Enzyme-linked immunosorbent assays with high sensitivity for antigen-specific and total murine IgE: a useful tool for the study of allergies in mouse models.
BACKGROUND
In studies on allergies in mouse models, IgE production is an essential parameter to be evaluated. Here, we examine the effect of commercially available immunoreaction enhancer solutions and different blocking reagents in enzyme-linked immunosorbent assay (ELISA) for total or antigen-specific murine IgE in order to improve the assays.


METHODS
Sera from mice immunized with recombinant house dust mite major allergens, Der f 1 and Der p 1, were used for the assays. Total IgE was measured by sandwich ELISA using monoclonal antibodies against murine IgE. Antigen-specific IgE was assayed using allergen-coated plates. Sensitivity or signal intensity in ELISA was compared among conditions differing in the use of enhancer solutions, blocking reagents, or monoclonal antibodies, and incubation time.


RESULTS
Use of enhancer solutions improved the sensitivity of ELISA for total IgE by approximately 30-fold of that using a conventional buffer. A blocking reagent caused more unwanted enhancement of the background signal in blank wells in ELISA for total IgE compared with another blocking reagent, however, improved signal intensity in ELISA for antigen-specific ELISA without significant enhancement of the background signal. Optimal assay conditions were determined.


CONCLUSIONS
Enhancer solutions are effective in improving ELISAs for total and antigen-specific murine IgE. Selection of blocking reagents was important to decrease unwanted enhancement of background signals and was effective in enhancing signals for positive samples. The ELISAs improved in this study are useful for the study of allergies in mouse models.
Takai
Toshiro
T
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan. t-takai@juntendo.ac.jp
Ochiai
Yuri
Y
Ichikawa
Saori
S
Sato
Emi
E
Ogawa
Takasuke
T
Tokura
Tomoko
T
Kuhara
Takatoshi
T
Kawai
Hiroshi
H
Hatanaka
Hideki
H
Takahashi
Seizo
S
Ogawa
Hideoki
H
Okumura
Ko
K
D000485:Allergens / Q000235:genetics / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000235:genetics / Q000276:immunology*; D039741:Antigens, Dermatophagoides / Q000235:genetics / Q000276:immunology; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006967:Hypersensitivity / Q000175:diagnosis / Q000276:immunology*; D007073:Immunoglobulin E / Q000032:analysis* / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification; D007202:Indicators and Reagents / Q000737:chemistry; D051379:Mice; D008808:Mice, Inbred CBA; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014611:Vaccination
D000485:Allergens; D000911:Antibodies, Monoclonal; D000941:Antigens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; C451162:Dermatophagoides pteronyssinus antigen p 1