Hybridoma technology is widely used to generate monoclonal antibodies, as the method can retain native heavy/light chain pairing and undergo in vivo affinity maturation [1,2], and the most common of these antibodies is mouse hybridoma [3,4]. After a positive hybridoma clone is obtained, purification from the culture supernatant must be performed to generate a relatively pure antibody for further analysis. The purification methods include nonchromatography methods such as precipitation, and chromatography methods such as size exclusion, ion exchange and affinity chromatography [5]. Compared to other methods, affinity chromatography is among the most widely employed methods due to its ease of operation and relatively high throughput [6].

Protein A is an important protein extracted from the cell wall of Staphylococcus aureus that can bind IgG at the junction between the CH2 and CH3 domains [7]. At present, the most commonly used ligand for antibody affinity chromatography is Protein A [8]. However, this ligand has some drawbacks when employed with the culture supernatant of hybridoma. In the classical hybridoma technique, 10–20 % fetal bovine serum (FBS) must be added to the culture medium to maintain cell growth [9,10]. Due to the lack of species specificity, Protein A retains 94 % reactivity with bovine IgG (bIgG) compared to mouse IgG [11]. Therefore, Protein A affinity chromatography is affected by bovine antibodies from FBS in the culture medium, resulting in an unsatisfactory purity for downstream applications by one-step purification. Although much effort has been dedicated to this topic [12], [13], [14], serum-free medium still has some shortcomings. For example, the medium must be optimized for different hybridoma cell lines [15,16]; furthermore, without serum, cell viability under mechanical shear stress decreases faster [17,18], and the cell growth rate is also decreased [19]. In addition, any hybridoma must endure a long adaptation process to serum-free medium. Therefore, serum-containing medium is still widely used, indicating a need to develop a purification method that is highly specific to mouse IgG (mIgG).

Immunoaffinity chromatography (IAC) is based on the binding between antibodies and antigens, which provides high specificity [20]. In mice, IgG is divided into five subclasses (IgG1, IgG2a, IgG2b, IgG2c, IgG3) depending on the heavy chain, while light chains are either kappa or lambda; moreover, kappa light chains are expressed in approximately 95 % of B cells [21]. Thus, the mouse antibody kappa constant region (mCK) is a suitable target to purify most mouse antibodies and antibody fragments. Compared to conventional antibodies, camelid heavy-chain only antibody variable domain (VHH) is suitable as a specific affinity ligand due to its high physicochemical stability, minimal size and great affinity for antigens [22]. Several studies have used VHHs as ligands to purify antibodies. For example, Klooster et al. [23] developed an affinity resin based on anti-human IgG VHH with a binding capacity of 12–15 mg/mL for human IgG. Tu et al. [24] prepared an IgG affinity resin coupling anti-Fc VHH, and the binding capacity was 15.04 ± 0.37 mg/mL for mouse IgG, but that for rabbit and human IgG also reached 3.40 ± 0.53 and 10.51 ± 0.53 mg/mL, respectively, indicating a relatively low specificity. To the best of our knowledge, there are relatively few literature reports on high specific affinity ligands for mIgG.

Magnetic beads can easily be separated from other suspended solids due to their magnetically responsive nature; thus, compared to packed bed chromatography columns, the magnetic bead method can eliminate the centrifugation and/or filtration of samples and fuse the purification and concentration steps [25,26]. As a result, the method is suitable for high-throughput purification in the early stages of antibody discovery and production [27].

In this study, we constructed an immune VHH phage display library and isolated the VHH that binds specifically to mCK but not to bovine IgG by panning and screening the library. Then, immunomagnetic beads were prepared by immobilizing VHH on magnetic beads. Finally, we evaluated several characteristics of the immunomagnetic beads. The results suggest that an immunomagnetic bead for specific affinity purification of mouse IgG from serum-containing hybridoma culture medium was successfully developed.