Preparation and identification of monoclonal antibodies against recombinant human thioredoxins

Thioredoxin (Trx) is a member of the thioredoxin protein family and has a conserved catalytic domain (-Trp-Cys-Gly-Pro-Cys-Lys-) with reduction/oxidation (redox) activity. There are two main members in this family, Trx-1, a cytosolic and nuclear form, and Trx -2, a mitochondrial form. Trx-1 is a 104 amino acid multifunctional protein that has been extensively studied. Here we report the preparation of monoclonal antibodies (MAb) against recombinant human Trx-1(hTrx). To enhance its immunogenicity, Trx-1 was coupled to carrier protein bovine serum albumin by a two-step glutaraldehyde method. Using conventional procedures, we prepared three stable hybridoma cell lines that can produce and secret anti-Trx MAbs. We further analyzed their isotypes, titer, and affinity and found that those MAbs belong to the G1 subclass with kappa light chains, respectively. The MAbs were capable of recognizing hTrx-1, as determined by Western blotting.