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 .