Modification of Periodate Oxidation Method to Produce HRP-IgG Conjugate and Test its Stability Overtime

Abstract

Goat anti-Rabbit Horseradish Peroxidase (HRP) secondary conjugate was produced using a modified periodate oxidation method. The obtained conjugate was tested in the quality control techniques of therapeutic proteins. To determine the working dilution, titration of the prepared conjugate was performed in Indirect Enzyme-Linked Immunosorbent Assay (ELISA) and found to be 1:5000. This dilution was further tested in Western Blot analysis. The secondary conjugate was kept at 4˚C for one month and its stability was verified by Western Blot and Indirect ELISA techniques.

Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme–Antibody Conjugation Reaction and Quality Control of a Final Product

“…The IgG-HRP conjugate was prepared by the modified periodate oxidation method, which was first described by Nakane and Kawaoi [6]. The protocols for the periodate oxidation method described in the literature vary according to the concentration of the IgG, HRP, and required reagents, the NaIO 4 /HRP and HRP/IgG molar ratio, the type of reducing agent, the duration of the reaction steps, the pH at which the reaction steps are performed, and the methods used to purify the IgG-HRP conjugate and remove the unconjugated enzyme [4,5,16,19].…”

“…However, the ultrafiltration step led to the increase of aggregate content (Table 2, Figure 2), possibly as a consequence of a combination of several factors, such as the high protein concentration, enhanced solution viscosity, and physical stress, which implies the filtration rate, fluid flow-induced shear stress, interfacial interactions, and extensive contact with the membrane surface [21][22][23][24]. Such a finding suggests that ultrafiltration might not be the best method for the matrix exchange and goes in favor of dialysis, as described in the majority of published protocols [4,6,16,17]. Namely, the ultrafiltration process involves a transient increase in the concentration of the sample in contrast to dialysis, which might be a trigger for aggregation.…”

“…The HRP activation was performed in the dark to prevent sodium periodate breakdown and only for a limited period of time to avoid the loss of enzymatic activity [4,5]. Self-coupling of the activated HRP was minimized through the removal of the periodate excess by ultrafiltration against 1 mM sodium acetate buffer, pH 4.4 [4,16]. Aliquots of the HRP solution before and after activation were examined by multi-detection SEC (Figure S3 and Table S2).…”

“…After the periodate oxidation of the polysaccharide residues, Schiff bases formed between the amino groups of the IgG and the active aldehyde groups of the HRP in an alkaline pH were further converted into stable alkylamine linkages by a reduction with sodium borohydride [4,17]. Finally, ultrafiltration of the resulting IgG-HRP conjugate was performed, giving the final product (UF IgG-HRP).…”

“…The polysaccharide residues of the HRP molecule may be oxidized with sodium periodate to generate reactive aldehyde groups that can react with the amino groups of an antibody (IgG) through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reductive amination with sodium cyanoborohydride or sodium borohydride [3][4][5][6].…”