allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease-rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18-26˚C, RT) or refrigerated (2-8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol nS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol nS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol nS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease-rich cockroach extract.

Tissue‐ferritin was homogeneously purified from the hemolymph‐free whole body of Galleria mellonella larvae. Tissue‐ferritin was composed of four subunits, 26, 30, 32 and 34 kDa. The 34 kDa subunit among them was specifically found in tissue ferritin while its N‐terminal sequence of 19 amino acid residues was identical to that of 32 kDa hemolymph‐ferritin subunit. It was observed that the 32 and 34 kDa subunit of tissue‐ferritin were glycosylated like 32 kDa subunit of hemolymph‐ferritin. It suggests that the 34 kDa subunit is an isoform of 32kDa ferritin subunit according post‐transcriptional modification. By semi quantitative RT‐PCR, the distribution of 32 kDa ferritin subunit mRNA was observed in fat body, Malpighian tubules, integument, and muscle, but found in a very small amount in silk gland or not found in gut. On the other hand, 26 kDa subunit mRNA was found in all organs tested although it was in a small quantity found in gut and silk gland. By dietary Hg treatment, 34 kDa subunit of tissue‐ferritin was clearly up‐regulated.

The effect of Hg treatment on hemolymph and tissue ferritin in the wax moth Galleria mellonella was examined by western blotting. At 48 h after feeding HgCl2, the level of hemolymph ferritin increased approximately 1.8‐fold over that of control insects that were not fed HgCl2, while there was a small increase in tissue ferritin. Time series experiments showed that tissue ferritin had a typically saturated pattern, with a maximum level from 24 to 72 h, although it decreased 12 h following HgCl2 feeding, while hemolymph ferritin first decreased but subsequently increased. Tissue ferritin in the fat body, gut and Malpighian tubules, the main tissues of ferritin expression, was upregulated over time following treatment with Hg, and in particular, tissue ferritin in the gut increased by a large amount at 12–48 h. The results suggest that in G. mellonella, the ferritin‐inducible mechanisms following treatment with HgCl2 are different for hemolymph and tissue ferritin, as are their biochemical properties.

The effects of seven heavy metals on hemolymph and tissue ferritin in Galleria mellonella were examined by western blotting. Hemolymph ferritin was clearly upregulated by Hg, while tissue ferritin was dramatically upregulated by Cd, Cu, Mn, Zn and Ni. The level of hemolymph ferritin for Hg and Cd increased approximately 1.5‐fold over normal controls, while the level of tissue ferritin for Cd, Cu, Mn, Zn and Ni increased more than twofold, indicating that the regulation of hemolymph and tissue ferritin differ with the different heavy metals. Compared to normal control insects not fed CdCl2, ferritin subunit mRNA from the Malpighian tubules, muscle, the gut and the integument was clearly downregulated, as determined by semi‐quantitative reverse transcription–polymerase chain reaction, but ferritin subunit mRNA from the silk gland and the fat body did not show any change. These results suggest that in G. mellonella, tissue ferritin is responsible for Cd stress and is thus important for survival of the moth, and that tissue and hemolymph ferritin have different ferritin‐inducible mechanisms in response to dietary CdCl2.