Detection of immunoglobulin isotypes from dried blood spots

Abstract

The study was designed to determine the sensitivity and reproducibility of recovering immunoglobulin (Ig) isotypes (IgG subclasses, IgA, IgE and IgM classes) from dried blood spots (DBS), a methodologic subcomponent of the Upstate KIDS Study. A multiplexed Luminex assay was used for IgG1/2/3/4, IgA and IgM analysis; an ELISA was used for IgE. Plasma samples from de-identified patients were used to compare the Luminex assay with nephelometry, which is routinely used to quantify IgA, IgG and IgM in clinical samples. The IgE ELISA was compared to an immunofluorescence assay. Prior to evaluation of punches from newborn dried blood spots (NDBSs), recoveries of Ig from punches of cord blood DBSs (CBDBSs) vs. plasma from the same cord bloods were compared. Although the recoveries of Ig from plasma and DBSs were not comparable, which could be due to cell lysates in the DBS samples, the analyses were reproducible. Additionally, the levels of IgA, IgG2, IgG4, and IgM recovered from CBDBSs positively correlated with those in plasma. The DBS data is a relative value since it is not equivalent to the plasma concentration. The majority of Ig concentrations recovered from 108 newborns of the Upstate KIDs Study were within the range of newborn plasma Ig levels with the exception of IgG3. The IgG4 values displayed the greatest variance with a wide range (0.01-319 mg/dl), whereas, IgG1 values had the narrowest range (85.2-960.4 mg/dl).

Keywords: Cord blood; Dried blood spots; IgE; Immunoglobulin isotypes; Newborn.

Figure 1

The Ig was extracted from punches taken from a dried blood spot on a Guthrie Card (A). A hanging drop of 50 μl of cord blood was absorbed onto an encircled area and a 3.2 mm punch is taken (B).

Figure 2

Correlation analysis of methods to assess Ig isotypes. IgA, IgM, IgG analysis by nephelometry vs. Luminex and IgE by an immunofluorescence assay vs. ELISA were compared using plasma samples. The methods showed significant positive correlations.

Figure 3

Correlation analysis of CB plasma Ig levels and the Ig concentrations recovered from the CBDBS.

Figure 4

SDS-PAGE of proteins eluated from Milliplex anti-IgA, anti-IgM, and anti-IgG beads and anti-IgE wells. The arrows indicated that the bands were identified by Western blot analysis to be the heavy (H) and light (L) chain bands of IgM, IgG and IgA. The arrow heads indicate the bands that did not react with goat anti-human IgA/IgG/IgM.

Figure 5

Ig recovered from NDBS. Ig isotypes eluted from 108 NDBSs were assayed by Luminex for IgA, IgM, and IgG subclasses (mg/dl) and by ELISA for IgE (ng/ml). The levels and ranges (log scale) are reported in relation to clinical plasma ranges for these Ig classes and subclasses. The boxes represent the 25^th and 75^th percentiles and the median values; the extended lines are the 10^th and 90^th percentile, and the dots are the samples beyond these percentiles, the outliers for this analysis.