Preparation of Recombinant Parvalbumins and ELISA
Protein sequences of parvalbumin from tuna (Thunnus albacares , Accession# CAQ72967), salmon (Salmo salar , β-1, Accession# CAA66403.1 and β-2, Accession# Q91483.3), cod (Gadus morhua , Accession# AAK63089.1), grouper (Epinephelus lanceolatus , Accession# XP_033500493.1), catfish (Ictalurus punctatus , Accession # AA025757.1), grass carp (Ctenopharyngodon idella , Accession# QCY53440.1) and tilapia (Oreochromis mossambicus , Accession #AAZ52553.1) were obtained from the NCBI database and reversed translated by MEGA X for cloning into His-tag expression vector pET30(A)+. His-tagged recombinant parvalbumins were then expressed inEscherichia coli BL21 (DE3) by culturing in MagicMedia (Invitrogen, Carlsbad, CA, USA) following the routine protocol in our laboratory16-18 . Allergens were then purified using the HisPur cobalt spin columns (Thermo Fisher Scientific, Rockford, IL, USA) as per manufacturer’s instructions. The concentration and purity of purified recombinant allergens were determined using the NanoDrop OneC spectrophotometer and SDS-PAGE, respectively. Purified recombinant parvalbumins were then coated onto MaxiSorp microtiter plates and incubated against diluted sera (1:10) from 37 fish-sensitized subjects for ELISA following our standard protocol as previously described 19 .