[Rapid Screening System for Paralytic Shellfish Toxins in Bivalves by Oligonucleotide Lateral Flow Immunoassay].

The mouse bioassay (MBA) for paralytic shellfish toxins (PSTs) in bivalves has been used as an official method in Japan. It is necessary to develop an alternative method to animal experiments in PSTs assay because 3Rs (Replacement, Reduction, and Refinement) of animal experiments are required from the animal welfare point of view. Various methods such as HPLC-FL, receptor binding assay, LC-MS/MS and ELISA have been established to detect PSTs without performing animal experiments. The present study was undertaken to develop a screening method using oligonucleotide lateral flow immunoassay (OLFIA) for detecting PSTs in bivalves. The screening level was defined as positive at 2 MU/g of MBA that is the half regulation limit of PSTs monitoring in Japan. All 20 positive (equal to or more than 2 MU/g) samples judged from MBA showed a positive reaction in the OLFIA. No positive samples resulted in a false negative reaction. The OLFIA exhibited high accuracy at 2 MU/g of screening criteria. The authors demonstrated here that the OLFIA can be useful for rapid detection of PSTs in bivalves.

[Detection of fish protein in food products by lateral flow immunoassay].

The major fish allergen is parvalbumin, a sarcoplasmic protein. In this study, a novel lateral flow immunoassay for the detection of fish protein in food products was developed using a polyclonal antibody raised against Pacific mackerel Scomber japonicus parvalbumin. The proposed lateral flow immunoassay showed high reactivity to various fish parvalbumins, but the reactivity to bullfrog parvalbumin was very low. The detection limit of the immunoassay for fish parvalbumin was estimated to be 2.0 µg protein/g, which matches the sensitivity required in the current Japanese food labeling system. Furthermore, the lateral flow immunoassay could detect fish parvalbumin without being affected by food matrices and was applicable even to heat-denatured parvalbumin. These results showed that the lateral flow immunoassay developed in this study is specific to fish parvalbumin, and should be useful as a rapid detection method for fish protein in processed food products.

A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 µg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods.

[Extraction method suitable for detection of unheated crustaceans including cephalothorax by ELISA].

When unheated whole samples of crustaceans (shrimp, prawn and crab) were analyzed with our ELISA kit (FA test EIA-Crustacean 'Nissui') using anti-tropomyosin antibodies, a remarkable reduction in reactivity was recognized. This reduction in activity was found to be due to the digestion of tropomyosin during the extraction process by proteases contained in cephalothorax. To avoid the digestion of tropomyosin by proteases, we developed an extraction method (heating method) suitable for the detection of tropomyosin in unheated crustaceans including cephalothorax. Experiments with unheated whole samples of various species of crustaceans confirmed that the heating method greatly improved the low reactivity in the standard method; the heating method gave extraction efficiencies of as high as 93-107%. Various processed crustaceans with cephalothorax, such as dry products (unheated or weakly heated products) and pickles in soy sauce (unheated products), that showed low reactivity with the standard method were confirmed to give superior results with the heating method. These results indicated that the developed heating method is suitable for detecting unheated crustaceans with cephalothorax by means of the ELISA kit.

[Simultaneous detection of hepatitis B virus surface antigen (HBsAg) and hepatitis C virus (HCV) related antibodies by an immunochromatographic test method using synthesized oligonucleotide-bound protein probes, OligoFast].

An immunochromatographic test using synthesized oligonucleotide-bound protein probes, OligoFast (Nissui Pharmaceutical Co., Ltd., Tokyo) was developed and evaluated for simultaneous detection of hepatitis B surface antigen (HBsAg) and antibodies related with hepatitis C virus (HCV). The color development of colloidal gold was visually read and easily interpretable for the respective antigen and antibody, positive or negative. When the performance panels of Boston Biomedica Inc. (BBI) for HBsAg and HCV-related antibodies were assayed, the results indicated; first, the most positive specimens with 1.2 IU/ml of HBsAg were correctly determined as positive, and secondly, all the positive specimens for HCV-related antibodies confirmed with Ortho RIBA 3.0 were consistently determined as positive and additional two undetermined specimens were interpreted as positive. However, when the seroconversion panel of BBI for hepatitis B virus (HBV) infection, the seroconversion was delayed 20 to 30 days when compared to HBV DNA detection. When the clinical serum specimens were tested in comparison with the automated AxSYM (Abbott Laboratories, Abbott Park, IL, U.S.A.), both sensitivity and specificity were estimated to be 100% for HBsAg, and 91.3% and 99.0% for HCV-related antibodies, respectively. With these results, we can conclude that this newly developed immunochromatographic test will be applicable to simultaneous detection of HBsAg and HCV-related antibodies in a single device, and will be expected to be widely applied in a clinical setting.