Immunoassays for detection of Alternaria: development and applications

Fungi of Alternaria genus are widespread in the nature. Most of them are saprophytes and grow on any organic substrates. The storage of Alternaria are decaying plants and the vegetative waste from which the fungi reaches soil. As well as other fungi, Alternaria takes part in decomposition and a mineralization of the vegetative waste. Up to now, more than 100 species were described, and only ca 25 of them are most common and have practical value. Phytopathogenical species often infect cereals, potatoes, tomatoes, citrus. Alternaria induces diseases of different agricultural crops. A. tenuis induces spottiness of cotton and citrus plants; A. brassicae affects cabbage, pepper, aubergines. A. dauci affects carrot, A. solani – potatoes and tomatoes. A. tenuis produces tentotoxin that inhibits production of chlorophyll. Alternaria is widely distributed among cereals – in wheat, in rye, in barley. Alternaria antigens may remain and accumulate in foods during food processing, especially if no high temperature heating is used. Besides of cereals, Alternaria also colonizes processed fruit and vegetables, eg juices, jams and tomato paste. Fungi from genus Alternaria can survive on different synthetic substrates and cause damage of furniture and clothes. Alternaria antigens are also detected in conditioning systems and refrigerators, because low temperature and high humidity create favorable conditions. Almost all species of Alternaria seeded from grains produce mycotoxins, such as alternariol and tenuazonic acid. The presence of Alternaria antigens in food may cause allergic symptoms. Spores of Alternaria are capable to induce chronic rhinoconjunctivitis or bronchial asthma after inhalation.

Alternaria alternata (Ag826) extract was used as the source of immunogen to immunize rabbits. Rabbit antiserum against Alternaria (AS826) was further enriched by affinity chromatography on immobilized antigen. Affinity purified antibodies (AS826 A) are available for research.

ELISA kit (K826) was constructed from AS826 antiserum as single site competition assay, see IFU
Additional kit information:
Test principle: competitive, polyclonal antibodies
Total time of analysis: 60/30/15 min
Sensitivity: 0.5 µg/ml of A.alternata antigens
Material: grain

Accumulation of antigens measured by K826 Alternaria ELISA in cultivation medium correlates well with the number of conidia (see figure below). Accumulation of antigen in culture fluid in general reflects the growth of fungal culture

Species specificity of K826 Alternaria EIA In spite of heterogeneity of Alternaria species and strains extracts (see the table below) most of them show good reactivity in K826 Alternaria EIA

Applications of of K826 Alternaria EIA – the examples

Material tested	Alternaria antigens, µg/ml	Fusarium antigens, µg/ml	Comments
Alternaria ALLERGEN	>500	<1	skin prick test, AllergopharmaTM
Fusarium ALLERGEN	<0.5	>500
Alternaria alternata infected wheat	>200	<5	Experimental infection in the field during florification
Fusarium graminearum infected wheat	<10	>500
25 lots of grain from fields	5	6	Infection background, µg/g
	12; 16; 17; 22	12; 33; 272	Positives, µg/g