Application of immunoreaction enhancer solutions to an enzyme-linked immunosorbent assay for antigen-specific IgE in mice immunized with recombinant major mite allergens or ovalbumin.
BACKGROUND
Weak signals for allergen-specific IgE are a problem in murine models for the study of allergies. It has been reported that the removal of IgG from murine sera enhances signal intensity. Very recently, buffer solutions designed to enhance signals in immunoassays have been developed and made commercially available.


METHODS
Sera from mice immunized either with a recombinant form of one of the major mite allergens Der p 1, Der f 1 and Der f 2, or with ovalbumin adsorbed to alum were used for the assays. Total IgE was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Allergen-specific IgE was assayed using plates coated with the allergens after the removal of IgG from sera with protein G-coupled sepharose beads in wells of other plates or with the use of commercially available enhancer solutions without the removal of IgG. IgE binding was detected with horseradish peroxidase-conjugated anti-murine IgE monoclonal antibody as the secondary antibody.


RESULTS
Significant levels of total IgE were produced after the immunizations. The in-well pretreatment of diluted sera (1/10 dilution) with protein G-coupled beads enhanced the signals for allergen-specific IgE. The use of the enhancer solutions for dilution of the sera and secondary antibody and prolonged incubation remarkably enhanced the signals at a more extensive dilution of sera (1/200 or less) without the removal of IgG.


CONCLUSIONS
An ELISA simply modified with the use of immunoreaction enhancer solutions has advantages in terms of signal intensity and ease of handling for the detection of allergen-specific murine IgE and would be useful for the study of allergies with murine models.
Kikuchi
Yuko
Y
Atopy Allergy Research Center, Juntendo University School of Medicine, Tokyo, Japan.
Takai
Toshiro
T
Ota
Mikiko
M
Kato
Takeshi
T
Takeda
Ken
K
Mitsuishi
Kouichi
K
Ikeda
Shigaku
S
Okumura
Ko
K
Ogawa
Hideoki
H
D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D060829:Arthropod Proteins; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007114:Immunization; D007073:Immunoglobulin E / Q000097:blood*; D007074:Immunoglobulin G / Q000302:isolation & purification; D007132:Immunoglobulin Isotypes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010047:Ovalbumin / Q000276:immunology*; D012680:Sensitivity and Specificity
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D001426:Bacterial Proteins; C451160:Dermatophagoides farinae antigen f 2; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007073:Immunoglobulin E; D010047:Ovalbumin; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; C451162:Dermatophagoides pteronyssinus antigen p 1