The influence of environmental temperature and humidity on temporal decomposition of cockroach allergens Bla g 1 and Bla g 2 in feces.

The aim of the study was to establish a model of the environmental fate of German cockroach (Blattella germanica L.) allergens Bla g 1 and Bla g 2 in feces under controlled and field conditions. Temporal decline (3, 6, and 9 mo) of allergens Bla g 1 and Bla g 2 in the feces protected from cleaning was measured under laboratory and experimental household conditions. The influence of environmental temperature (15, 20, 25, 30, and 35 degrees C) and moisture (53, 75, 85, and 100% RH) on allergen degradation was estimated for 3, 6, and 9 mo. Bla g 1 was more stable than Bla g 2 and the proteins. The proteins and Bla g 2 contents were correlated negatively with the decomposition time; Bla g 1 was not. However, when the content of Bla g 1 in control and exposed tubes was compared, the decrease after exposure was significant at exposure in 35 degrees C, 53 and 100% RH. In laboratory, the shortest half-life (16-38 d) of Bla g 2 was at high temperature and humidity (100% RH at 35 degrees C), whereas the longest half-life (340 d) was at 25 degrees C and 85% RH. In the apartment, the half-life was 406 d. The results indicate that Bla g 1 and Bla g 2 allergens can persist in feces for several months under usual household humidity and temperature.

Protein chips for detection of mite allergens using Kunitz-type protease inhibitors.

Stored-food and house-dust arthropods include many species of mites and beetles that affect human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and -B4. We manufactured a protein chip with immobilized KPI-B1 and -B4 and showed trypsin/chymotrypsin-binding specificity, indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens.

Development of polyclonal antibodies for the detection of Tribolium castaneum contamination in wheat grain.

Two polyclonal antibodies (Pab) were developed for the detection of Tribolium castaneum, which is a stored product pest of medical and economical importance. Selected Pab anti- T. castaneumK51 showed low cross-reactivity to other stored product arthropods but revealed high reactivity to T. destructor, T. confusum, and partly to Tenebrio molitor. PTA-ELISA was used to detect adults, larva, and feces of T. castaneum in artificially contaminated grain samples. Calibration methods were applied to determine detection limits for each type of contaminants. Anti- T.castaneumK51 enabled detection of T. castaneum in grain samples; detection limits reached 60 and 640 individuals/kg of grain for larvae and adults, respectively, and 4 mg of feces/kg of grain. After recalculation, the detection limit for feces enables detection of 30 larvae after 5 days of feeding in optimal conditions. The main advantage of the developed assay is traceability of T. castaneum contamination, especially when the adults and larvae are removed from contaminated material, based on the detection of feces that persist in the grain.

Comparison of detection methods for Acarus siro (Acari: Acaridida: Acarididae) contamination in grain.

Acarus siro L. 1758 (Acari: Acaridida: Acarididae) is an important pest of stored grain because it contaminates the grain by allergens and transfers pathogenous microorganisms. Rapid detection of contamination enables to intercept an early grain infestation by the pest. In this study, we compared the usability and efficiency of various detection approaches. Under laboratory conditions, grain samples of various sizes were infested by different levels of the following contaminants: eggs, adults, and feces of A. siro. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA) by using anti-A. siro polyclonal antibody (immunochemical method), extracted in Berlese-Tullgren funnels, sieved, and processed by filth-flotation (conventional methods). The adults or juveniles of A. siro could be detected by all the three tested conventional methods and ELISA with detection limits in the range from 221 to 1,157 mites/kg grain. Eggs were detected by filth-flotation only; the detection limit was 1,950 eggs/kg grain. The feces of A. siro were detectable by ELISA test, only. ELISA enabled the detection of the feces with the minimal threshold level of 1.04 microg feces/g grain; it means the assay allowed to trace less than one metabolically active mite per gram of grain. The study thus demonstrated that reliable A. siro detection in grain can only be achieved by combining different detection methods. European Union and U.S. administratives dictate zero or near-zero tolerance level for mite infestation in stored products. This demand is difficult to fulfill, because every detection method is limited by its detection limit; thus, it is hard to reliably detect infestation levels lower than obtained detection limits. This methodical limitation is discussed in context with the determined detection limits of the tested methods.