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.

Evaluation of a novel rapid detection system for yeasts and molds.

A novel system, the NISSUI rapid detection system, has been developed to rapidly detect yeasts and molds in food. This system consists of a liquid medium containing resazurin as a redox indicator, a unique original micro-well dish containing 676 micro-wells, and a fluorescence-monitoring instrument with an incubator. To evaluate this system, orange juice, milk, and physiological saline solutions artificially inoculated with yeasts or molds were used as samples. Comparison of the new system used at 28ºC for 48 h with a spread-plate method using a potato-dextrose-agar plate at 25ºC for 120 h yielded a correlation coefficient (r) of 0.95. Our data reveal that the new method considerably shortens the time required for detection of yeasts and molds in food.

[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.

[Laboratory-based evaluation of a newly developed immunochromatographic test method by using synthesized oligonucleotide-bound protein probes for simultaneous detection of hepatitis B surface antigen (HBsAg) and specific antibodies to Treponema pallidum].

An immunochromatographic test method using synthesized oligonucleotide-bound protein probes was newly developed and evaluated for simultaneous detection of hepatitis B surface antigen (HBsAg) and specific antibodies to Treponema pallidum (TP). The test principle includes, first, antigen-antibody reaction, and secondly, DNA (oligonucleotide)-DNA interaction. The test device is composed of colloidal gold-labeled HBs antibody and TP antigen, and oligonucleotide-labeled HBs antibody and TP antigen. When the test sample contains HBsAg and/or TP specific antibody, the colloidal gold-labeled probes and oligonucleotide-labeled probes will make a sandwich complex with the target. Then, the formed complex migrates and is immobilized by the respective complementary oligonucleotide fixed on the different lines of the membrane. The color development of colloidal gold was visually read after 20 min and/or 60 min incubation, and easily interpretable, positive or negative. When the performance panels of Boston Biomedica Inc. (BBI) for HBsAg and TP-specific antibody, the results indicated; first, the most positive serum and plasma specimens with 1.2 IU/ml of HBsAg were correctly determined as positive, and secondly, all the test results for TP-specific antibody were comparable to the results of fluorescent treponemal antibody test (FTA-ABS). 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. Also, when the clinical serum and plasma samples were tested, sensitivity and specificity were estimated to be 87.0% and 100% for HBsAg, and both 100% for TP-specific antibody, respectively. With these results, we can conclude that this newly developed immunochromato-graphic test method will be applicable to simultaneous detection of multiple antigen and/or specific antibody in a single device, and will be expected to be widely applied in a clinical setting.

[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.