Mario A.J. Aldao

Determination of Aflatoxin B 1 in Highly Contaminated Peanut Samples Using HPLC and ELISA

The precision, accuracy, detection limit and peanut matrix influence of an ELISA were analysed in the determination of aflatoxin B 1 . The assay was performed on two different reference samples: peanut extract and peanut paste, that were spiked with known amounts of aflatoxins. The lower detectable level was 0.5 w g kg -1 . The average intra-assay precision expressed as coefficient of variation (CV) was 11.7% for concentrations between 2.54 and 901 w g kg -1 and the average inter-assay precision was 29.7% for the same range of concentrations. The average accuracy measured by a recovery assay in samples that contained only aflatoxin B 1 was 107%. The correlation between the ELISA and high-performance liquid chromatography (HPLC) applied to 28 peanut samples artificially contaminated with Aspergillus flavus and A. parasiticus spores showed a high correlation ( r = 0.977, P < 0.0001). The detection limits and matrix influence associated with our ELISA procedure were more sensitive than those reported for other ELISA procedures due to specific antiserum treatment.

Aflatoxin production in six peanut (Arachis hypogaea L.) genotypes infected with Aspergillus flavus and Aspergillus parasiticus, isolated from peanut production areas of Cordoba, Argentina.

Aflatoxin contamination is one of the main factors affecting peanut seed quality. One of the strategies to decrease the risk of peanut aflatoxin contamination is the use of genotypes with resistance to Aspergillus infection. This laboratory study reports the resistance to Aspergillus infection and aflatoxin contamination of six peanut genotypes inoculated with 21 Aspergillus isolates obtained from the peanut production region of Cordoba, Argentina. The resistance was investigated in the seed coat and cotyledons of three resistant genotypes (J11, PI 337394, and PI 337409) and three breeding lines (Manfredi 68, Colorado Irradiado, and Florman INTA) developed at the Instituto Nacional de Tecnologia Agropecuaria (INTA), Manfredi Experimental Station, Cordoba, Argentina. Resistance to fungal colonization and aflatoxin contamination was found to be associated with seed coat integrity in the PI 337394, PI 337409, and J11 genotypes, whereas the INTA breeding lines such as Colorado Irradiado showed a moderate resistance and the Manfredi 68 and Florman INTA genotypes the least resistance. Furthermore, another type of resistance associated with cotyledons was found only in the PI 337394 genotype.

Analysis of protease activity in Aspergillus flavus and A. parasiticus on peanut seed infection and aflatoxin contamination.

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.

Competitive Elisa for quantifying small Amounts of aflatoxin B1

An indirect ELISA was developed to quantify aflatoxin B1 (AFB1). The detection limit was 0.025 ng ml‐1. The test used polyvinyl chloride (PVC) plates, activated with AFB1 bound to bovine serum albumin (BSA). Polyclonal antibodies were raised in rabbits against AFB1‐BSA. The specific anti‐AFB1antibodies were recovered from the crude antiserum by affinity chromatography from a column containing immobilized BSA on Nylon 6–6. Goat anti‐rabbit IgG antibodies bound to peroxidase were used to detect the rabbit IgG anti‐AFB1 antibodies bound to PVC plates. The colour developed by the subsequent enzyme conversion of the substrate was detected by spectrophotometry. The developed colour gave clear absorbance differences at varying doses of AFB1. Cross‐reactivity with aflatoxin B2, aflatoxin G1 and aflatoxin G2 was measured, showing percentages of 5.43, 64.5 and 5.07 respectively.

Pregnancy-associated α2-glycoprotein in children with acute lymphocytic leukemia, Hodgkin's disease and non-Hodgkin's lymphomas

Pregnancy-associated q-glycoprotein ((rz-PAG) is a protease-inhibiting glycoprotein which consists in two sub-units (180 kDa each) containing 10% carbohydrates [I]. Serum levels are low (< 10 pg/ml) in normal males and in non-pregnant females but increase considerably during pregnancy, after the administration of oestrogens and in relation to some neoplastic and non-neoplastic disorders [2-81. In addition, there is a possible association between q-PAG and leucocyte populations [9-l 11. In this work, we developed an EIA to measure circulating ot-PAG levels in children suffering from three different malignant hematolo~~l disorders in order to evaluate its usefulness as a diagnostic or prognostic tumor marker.

Parameters affecting the adsorption of ligands to polyvinyl chloride plates in enzyme immunoassays

In the present work, we studied the efficacy of three blocking agents (HSA, BSA and OVA) in the inhibition of non-specific binding to PVC plates. According to the inhibition data, 1% OVA was the most effective blocking agent. On the other hand, the presence of detergents in all of the blocking solutions drastically decreased the percent inhibition of the non-specific binding. Furthermore, the effect of ligand concentration on adsorption and the kinetics of ligand adsorption to PVC plates were also investigated. Ligand adsorption is a linear function of input up to a limit (around 8.70 ng/mm2) where saturation is reached. The rate of adsorption of pure human IgG to PVC plates was proportionally increased with the temperature, as shown by proportional rate constants almost 2 times faster at 37 degrees C than at 4 degrees C. These results have practical implications for investigators using PVC for immunoassays and should be taken into consideration when designing such assays.