Yoshihiko Uesaka

Development of a highly sensitive bead-ELISA to detect bacterial protein toxins

A highly sensitive sandwich enzyme‐linked immunosorbent assay to detect bacterial toxins was developed. Fab of anti‐toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti‐toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead‐ELISA was found to be quite high with various bacterial toxins: less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga‐like toxin II) of Escherichia coli, less than 200 pg/ml for heat‐labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.

Development of oligonucleotide lateral-flow immunoassay for multi-parameter detection

We have developed a highly sensitive and rapid oligonucleotide lateral-flow immunoassay (OLFIA), using a colloidal gold as an indicator, for the simultaneous detection of antigens and/or antibodies in specimen. This system can detect more than two types of antigens and/or antibodies in a single assay device at the same time. The device is basically composed of colloidal gold-labeled antibodies and oligonucleotide-labeled antibodies fixed in a conjugate pad, and the complementary oligonucleotide-labeled proteins are immobilized on a nitrocellulose membrane. If the target antigen is present in a specimen, the colloidal gold-labeled antibody and oligonucleotide-labeled antibody will make a complex with the antigen. Subsequently, the formed complex migrates to the place where complementary oligonucleotide is immobilized and is bound to the solid phase via the DNA-DNA interaction. As a result, more than two types of reactions can be detected on a single assay device by the combination of colloidal gold-labeled antibodies, different oligonucleotide-labeled antibodies and complementary oligonucleotide-labeled proteins immobilized at different places on a nitrocellulose membrane.

Detection of Cholera Toxin by a Highly Sensitive Bead-Enzyme Linked Immunosorbent Assay

A bead‐enzyme linked immunosorbent assay (bead‐ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead‐ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead‐ELISA. Evaluation of the sensitivity and specificity of the bead‐ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead‐ELISA was more sensitive than the RPLA. Quantification of CT by the bead‐ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead‐ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.

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.