New extraction method suitable for immunoblotting analysis of fish allergens

Abstract

Immunoblotting is a simple method to analyze allergens in biological samples. In previous immunoblotting studies on fish allergens, however, collagen, an important allergen next to parvalbumin (major fish allergen), has not been detected in fish muscle extracts probably due to its unique chemical properties. This study was aimed to develop an extraction method suitable for immunoblotting analysis of fish allergens including collagen as well as parvalbumin. When various extracts from the Japanese eel white muscle were analyzed by SDS–PAGE, heating of the muscle homogenate at 80 °C for 20 min was found to be the most effective method to extract collagen as well as parvalbumin. The same extraction method was also effective for the other five species of fish analyzed (rainbow trout, Japanese horse mackerel, crimson sea bream, Pacific mackerel, and Japanese flounder). Furthermore, parvalbumin and/or collagen were successfully identified as allergens in the six species of fish by immunoblotting using the heated extracts prepared by the method described above. It can be concluded that the extraction method (heating of the muscle homogenate at 80 °C for 20 min) developed in this study is useful not only for analyzing fish allergens by immunoblotting but also for preparing antigens for diagnosis of fish allergy by RAST (radioallergosorbent test).