in a nonmucosal vaccine setting. This monomeric immunoglobulin
is the predominant isotype in blood and extracellular fluid
[8]. While antigen-specific IgM dominates the humoral response
immediately following antigen exposure, it is succeeded by IgG as
antibody isotype class-switching occurs during maturation of the
antibody response [9]. Herein, using influenza hemagglutinin as a
model antigen, we describe an ELISA-based method to measure
antigen-specific IgG titers in mouse samples. While a sandwich
ELISA generally relies on two different antibodies to capture and
detect a protein of interest, this direct antibody ELISA utilizes the
antigen of interest and an enzyme-linked secondary antibody to
capture and detect the IgG molecules (Fig. 1). This method has
allowed for the determination of the impact of (1) various adjuvants
on the immunogenicity of vaccine formulations, (2) repeated vaccine administration, and (3) longevity of the immune response. It
can be easily adapted to different antigens or antibody types (different isotypes, subtypes and/or species).
2 Materials
Use ultrapure water to prepare all necessary buffers/solutions. The
Wash buffer, Citrate buffer and Stop solution may be prepared in
advance. However, the Blocking buffer, Diluent buffer, and Substrate solution are to be prepared on the day of the assay as
described below.
2.1 Antigens
and Antibodies
1. Influenza A H1N1 hemagglutinin: Dissolved and dispensed
into single use aliquots, which are then frozen at <À15
C.
2. Goat anti-mouse IgG-horseradish peroxidase (Southern Biotech, Birmingham, AL, USA): Stored at 2–8
C (see Note 1).
3. Goat anti-mouse IgG1-horseradish peroxidase (Southern Biotech): Stored at 2–8
C.
4. Goat anti-mouse IgG2a-horseradish peroxidase (Southern
Biotech): Stored at 2–8
C.
2.2 Solutions
and Reagents
1. Fetal Bovine Serum (FBS).
2. Wash Buffer (10Â stock solution): Add indicated amounts of
following chemicals to 3 L of ultrapure water: 8.0 g monopotassium phosphate (KH 2 PO 4 ), 24.4 g disodium phosphate
(Na 2 HPO 4 ), 8.0 g potassium chloride (KCl), and 320.0 g
sodium chloride (NaCl). Once dissolved, add 20 mL of
Tween 20 and bring up volume to 4 L with ultrapure water.
Dilute this 10Â stock solution tenfold in ultrapure water to
achieve the 1Â solution used below for preparation of diluent
538
Bassel Akache et al.
is the predominant isotype in blood and extracellular fluid
[8]. While antigen-specific IgM dominates the humoral response
immediately following antigen exposure, it is succeeded by IgG as
antibody isotype class-switching occurs during maturation of the
antibody response [9]. Herein, using influenza hemagglutinin as a
model antigen, we describe an ELISA-based method to measure
antigen-specific IgG titers in mouse samples. While a sandwich
ELISA generally relies on two different antibodies to capture and
detect a protein of interest, this direct antibody ELISA utilizes the
antigen of interest and an enzyme-linked secondary antibody to
capture and detect the IgG molecules (Fig. 1). This method has
allowed for the determination of the impact of (1) various adjuvants
on the immunogenicity of vaccine formulations, (2) repeated vaccine administration, and (3) longevity of the immune response. It
can be easily adapted to different antigens or antibody types (different isotypes, subtypes and/or species).
2 Materials
Use ultrapure water to prepare all necessary buffers/solutions. The
Wash buffer, Citrate buffer and Stop solution may be prepared in
advance. However, the Blocking buffer, Diluent buffer, and Substrate solution are to be prepared on the day of the assay as
described below.
2.1 Antigens
and Antibodies
1. Influenza A H1N1 hemagglutinin: Dissolved and dispensed
into single use aliquots, which are then frozen at <À15
C.
2. Goat anti-mouse IgG-horseradish peroxidase (Southern Biotech, Birmingham, AL, USA): Stored at 2–8
C (see Note 1).
3. Goat anti-mouse IgG1-horseradish peroxidase (Southern Biotech): Stored at 2–8
C.
4. Goat anti-mouse IgG2a-horseradish peroxidase (Southern
Biotech): Stored at 2–8
C.
2.2 Solutions
and Reagents
1. Fetal Bovine Serum (FBS).
2. Wash Buffer (10Â stock solution): Add indicated amounts of
following chemicals to 3 L of ultrapure water: 8.0 g monopotassium phosphate (KH 2 PO 4 ), 24.4 g disodium phosphate
(Na 2 HPO 4 ), 8.0 g potassium chloride (KCl), and 320.0 g
sodium chloride (NaCl). Once dissolved, add 20 mL of
Tween 20 and bring up volume to 4 L with ultrapure water.
Dilute this 10Â stock solution tenfold in ultrapure water to
achieve the 1Â solution used below for preparation of diluent
538
Bassel Akache et al.