Anticorpo monoclonal anti-AFB1 : coluna de imunoafinidade e espectrofluorimetria para controle de qualidade de alimentos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

The frequent occurrence of aflatoxin in foodstuffs causes a potential negative impact on Brazilian agribusiness. Aflatoxin has been classified in group 1 by International Association in Research on Cancer. The production of immunoreagents associated with immunochemical assay developments could allow accessible and low cost quality control aiming prevention of contamination and safe food supply to consumers. Considering the need in minimizing the dependence on immunochemical kits imports, which are essential to carry out advanced toxic residues analysis routinely in laboratories, the technology of monoclonal antibody (mAb) using hybridoma cell line AF4 that produces IgG against aflatoxin B1 (AFB1) was introduced. Cell culture in synthetic medium raised 632.63 mg of purified IgG (82.18 mg/mL), overcoming the expectation described in literature, i.e. 50.00 mg/mL of mAb, due to the use of L-glutamine and cell culture until death. Semi-purified and lyophilized mAb against AFB1 was used to prepare the immunoaffinity column (IAC) using activated Affi-Gel 10 immunoaffinity support. IAC Affi-Gel 10 coupled with 0.5 mL of IgG-gel showed an AFB1 retention capacity of 25.32 ng, with further toxin elution from IAC with 15 mL of methanol. Spectrofluorimetry was used to quantify the AFB1, where the excitation and emission wavelengths were standardized to 360 and 420 nm, respectively, which were selected based on the best sensibility and stability through consecutive readings of pure AFB1. The IAC Affi-Gel 10 developed was not totally efficient concerning the cleaning-up of artificially contaminated corn with AFB1. The end-capping procedure must be continued to reach the effectiveness which is equivalent to the commercial IAC Aflatest-P (VICAM) (recovery of 72.40 to 79.77 %). Nevertheless, taking into account the specific activity of mAb against AFB1, checked by the AFB1 retention capacity test, the following study should insert new conditions concerning column end-capping and standardizing assays. The reusability of IAC Aflatest-P was evaluated after column regeneration, but the data were variable, reaching 34.15 to 82.88 % for AFB1 recovery. Such data showed that the reuse of commercial IAC may lead to analysis error. In short, the production of mAb against AFB1 was successfully reached. It is also recommended a further procedure using ultrafiltration at the concentration step and antibody maintenance at 4º C to keep its integrity and biological activity. Therefore, the availability of specific mAb, cleaning-up of food matrices extract in a single chromatographic step and also toxic solvents reduced usage and their possible reusability justify the IAC development bearing in mind the reduction in quality control costs.

ASSUNTO(S)

alimentos - qualidade aflatoxins aflatoxina food - quality

ACESSO AO ARTIGO

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