Development of a Rapid and Versatile Method of Enzyme-Linked Immunoassay Combined with Immunoaffinity Column for Aflatoxin Analysis.

We combined immunoaffinity column (IAC) and enzyme-linked immunosorbent assay (ELISA) methods to develop a rapid method to analyze total aflatoxin (AF) in foods, using a large number of samples. Using newly developed monoclonal antibodies, with high cross-reactivity and high organic solvent tolerance, we developed the IAC-ELISA method. Our IAC-ELISA method showed a good correlation with the high-performance liquid chromatography method for corn samples spiked with total AF. Certain food samples, such as chili powder, chocolate, green coffee beans, and roasted coffee beans, are difficult to measure owing to their matrices, which affect ELISA adversely. Our IAC-ELISA method clearly improved the recovery rates (79 to 109%) compared with the ELISA method (97 to 164%) for four food samples. Our method is simple and quick; thus, it may be ideal for routine inspections.

The validation of the NH Immunochromato O157 test kit.

NH Immunochromato O157 is an immunochromatographic test for detection of Escherichia coli O157:H7 and O157:NM in food. It enables simple and rapid testing for the target organism after 18-24 h enrichment. In inclusivity and exclusivity testing, all 50 O157:H7 strains and 15 O157:NM strains tested positive, while all 33 exclusivity strains yielded negative results. Taken together, all 98 strains tested in inclusivity/exclusivity testing were identified correctly. NH Immunochromato O157 method was compared to U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, Chapter 5.08, reference method for detection of E. coli O157:H7 in 25 g of raw ground beef. The performance of both methods was revealed to be statistically equivalent. Autoclaved and non-autoclaved sample enrichments yielded the same results, showing sterilization is not mandatory for testing with NH Immunochromato O157. The results of the robustness study were not statistically different in all conditions, suggesting that NH Immunochromato produce reliable results under various conditions. However, the users are recommended to follow the instruction when applying sample to the test strip, because a smaller sample volume may produce invalid result.

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods.

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.

[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.