A method for measuring specific IgE in sera by direct ELISA without interference by IgG competition or IgG autoantibodies to IgE.
BACKGROUND
In measuring specific IgE levels in sera by direct ELISA, competition with coexisting IgG often impedes an exact IgE determination; additionally, IgG autoantibodies to IgE (IgG-IgE) in sera affect the assay. In this paper, we attempt to determine accurate specific IgE levels by selective removal of IgG with a protein G-immobilized gel (PG) and by acid treatment of the PG to compensate for the unintended removal of IgE, probably due to the PG binding IgG-IgE.


METHODS
IgG in sera was removed using PG at pH 7.0. Then, the PG was treated with citrate buffer at pH 3.0 for 5 min to liberate IgE from IgG-IgE complexes, after IgG-binding sites on the PG were saturated with bovine IgG, since PG came to bind IgE at acidic pHs. IgE levels were then measured by ELISA.


RESULTS
The PG treatment of sera removed the effect of inhibitory competition by coexisting IgG, especially at higher concentrations of sera, to improve specific IgE detection by direct ELISA. However, PG treatment alone sometimes reduced IgE levels (39% of sera tested), even though PG does not bind IgE at pH 7.0, which indicated the presence of IgG-IgE complexes. The reduction in IgE returned almost to their original levels in the sera by acid treatment of the PG. By combining the PG treatment with acid treatment, specific IgE measurement in sera was improved significantly (p < 0.01, Wilcoxon signed rank test).


CONCLUSIONS
Measurement of specific IgE in sera by direct ELISA was improved by using the PG and acid treatment technique.
Kadooka
Y
Y
Nutritional Science Laboratory, Snow Brand Milk Products Co. Ltd., Kawagoe, Saitama, Japan.
Idota
T
T
Gunji
H
H
Shimatani
M
M
Kawakami
H
H
Dosako
S
S
Samori
T
T
D000818:Animals; D001323:Autoantibodies / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000097:blood* / Q000276:immunology* / Q000378:metabolism; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D016269:Milk Hypersensitivity / Q000175:diagnosis* / Q000276:immunology; D011852:Radioallergosorbent Test
D001323:Autoantibodies; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D007073:Immunoglobulin E