Chapter 31
Measurement of Antigen-Specific IgG Titers by Direct ELISA
Bassel Akache, Felicity C. Stark, and Michael J. McCluskie
Abstract
Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from
the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of
interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification of antigen-specific IgG titers in mouse serum to
measure vaccine-induced humoral responses.
Key words Vaccines, Immunoglobulin, Antibody, Humoral response, ELISA
1 Introduction
For many infectious disease indications, effective vaccines rely on
the generation of antibodies capable of binding and neutralizing
the pathogen [1]. As such, methods to accurately measure
antigen-specific antibodies are widely employed during vaccine
development, whether in (1) the preclinical setting when optimizing vaccine formulations or (2) clinically, when correlating immunological readouts to vaccine efficacy in human subjects [2–5]. In
addition, such assays are used widely for infectious disease diagnosis
or the detection of autoantibodies in an autoimmune disease
setting [6, 7]. The levels of antigen-specific antibodies are generally
assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent bead-based immunoassays. While bead-based assays offer
many advantages to ELISA such as a wider dynamic range and the
ability to multiplex (i.e., simultaneous measurement of antibodies
to multiple antigens in a single sample) [5], they are more laborious
to optimize and require specialized equipment. Due to its relative
ease to execute and adopt, the ELISA-based method is more generally employed to quantify vaccine-specific responses in a preclinical setting.
Of the various immunoglobulin types (i.e., A, D, E, G, M),
immunoglobulin G (IgG) is most commonly the isotype of interest
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_31, © Springer Science+Business Media, LLC, part of Springer Nature 2021
537
Measurement of Antigen-Specific IgG Titers by Direct ELISA
Bassel Akache, Felicity C. Stark, and Michael J. McCluskie
Abstract
Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from
the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of
interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification of antigen-specific IgG titers in mouse serum to
measure vaccine-induced humoral responses.
Key words Vaccines, Immunoglobulin, Antibody, Humoral response, ELISA
1 Introduction
For many infectious disease indications, effective vaccines rely on
the generation of antibodies capable of binding and neutralizing
the pathogen [1]. As such, methods to accurately measure
antigen-specific antibodies are widely employed during vaccine
development, whether in (1) the preclinical setting when optimizing vaccine formulations or (2) clinically, when correlating immunological readouts to vaccine efficacy in human subjects [2–5]. In
addition, such assays are used widely for infectious disease diagnosis
or the detection of autoantibodies in an autoimmune disease
setting [6, 7]. The levels of antigen-specific antibodies are generally
assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent bead-based immunoassays. While bead-based assays offer
many advantages to ELISA such as a wider dynamic range and the
ability to multiplex (i.e., simultaneous measurement of antibodies
to multiple antigens in a single sample) [5], they are more laborious
to optimize and require specialized equipment. Due to its relative
ease to execute and adopt, the ELISA-based method is more generally employed to quantify vaccine-specific responses in a preclinical setting.
Of the various immunoglobulin types (i.e., A, D, E, G, M),
immunoglobulin G (IgG) is most commonly the isotype of interest
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_31, © Springer Science+Business Media, LLC, part of Springer Nature 2021
537