MAB1304 Sigma-AldrichMouse Anti-Human IgG Antibody, Fd, clone HP6045

Although intravenous immunoglobulin (IVIG) is highly effective in Kawasaki disease (KD), mechanisms are not understood and 10-20% of patients are treatment-resistant, manifesting a higher rate of coronary artery aneurysms. Murine models suggest that α2-6-linked sialic acid (α2-6Sia) content of IVIG is critical for suppressing inflammation. However, pro-inflammatory states also up-regulate endogenous levels of β-galactoside:α2-6 sialyltransferase-I (ST6Gal-I), the enzyme that catalyzes addition of α2-6Sias to N-glycans. We asked whether IVIG failures correlated with levels of α2-6Sia on infused IVIG or on the patient's own endogenous IgG.We quantified levels of α2-6Sia in infused IVIG and endogenous IgG from 10 IVIG-responsive and 10 resistant KD subjects using multiple approaches. Transcript levels of ST6GAL1, in patient whole blood and B cell lines were evaluated by RT-PCR. Plasma soluble (s)ST6Gal-I levels were measured by ELISA.There was no consistent difference in median sialylation levels of infused IVIG between groups. However, α2-6Sia levels in endogenous IgG, ST6GAL1 transcript levels, and ST6Gal-I protein in serum from IVIG-resistant KD subjects were lower than in responsive subjects at both pre-treatment and one-year time points (p less than 0.001, respectively).Our data indicate sialylation levels of therapeutic IVIG are unrelated to treatment response in KD. Rather, lower sialylation of endogenous IgG and lower blood levels of ST6GALI mRNA and ST6Gal-I enzyme predict therapy resistance. These differences were stable over time, suggesting a genetic basis. Because IVIG-resistance increases risk of coronary artery aneurysms, our findings have important implications for the identification and treatment of such individuals.

We developed quantitative immunoenzymometric assays for human IgG and its subclasses by using monoclonal antibodies, an avidin-biotin detection system and, as the calibrant, the U.S. National Reference Preparation for Specific Human Proteins. The assays are sensitive (detecting as little as 6 micrograms/L), precise (average inter-assay CV less than 11%), and vary linearly with concentrations over a five- to 10-fold range, depending on the monoclonal antibody. We evaluated 22 different monoclonal antibodies, many of which remained highly reactive when immobilized in wells of microtiter plates coated with bovine serum albumin-glutaraldehyde to "capture" total IgG or subclasses of IgG in the sample. We demonstrated the specificity of the most reactive antibodies by using a panel of 20 purified myeloma proteins. The sum of IgG subclass concentrations correlated well (r = 0.84, p less than 0.001) with the total IgG measured in sera from 63 apparently healthy adults (26 men, 37 women). We estimated 95 percentile reference intervals for the immunoglobulins in these subjects and determined the following mean percentage distributions of IgG subclasses: IgG1 49, IgG2 33, IgG3 9, and IgG4 7. The availability of these assays should facilitate studies of the clinical significance of the subclasses.