IMMUNOLOGICAL DETECTION OF FUNGAL CONTAMINATION IN FOODS AND CHARACTERIZATION OF ANTIGENS (IMMUNOASSAY, ELISA)

HER HELEN LIN, Purdue University

Abstract

A double-sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of molds (Alternaria alternata, Geotrichum candidum and Rhizopus stolonifer) in food products, including tomato puree, applesauce, apricot nectar, peach puree, bread and cottage cheese. Detection limits for G. candidum, R. stolonifer and A. alternata were 100, 1000, and 100 ng dried mold/g of sample, respectively. Cross reactivity among the three species was less than 10%. Specificity of the ELISA was studied using twenty-eight other strains or isolates of molds and yeasts. Of the strains studied, the antiserum to A. alternata cross reacted strongly with A. cucumerina, Epicoccum sp. and Leptosphaerulina briosiana, and weakly with several other strains; the antiserum to R. stolonifer only cross reacted with Mucor sp. which is in the same family (Mucoraceae) as Rhizopus; while the antiserum to G. candidum was very specific and essentially only reacted with G. candidum. Five isolates of G. candidum other than the one (ATCC 7115) that was used to induce antibody were studied. All of them reacted positively but at different degrees with the antisera. The ELISA was able to detect both biologically viable and non-viable molds. Positive relationships between ELISA absorbances and the amount of mold added to food samples were observed in every case. The food components in general did not cross react with fungal antigens; therefore, low background was observed. But their presence did affect the efficiency of the antigen-antibody reaction. The effect was lower for fruit products but higher for bread and cottage cheese samples. For the latter, the dilution factor of the samples was very important in determining the efficiency of the assay, since higher dilution rates usually gave more satisfactory results. Effects of modification in the ELISA procedure were studied to eliminate the effects due to foods, but none of them seemed advantageous. A comparison was made between ELISA and Howard mold count (HMC). Closely related results were obtained from the two methods. While HMC detected mold in general, ELISA was more specific. The antigenic components of the molds were studied. High molecular weight, phosphate buffered saline (PBS)-extractable and heat stable antigens were isolated by gel filtration. Further analysis suggested that they were polysaccharides in nature with glucose as the major sugar residue.

Degree

Ph.D.

Subject Area

Food science

COinS