Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA

Abstract

Tropomyosin (TPM) is considered as a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products iscrucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDSPAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content.

Tropomiozin (TPM) se smatra glavnim alergenom morskih plodova. Razvoj metoda za kvantifikaciju TPM-a u prehrambenim proizvodima je ključan za alergične osobe. Nekoliko pufera za ekstrakciju je upoređeno u pogledu efikasnosti ekstrakcije protein iz sveže zamrznutih i kuvanih gambora, tokom 2 i 24 časa. Sadržaj proteina u solubilnim ekstraktima je određen Bredfordovom metodom. Proteinski profil ekstrakata je analiziran SDS-PAGE metodom. Za kvantifikaciju TPM-a je razvijena sendvič ELISA metoda. Nijedan puffer nije pokazao značajnu razliku u količini ekstrahovanih protein nakon 2 i 24 časa ekstrakcije. U poređenju sa sirovim, značajno manje proteina je ekstrahovano iz kuvanih gambora. Kvantifikacija TPM-a ELISA metodom je pokazala da se fosfatom puferisanim fiziološkim rastvorom (PBS) koji sadrži 1 M NaCl (PBSN) i karbonatnim puferom, pH 10, ekstrahuje oko 6 puta više TPM-a u poređenju sa PBS-om. Ovo ukazuje na značaj odabira adekvatnog pufera za ekstrakciju TPM-a, jer se upotrebom tradicionalno korišćenih pufera može potceniti koncentracija TPM-a u gamborima.