[Distribution of Mycotoxins and Usnic Acid in the Thalli of Epigenous Lichens].

The distribution of mycotoxins anc unic acid in the thallus of epigenous fruticose lichens Alectoria ochroleuca; several species of the genera Cladonia, Cotraria, and Flavocetraria; foliose lichens Nephroma arcticum; and six species of the genus Peltigera were studied by mnzyme-linked immunosorbent assay. The mycotoxin content was lower in the top or apical parts of the thalli than in the basal parts and in dead areas. Differences in the distribution of usnic acid were insignificant in the majority of species, but in Cladonia stellaris and N. arcticum lichens the content of this metabolite in the upper and pc ripheral areas was higher than in the basal parts.

[Metabolites of Toxigenic Fungi in Lichens of Genera Alectoria, Bryoria, Evernia, Pseudevernia, and Usnea].

Complexes of mycotoxins in fruticose lichens of 14 species belonging to five genera of the family Parmeliaceae were characterized by size, composition, and content of individual components. It was shown that species of the genus Bryoria always contain five mycotoxins (sterigmatocystin, mycophenolic acid, citrinin, emodin, and alternariol). In Evernia and Pseudevernia, this list is supplemented with zearalenone, diacetoxyscirpenol, and cyclopiazonic acid or fumonisins. It was noted that Alectoria and Usnea are distinguished by a peculiar set of toxic metabolites and occupy an intermediate position according to their number. The similarities and distinctions of the mycotoxin profile in species belonging to the same genus and in specimens from different habitats are discussed.

[Metabolites of Toxigenic Fungi in Lichens of the Genera Nephroma, Peltigera, Umbilicaria, and Xanthoria].

The component composition of mycotoxin complexes is characterized in foliose lichens of the genera Nephroma, Peltigera, Umbilicaria, and Xanthoria. The interspecies differences in the genus Peltigera are expressed by the number of metabolites detected, from seven in P. aphthosa to three in P. canina, P. didactyla, P. praetextata, and P. rufescens. In Nephroma arcticum eight mycotoxins occurred regularly, with mycophenolic acid in especially high quantities in comparison with other lichens. In Umbilicaria, of six permanent components the content of alternariole is the highest, and in Xanthoria the content of emodine is the highest. Variation of the quantitative content of mycotoxins in general and of species of lichens is discussed, as is expansion of the background spectrum of these metabolites in collections from different territories.

[Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups].

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were identified determined by enzyme immunoassay enzyme-linked immunosorbent assay. The following mycotoxins were identified found in these lichens in a broad concentration range with a frequency of 70-100%: sterigmatocystin (7-2090 ng/g), alternariol (20-6460 ng/g), and emodin (45-94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B1 was detected in only six species with a frequency of 2-42%, whereas roridin A was identified present in 10% of Hypogymnia physodes samples.

[Enzyme immunoassay of usnic acid in lichens].

An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families.

[Characteristics of mycotoxin accumulation in lichens].

The levels and frequencies of mycotoxin accumulation in lichens belonging to 20 genera of the families Cladoniaceae, Nephromataceae, Parmeliaceae, Peltigeraceae, Telosshistaceae, and Umbilicari- aceae were characterized using enzyme immunoassay. Alternariol, sterigmatocystin, mycophenolic acid, cit- rinin, cyclopiazonic acid, and emodin were regularly detected in all genera, except for Peltigera, at an average level of more than 1000 ng/g (i.e., 0.0001%). The necessity for the safety monitoring of drugs based on lichen extractives is discussed.

[Enzyme immunoassay of the secondary metabolites of micromycetes as components of lichen substances].

The composition of low-molecular biologically active metabolites typical of microscopic fungi has been studied in blastemas of fruticose lichens of the genera Cladonia, Cetraria, Evernia, Bryoria, and Usnes. The enzyme immunoassay method showed the presence of sterigmatocystin, emodin, mycophenolic acid, citrinin, alternariol, and diacetoxyscirpenol, which occurred regularly and, in most cases, at a frequency of 55 to 100%. The highest levels of accumulation were 0.001-0.003% for emodin, 0.0002% for alternariol and citrinin, 0.0001% for sterigmatocystin and mycophenolic acid, and 0.00005% of the weight of air-dry material for diacetoxyscirpenol. Other metabolites (cyclopiazonic acid, ergot alkaloids, ochratoxin A, PR toxin, deoxynivalenol, zearalenone, and fumonisins) were detected in these lichens less frequently (sometimes only upon the expansion of the territory of sampling), and their content was no more than 0.00005%. The peculiarities of the component composition and the levels of accumulation of fungal metabolites in lichens of different taxonomic affiliation were discussed.

[Enzyme immunassay of alternariol for the assessment of risk of agricultural products contamination].

A highly specific test system based on polyclonal rabbit antibodies targeting a conjugate of alternariol and bovine serum albumin prepared by formaldehyde condensation has been developed for the first time and shown to have the sensitivity of 0.4 ng/ml in the indirect competitive determination of alternariol. The possibility of applying the assay for assessment of the intensity of alternariol contamination of corn, animal feed, and raw materials used for the preparation of the latter has been demonstrated.

[Producers of mycophenolic acid in ensiled and grain feeds].

Using the reaction of activated N-hydrooxisuccinimide ester of mycophenolic acid, a series of immunoreactive conjugated antigens with albumins, gelatin, and glucosoxidase is obtained. On the basis of polyclonal rabbit antibodies, a test-system for indirect competitive immunoenzyme analysis is elaborated, which has the sensitivity 0.4 ng/ml. By immunoanalysis, the ability for active biosynthesis of mycophenolic acid in strains of Byssochlamys nivea (44/44, 4100-68400 ng/ml) and Penicillium roqueforti (7/16, 204-25120 ng/ml) from the mycobiota of ensiled feeds is confirmed. The correspondence between weakly expressed producing capacity of most species of fungi of the genera Penicillium and Aspergillus prevailing in grain feeds and the data on low occurrence of this metabolite in grain (8.0%) and combined feeds (11.9%) is confirmed. A potential relationship between particular cases of a significant accumulation of mycophenolic acid (from 500 to 1500 microg/kg) in grains of wheat, corn, and combined feeds and a high biosynthetic activity in rare species P. puberulum, P. stoloniferum, and P. gladioli is discussed.

A survey on the occurrence of citrinin in feeds and their ingredients in Russia.

More than 1700 samples of forage grain, sunflower and soy-bean oil-seed meal and cakes, gluten, and compounded feeds were analyzed for citrinin by indirect ELISA with a minimum detectable level of 10 ppb. Out of 829 compounded feeds (rations and concentrates) 8.8 per cent samples were positive and the amount of citrinin ranged between 12 and 182 ppb. Only 4.5% of wheat and 3.6% of barley contained citrinin at 50-998 ppb. 1.9% of maize grain samples were positive at levels of 218-953 ppb. Among the other ingredients the highest incidence (28.9%) at the levels of 14-397 ppb was found for sunflower oil-seed meal and cakes. Three positive cases of 148 processed soy-bean samples contained citrinin in a range of 14-30 ppb.

[Enzyme-linked immune sorbent assay for PR-toxin in taxonomical assessment of fungi belonging to the genus Penicillium Link].

The use of an indirect competitive enzyme-linked immune sorbent assay (ELISA) involving polyclonal rabbit antibodies against BSA-conjugated PR-toxin (sensitivity, 1 ng/ml) established the ability to synthesize PR-toxin in 18 out of 35 morphologically identified strains of Penicillium roqueforti and P. chrysogenum. The results indicate that ELISA for PR-toxin may be used in assessing the taxonomical position of terverticillate penicillia in the presence of other micotoxins.

[Products of spontaneous conjugation of aflatoxins with bovine serum albumin: immunochemical properties]. / Immunokhimicheskie svoistva spontannykh kon"iugatov aflatoksinov s bych'im syvorotochnym al'buminom.

Products of spontaneous conjugation of aflatoxins B1, G1, and G2 with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by anti-aflatoxin antibodies. Antisera against BSA-B1, BSA-G1, and BSA-G2 were obtained and their specificity, determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.

[Specific features of albumin interactions with hemiacetals of aflatoxins and sterigmatocystin]. / Osobennosti vzaimodeistviia gemiatsetalei aflatoksinov i sterigmatotsistina s albuminom.

Aflatoxin B2a (AB2a), aflatoxin G2a (AG2a), and hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB2a antibodies reacted with AB2a, aflatoxin B1, and aflatoxin AB2 (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG2a antibodies reacting with AG2a, aflatoxin G1, and aflatoxin AG@2 (100, 2.5, and < 1.0%, respectively). Binding of anti-AB2a and anti-AG2a antibodies to solid-phase conjugates of AB2a or AG2a exhibited similar analytical characteristics.

[Comparative characteristics of immunoreagents based on aflatoxin B1 hemiacetals and sterigmatocystine]. / Sravnitel'naia kharakteristika immunoreagentov na osnove gemiatsetalei aflatoksina B1 i sterimatotsistina.

Aflatoxin B1 and sterigmatocystine hemiacetal derivatives were synthesized, and their conjugation to albumins and gelatin and also spectral and immunochemthe specificity and analytical properties of the antibodies produced by immunization with conjugated antigens. The possible mechanism of hemiacetal interaction with proteins is discussed. Based on immune reagents to sterigmatocystine hemiacetal, a test system was developed for determination of sterigmatocystine at the sensitivity of 0.1 ng/ml.

[Production and analytical properties of antibodies with high specificity to zearalenone]. / Poluchenie i analiticheskie svoistva antitel s vysokoi spetsifichnost'iu k zearalenonu.

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.

[Group-specific antibodies against zearalenone and its metabolites and synthetic analogs]. / Poluchenie antitel s gruppovoi spetsifichnost'iu k zearalenonu, ego matabolitam i sinteticheskim analogam.

Formation kinetics, specificity, and analytical potential of polyclonal antibodies raised in rabbits against BSA conjugates of carboxymethyloxime-zearalanone (CMO-ZAN) and carboxymethyloxime-zearalenone (CMO-ZEN). Preparation of the conjugates involved conversion of CMO-ZAN and CMO-ZEN into activated esters and carbodiimide condensation. Two versions of a group-specific enzyme immunoassay (for zearalenone/alpha-zearalenone and zearalanol/alpha-zearalanol) based on heterologous combination of solid-phase antigens are described (sensitivity, 0.01 ng/ml).

[Comparative immunochemical characteristics of polyclonal and monoclonal antibodies to aflatoxin B1]. / Sravnitel'naia immunokhimicheskaia kharakteristika poliklonal'nykh i monoklonal'nykh antitel k aflatoksinu B1.

Four hybrid clones (MM-(AB1)-1, MM-(AB1)-2, MM-(AB1)-3, and MM-(AB1)-4) were obtained by hybridoma technology with immunization of BALB/c mice with a BSA conjugate of aflatoxin B1 carboxymethyloxime derivative. Antibodies produced by these clones varied in the ability to recognize the aflatoxin B1 analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively).

[Preparation of antibodies to roridin A and their applications]. / Poluchenie antitel k roridinu A i ikh primenenie.

Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues--roridin A, verrucarin, and verrucarol--amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 micrograms/kg.

[Ochratoxin A: study of grain contamination]. / Okhratoksin A: issledovanie kontaminatsii zerna.

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9-291.7 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.

[Immunoenzyme test system for detection of aflatoxin B1]. / Immunofermentnaia test-sistema opredeleniia aflatoksina B1.

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B1 carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B1 with a low relative cross-reactivity against aflatoxin B2, G1, G2, M1, B2a and G2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.