A comparison between different chemical fractionation methods for immunoglobulin preparation.

Background: Application of antibodies in therapeutics and diagnostics are growing Continually. Herein, we aimed to find the most qualified immunoglobulin (Ig) chemical preparation method.

Methods: A rabbit was immunized against recombinant SARS-CoV-2 nucleocapsid (NP) and reactive polyclonal antibodies were prepared using the ammonium sulfate (AS), caprylic acid (CA), polyethylene glycol (PEG), and caprylic acid/ammonium sulfate (CA/AS) methods. Different antibody solutions were analyzed by SDS-PAGE and subsequently quantified by ImageJ software for further analysis in terms of Ig purity, Ig recovery, and albumin impurity. Ultimately, the prepared antibodies were assessed via Western blotting and ELISA to evaluate their ability to bind NP.

Results: Prepared Ig solutions via the CA/AS method had the highest Ig purity (followed by CA, PEG, and AS) and lowest albumin impurity (followed by CA, AS, and PEG). The PEG method had the highest recovery followed by AS, CA, and CA/AS methods. Moreover, antibodies prepared via different methods demonstrated comparable binding capacities to NP in ELISA and Western blotting.

Conclusions: CA/AS, closely followed by CA, proved to be the most qualified method for the preparation of Ig yielding the highest Ig purity while the PEG method resulted in the highest Ig recovery rate.