Abstract
To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. Here, we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. The application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. Our workflow consists of the following two steps: 1) the use of surface plasmon resonance to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and 2) the recovery of serum IgGs using an automated small scale purification system, followed by the determination of their epitope diversity using hydrogen deuterium exchange coupled with mass spectrometry. We showed that these methods were sensitive enough to detect antigen-specific IgGs in the nanogram/μl range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. We also showed that this workflow can guide the selection of an animal that produces high affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. We postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens.
Highlights
To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses
We showed that these methods were sensitive enough to detect antigen-specific IgGs in the nanogram/l range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods
To increase the efficiency of our therapeutic antibody generation process to achieve long term success, we developed alternative methods for assessing the quality of polyclonal antibodies in immune sera, surface plasmon resonance (SPR)2 and hydrogen deuterium exchange (HDX) coupled with mass spectrometry
Summary
Serum IgG Purification Efficiency—To enable analysis by mass spectrometry, serum IgGs were purified using the PhyNexus automated purification system (Table 1). As shown in the results summary (Table 3), our analysis showed that the serum samples contained different percentages of human IL-13-specific antibodies, and exhibited significantly different average apparent affinities (Fig. 5). Among the 12 serum samples, only five (P580101, P5801-02, P5811-01, P5821-01, and P5821-02) showed binding responses toward at least one of the prioritized epitope regions Four of these samples exhibited similar double digit picomolar or better affinities and less than ϳ2-fold shifts in binding to cyno-BAFF, the serum sample from mouse P5801-02 contained the highest amounts (Ͼ30%) of human. Blockade of receptor binding was confirmed, indicating that the selection of mice based on the epitope mapping of serum IgGs is an effective strategy
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