Osamu Kawamura

A sensitive enzyme-linked immunosorbent assay of ochratoxin A based on monoclonal antibodies.

We prepared seven monoclonal antibodies (mAbs, OTA.1, 2, 3, 4, 5, 6 and 7) which were reacted with ochratoxin A (OTA), and have developed a specific and highly sensitive enzyme-linked immunosorbent assay (ELISA) for detection of OTA. The mAbs, OTA.1, 3, 4, 5 and 7, specifically reacted with OTA but much less with its analogs, ochratoxin B (OTB, about 1% of OTA) and ochratoxin alpha (OT alpha, less than 0.1% of OTA). One of the mAbs, OTA.2, equally reacted with OTA and OTB but hardly at all with (4R)-4-hydroxyochratoxin A or OT alpha (less than 0.1% of OTA). All of the mAbs reacted with ochratoxin C. None of the mAbs reacted with coumarin, 4-hydroxycoumarin or L-beta-phenylalanine. In the competitive ELISA with OTA.1 and OTA.7, the lowest detectable amount of standard OTA in solution was 50 pg/ml (2.5 pg per assay). This assay was applied for the quantitation of OTA added to chicken meat, wheat flour, porcine plasma and bovine serum. With minimal sample preparation, reliable and reproducible determinations were possible when concentrations of OTA were higher than 0.1-1 ng/g.

Determination of Ochratoxin a in coffee beans and coffee products by monoclonal antibody affinity chromatography

A clean‐up method using a monoclonal antibody affinity column was developed for the analysis of ochratoxin A (OTA) in coffee products. Monoclonal antibody specific for OTA was covalently bound to Sepharose‐4B and the resultant affinity column was used for the clean‐up of sample extracts. OTA was quantitatively recovered from the affinity column. The subsequent high performance liquid chromatography (HPLC) of the eluted sample revealed no marked interference peaks when compared with those extracts obtained by conventional approaches. The HPLC peak coinciding with OTA was confirmed by derivatization into ethyl and n‐propyl esters. Furthermore, the present affinity column could be regenerated at least 30 times without significant loss of the binding activity. The detection limit of OTA in the present affinity column‐HPLC procedure was 0.5 μg/kg for coffee beans and instant coffee powder, and 0.025 μg/kg for a canned coffee drink. The recoveries of OTA from coffee products were more than 98%, with coefficient...

A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B1 (AFB1) in a peanut product. AFB1 was converted to AFB1-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB1-BSA conjugates were fused with myeloma cells. After double selection with AFB1-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB1 MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB1 MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) — labeled sheep anti — mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker.The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB1 per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB1 per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB2 as well as AFB1, weakly with AFG2 > AFG1 > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB1, weakly with COLI > AFQ1 and less weakly with others; (c) AF 5 was AFQ1 as well as AFB1 weakly with COL II > AFG2 > COL I and less weakly with others.The 60% aqueous methanol extracts of oil-roasted blanched peanuts (“butter peanut”), naturally contaminated with AFB1, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB1 per g samples.

Enzyme‐linked immunosorbent assay for detection and survey of ochratoxin a in livestock sera and mixed feeds

By a combination of monoclonal antibody (mAb)‐based enzyme‐linked immunosorbent assay (ELISA) and a simplified extraction procedure, the contents of ochratoxin A (OTA) in pig, cow and horse serum and in mixed feeds for pigs were analyzed. Preliminary ELISA with anti‐OTA mAb. 7/alkaline phosphatase (ALP)‐labelled second antibody has revealed that all 19 sera of pigs contained OTA at an average of 0–4 ngml‐1, and these data were closely correlated with HPLC analysis (r = 0.903). OTA in the sera was confirmed by both chemical methylation and enzymatic hydrolysis. The levels of OTA in one fetal pig serum sample and in five cow serum samples were below the detection limit of the ELISA (<0.1 ngml‐1). HPLC analysis revealed 0.3 and 0.7 μg kg‐1 of OTA in two samples of mixed feeds. The second survey on 139 serum samples with an improved ELISA utilizing horseradish peroxidase (POD) as enzyme label giving a detection limit of 0.01 ngml‐1) has demonstrated average levels of 0.36, 0.02 and 0–11 ng ml‐1 for OTA in ser...

Survey of T‐2 toxin in cereals by an indirect enzyme‐linked immunosorbent assay

A survey of T‐2 toxin in cereals randomly sampled in 11 countries was carried out, employing an indirect competitive enzyme‐linked immunosorbent assay (ELISA). Among 540 samples, 72 (13%) contained over 10 ng/g of T‐2 toxin with an average of 91.5 ng/g. The content of T‐2 toxin was highest (500 ng/g) in an oat sample from Western Germany, and a relatively high content was observed in barley, wheat, oat and rye produced in Finland, Norway, Western Germany and the USSR. The ELISA method combined with a one‐step extraction procedure is proposed for the mass screening of T‐2 toxin in cereals.

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.

Improved preparation of T-2 toxin-protein conjugates

T-2 toxin (T-2) was converted into T-2 hemisuccinate (T-2 HS), T-2 hemiglutarate (T-2 HG) and T-2 hemiphthalate (T-2 HP), and conjugated with bovine serum albumin (BSA), ovalbumin (OVA) and keyhole limpet haemocyanin (KLH) using carbodiimide under various conditions. The recovery of T-2 conjugated proteins and the amount of T-2 bound were largely dependent on the amount of coupling reagent, pH, reaction temperature, buffer and reaction time. In regard to T-2 derivatives binding with a protein, T-2 HG was the most efficient, followed by T-2 HS and then T-2 HP. With regard to T-2 to protein binding, KLH was the most efficient, followed by BSA and then OVA. To promote T-2-protein binding, phosphate-buffered saline was more effective than cacodylate buffer. Within a pH range of 5.5-7.0, weakly acidic conditions were more efficient than neutral conditions for promoting T-2-protein binding.

Immunohistochemistry of fumonisin in poultry using avidin–biotin–peroxidase system

Using monoclonal anti-fumonisin B1 antibody (anti-FB1) and avidin-biotin-peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB1 or culture extract of Fusarium moniliforme str. 113F containing 150 microg of FB1 and 4 microg of FB2 were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues.

Safety and Quality in the Agricultural Product Chain in Brazil

An agriculture-intensive country should be aware of natural toxins, including both mycotoxins and cyanotoxins, which are closely associated with the quality of raw materials, for food safety and industry. The major production chains – corn, wheat, beef, and broiler chicken – are the top components of agribusiness, and they should be tracked by reliable and practical tools. The corn chain is of particular concern in food production; intensive controls, multi-year mycotoxin monitoring, and improved harmless/sustainable management methods for uninterrupted farming in the tropicsubtropics are needed to achieve a long-lasting trend. The rapid control of natural toxins (mycotoxin and cyanotoxin) has focused on immunochemical methods developed with highly specific monoclonal antibodies (mAb) matched with chroma‐ tographic methods. In parallel, the promising widespread application of nondestructive analytical methods based on NIR (Near Infrared Reflectance)