Requirement of a Blocking Step in Affinity Purification of Polyclonal Antibodies

∗ Corresponding author: Department of Medical Biotechnology, Faculty of Allied Medical Sciences & Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, PO Box: 14155-6183, Iran. E-mail: [email protected] ntibodies play an essential role in medicine. Today diagnosis and treatment of many diseases require these “magic bullets”. Furthermore, antibodies are essential components of a myriad of biomedical research. Hence, producing high quality premium antibodies is a great concern in molecular medicine and biotechnology. Since antibodies are produced in complex matrices i.e. serum for polyclonal antibody and cell culture media for monoclonal antibody, efficient purification of antibodies becomes imperative. Affinity purification is among the most extensively used methods for antibody purification. Affinity purification offers high selectivity and purity levels often above 95% in one step (1). Due to high specificity, antigen-specific affinity purification is the most popular method for affinity purification (2). Non-specific bindings (NSB) in affinity purification of antibodies are required to be minimized if antibodies are to achieve high analytical sensitivity and specificity. Various measures are taken to reduce NSB in affinity purifications. For instance, it is routine to use wash buffer containing additional salt or detergent to disrupt any weak interactions. Non-specific binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be reduced by pretreatment with glycine or ethanolamine (3). A recent approach to minimizing non-specific protein interactions in high throughput screens has utilized pre-equilibration of affinity surfaces with thiocyanate (4). In the present study, we examined whether blocking the affinity resin with bovine serum albumin (BSA) significantly reduces NSB in antigen-specific affinity purification of antibodies. TNFAIP8 peptide [tumor necrosis factor-alpha (TNFα)-induced protein 8] and rabbit serum containing anti-TNFAIP8 antibody both obtained from Sigma were used to conduct immunoaffinity purification in two methods: conventional and modified. Both immunoaffinity purifications were performed using NHS-Activated Agarose dry resin (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol.The modified method, however, included an extra step, a blocking step with BSA, performed after quenching with 1 M A Submmited 3 August 2015; Accepted 22 September 2015; Published 11 October 2015