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. 2015 Mar 25;10(3):e0119298.
doi: 10.1371/journal.pone.0119298. eCollection 2015.

Competition between serum IgG, IgM, and IgA anti-glycan antibodies

Saddam M Muthana  1 , Li Xia  1 , Christopher T Campbell  1 , Yalong Zhang  1 , Jeffrey C Gildersleeve  1
Affiliations

Competition between serum IgG, IgM, and IgA anti-glycan antibodies

Saddam M Muthana et al. PLoS One. .

Abstract

Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Competition between serum IgG, IgA, and IgM anti-glycan antibodies.
(A) Addition of IgM and IgA to IgG. Polyclonal IgG isolated from serum was first profiled on the array alone. Separately, IgG was premixed with varying amounts of IgM or IgA and then profiled on the array. For each array component, the change in IgG signal in the presence of IgM or IgA was determined. The box plots depict the range of IgG changes (on a log base 2 scale) measured on the array upon addition of 4 serum equivalents of IgM or IgA. The line in the middle of the box is the median, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. (B) Addition of IgG and IgA to IgM. An analogous protocol as above was used to evaluate effects of IgG and IgA on IgM signals. (C) Addition of IgM and IgG to IgA. An analogous protocol as above was used to evaluate effects of IgG and IgM on IgA signals. The box plots demonstrate significant decreases in IgG and IgA signals in the presence of IgM for the vast majority of array components.
Fig 2
Fig 2. Dose-dependent inhibition of IgG and IgA signals upon addition of IgM.
(A) Decrease in IgG signals with increase in IgM concentration. (B) Decrease in IgA signals with increase in IgM concentration. Polyclonal IgG and IgA isolated from serum were first profiled on the array alone. Separately, they were pre-mixed with varying amounts of IgM and then profiled on the array. The box plots depict the range of IgG or IgA signals (on a log base 2 scale) alone or in the presence of varying amounts of IgM (50–400 μg/mL). The line in the middle of the box is the median signal, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. The box plots demonstrate decreasing IgG and IgA signals in the presence of increasing amounts of IgM across the entire array.
Fig 3
Fig 3. Dependence of inhibition on carbohydrate antigen structure.
Measured IgG signals to Forssman disaccharide and tetrasaccharide (A) and four blood group A antigens (B) in the absence of added IgM (0 μg/mL) or in the presence of varying amounts of IgM (50–400 μg/mL).
Fig 4
Fig 4. Changes in serum IgG and IgM signals in a pooled serum sample in the presence of varying amounts of added IgG, IgA, or IgM.
(A) Changes in IgG signals upon addition of IgM. (B) Changes in IgG signals upon addition of IgA. (C) Changes in IgM signals upon addition of IgG. (D) Changes in IgM signals upon addition of IgA. IgG and IgM signals in a pooled serum sample were first profiled on the array alone. Separately, the serum sample was pre-mixed with varying amounts of IgG, IgA, or IgM and then profiled on the array. The box plots depict the range of IgG or IgM signals (on a log base 2 scale) alone or in the presence of varying amounts of IgG, IgA, or IgM. The line in the middle of the box is the median signal, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. The only significant decreases observed are for IgG signals upon addition of IgM.
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