Development of a bead immunoassay to measure Vi polysaccharide-specific serum IgG after vaccination with the Salmonella enterica serovar Typhi Vi polysaccharide

Abstract

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 microg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.

FIG. 1.

Comparison of anti-Vi IgG standard curves using the ELISA or bead assay. A human serum standard (pooled) containing anti-Vi IgG (200 ELISA units/ml in undiluted serum) was tested over various dilutions by ELISA or bead assay, and the raw data values from each assay platform were plotted against the anti-Vi IgG EU/ml in the diluted serum. The average values for sample diluent controls included in each assay are indicated. The results shown in Fig. 1 represent a total of 32 standard curves generated using ELISA and 46 standard curves generated using the bead assay.

FIG. 2.

The Vi bead assay provides a superior signal-to-noise ratio versus ELISA. The signal-to-noise ratio was calculated for the Vi standard curve raw data for each assay platform by dividing the raw data value at each standard concentration by the raw data for the diluent control. Results shown in Fig. 2 represent a total of 32 standard curves generated using ELISA and 46 standard curves generated using the bead assay.

FIG. 3.

Measurement of anti-Vi IgG in serum samples from vaccinated subjects (ELISA versus bead). Anti-Vi IgG EU/ml was measured in 51 serum samples collected before vaccination with Vi (day 0) or 28 days after vaccination (day 28). Response to vaccination was calculated by subtracting the day 0 anti-Vi IgG EU/ml from the day 28 value (day 28 − day 0).

FIG. 4.

Serum anti-Vi IgG EU/ml before and after vaccination with Vi polysaccharide, as measured with the Vi bead assay. Serum samples from 996 participants were collected before (day 0) and after (day 28) vaccination with the Vi polysaccharide vaccine and tested for the presence of anti-Vi IgG with a bead immunoassay. The box represents the median and the 25th and 75th percentiles, while the whiskers the represent the largest and smallest values. *, day 28 values significantly increased compared to the day 0 values (P < 0.0001; two-tailed Mann-WhitneyU test).

FIG. 5.

Estimation of anti-Vi IgG μg/ml. (A) A human immunoglobulin isotyping kit (catalog no. 48-302; Millipore, Billerica, MA) was performed according to manufacturer's instructions except that the anti-human IgG-rPE detection reagent utilized in the Vi bead assay was used as the detection reagent. (B) MFI values for the anti-Vi IgG standard curves that were within range of the total IgG standard curve were used to estimate the median IgG concentration of the Vi-specific IgG in the standard. This was used to plot anti-Vi IgG EU/ml versus anti-Vi IgG ng/ml to estimate protective levels of anti-Vi IgG (i.e., 1 μg/ml).