1respectively.

An improved indirect competitive Elisa for aflatoxin m1 in milk powders using novel monoclonal antibodies

Among three newly prepared monoclonal anti‐aflatoxin M1 antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M1, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml‐1 of aflatoxin M1 in solution. The contents of aflatoxin M1 in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M1. A limited ELISA survey showed the presence of aflatoxin M1 in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g‐1, and it was confirmed by an improved HPLC analysis.