Efficient Qualitative and Quantitative Determination of Antigen-induced Immune Responses.
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.
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Danlin
0000-0002-5085-5950
D
Yang
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Lee
0000-0002-0225-3410
L
Frego
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Marcio
0000-0001-9384-0794
M
Lasaro
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Kristopher
0000-0002-2797-2622
K
Truncali
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Rachel
0000-0003-1413-1223
R
Kroe-Barrett
From the Department of Biotherapeutics Discovery, Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877 sanjaya.singh@boehringer-ingelheim.com.
Sanjaya
0000-0002-9119-7966
S
Singh
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D020349:Surface Plasmon Resonance / Q000379:methods*
D000936:Antigen-Antibody Complex; D000941:Antigens; D007074:Immunoglobulin G
drug discovery; epitope mapping; immunology; kinetics; mass spectrometry (MS); serum; surface plasmon resonance (SPR); vaccine development